Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

Energy Technology Data Exchange (ETDEWEB)

Orcutt, Kelly Davis; Slusarczyk, Adrian L. [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Cieslewicz, Maryelise [Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Ruiz-Yi, Benjamin [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Bhushan, Kumar R. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Frangioni, John V. [Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Department of Radiology, Beth Israel Deaconess Medical Center, Boston, MA 02215 (United States); Wittrup, K. Dane, E-mail: wittrup@mit.ed [Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States); Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139 (United States)

2011-02-15

Introduction: In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelates for use in PRIT applications. Methods: We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to DOTA, reformatted as a single chain variable fragment (scFv). Results: Modeling predicts that for high antigen density and saturating bsAb dose, a hapten-binding affinity of 100 pM is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nM to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2{+-}1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen, pretargeted high-affinity scFv results in significantly higher tumor retention of a {sup 111}In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions: We have engineered a versatile, high-affinity, DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.

DOTA-PESIN, a DOTA-conjugated bombesin derivative designed for the imaging and targeted radionuclide treatment of bombesin receptor-positive tumours

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Zhang, Hanwen; Maecke, Helmut R. [University Hospital Basel, Division of Radiological Chemistry, Department of Radiology, Basel (Switzerland); Schuhmacher, Jochen; Eisenhut, Michael [German Cancer Research Centre, Department of Radiopharmaceutical Chemistry, Heidelberg (Germany); Waser, Beatrice; Reubi, Jean Claude [University of Berne, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, P.O. Box 62, Berne (Switzerland); Wild, Damian [University Hospital, Clinic and Institute of Nuclear Medicine, Department of Radiology, Basel (Switzerland)

2007-08-15

We aimed at designing and developing a novel bombesin analogue, DOTA-PEG{sub 4}-BN(7-14) (DOTA-PESIN), with the goal of labelling it with {sup 67/68}Ga and {sup 177}Lu for diagnosis and radionuclide therapy of prostate and other human cancers overexpressing bombesin receptors. The 8-amino acid peptide bombesin (7-14) was coupled to the macrocyclic chelator DOTA via the spacer 15-amino-4,7,10,13-tetraoxapentadecanoic acid (PEG{sub 4}). The conjugate was complexed with Ga(III) and Lu(III) salts. The GRP receptor affinity and the bombesin receptor subtype profile were determined in human tumour specimens expressing the three bombesin receptor subtypes. Internalisation and efflux studies were performed with the human GRP receptor cell line PC-3. Xenografted nude mice were used for biodistribution. [Ga{sup III}/Lu{sup III}]-DOTA-PESIN showed good affinity to GRP and neuromedin B receptors but no affinity to BB3. [{sup 67}Ga/{sup 177}Lu]-DOTA-PESIN internalised rapidly into PC-3 cells whereas the efflux from PC-3 cells was relatively slow. In vivo experiments showed a high and specific tumour uptake and good retention of [{sup 67}Ga/{sup 177}Lu]-DOTA-PESIN. [{sup 67}Ga/{sup 177}Lu]-DOTA-PESIN highly accumulated in GRP receptor-expressing mouse pancreas. The uptake specificity was demonstrated by blocking tumour uptake and pancreas uptake. Fast clearance was found from blood and all non-target organs except the kidneys. High tumour-to-normal tissue ratios were achieved, which increased with time. PET imaging with [{sup 68}Ga]-DOTA-PESIN was successful in visualising the tumour at 1 h post injection. Planar scintigraphic imaging showed that the {sup 177}Lu-labelled peptide remained in the tumour even 3 days post injection. The newly designed ligands have high potential with regard to PET and SPECT imaging with {sup 68/67}Ga and targeted radionuclide therapy with {sup 177}Lu. (orig.)

Improved tumor-targeting MRI contrast agents: Gd(DOTA) conjugates of a cycloalkane-based RGD peptide

International Nuclear Information System (INIS)

Park, Ji-Ae; Lee, Yong Jin; Ko, In Ok; Kim, Tae-Jeong; Chang, Yongmin; Lim, Sang Moo; Kim, Kyeong Min; Kim, Jung Young

2014-01-01

Highlights: • Development of improved tumor-targeting MRI contrast agents. • To increase the targeting ability of RGD, we developed cycloalkane-based RGD peptides. • Gd(DOTA) conjugates of cycloalkane-based RGD peptide show improved tumor signal enhancement in vivo MR images. - Abstract: Two new MRI contrast agents, Gd-DOTA-c(RGD-ACP-K) (1) and Gd-DOTA-c(RGD-ACH-K) (2), which were designed by incorporating aminocyclopentane (ACP)- or aminocyclohexane (ACH)-carboxylic acid into Gd-DOTA (gadolinium-tetraazacyclo dodecanetetraacetic acid) and cyclic RGDK peptides, were synthesized and evaluated for tumor-targeting ability in vitro and in vivo. Binding affinity studies showed that both 1 and 2 exhibited higher affinity for integrin receptors than cyclic RGDyK peptides, which were used as a reference. These complexes showed high relaxivity and good stability in human serum and have the potential to improve target-specific signal enhancement in vivo MR images

Improved tumor-targeting MRI contrast agents: Gd(DOTA) conjugates of a cycloalkane-based RGD peptide

Energy Technology Data Exchange (ETDEWEB)

Park, Ji-Ae, E-mail: jpark@kirams.re.kr [Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Lee, Yong Jin; Ko, In Ok [Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Kim, Tae-Jeong; Chang, Yongmin [Institute of Biomedical Engineering, Kyungpook National University, Daegu (Korea, Republic of); Lim, Sang Moo [Department of Nuclear Medicine, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Kim, Kyeong Min [Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Kim, Jung Young, E-mail: jykim@kirams.re.kr [Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2014-12-12

Highlights: • Development of improved tumor-targeting MRI contrast agents. • To increase the targeting ability of RGD, we developed cycloalkane-based RGD peptides. • Gd(DOTA) conjugates of cycloalkane-based RGD peptide show improved tumor signal enhancement in vivo MR images. - Abstract: Two new MRI contrast agents, Gd-DOTA-c(RGD-ACP-K) (1) and Gd-DOTA-c(RGD-ACH-K) (2), which were designed by incorporating aminocyclopentane (ACP)- or aminocyclohexane (ACH)-carboxylic acid into Gd-DOTA (gadolinium-tetraazacyclo dodecanetetraacetic acid) and cyclic RGDK peptides, were synthesized and evaluated for tumor-targeting ability in vitro and in vivo. Binding affinity studies showed that both 1 and 2 exhibited higher affinity for integrin receptors than cyclic RGDyK peptides, which were used as a reference. These complexes showed high relaxivity and good stability in human serum and have the potential to improve target-specific signal enhancement in vivo MR images.

The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule

DEFF Research Database (Denmark)

Garousi, Javad; Andersson, Ken G; Dam, Johan H

2017-01-01

-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-ZEGFR:2377. DOTA-ZEGFR:2377 was labelled with (57)Co (T1/2 = 271.8 d......), (55)Co (T1/2 = 17.5 h), and, for comparison, with the positron-emitting radionuclide (68)Ga (T1/2 = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope (57)Co was used in animal studies. Both (57)Co-DOTA-ZEGFR:2377 and (68)Ga-DOTA......Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The (111)In-labelled DOTA-conjugated ZEGFR:2377 Affibody molecule was successfully used for imaging of EGFR...

Standardization of methodology to derivatization and radiolabeling of the anti-CD20 monoclonal antibody from bifunctional chelator DOTA-NHS-Ester

International Nuclear Information System (INIS)

Massicano, Adriana V.F.; Akanji, Akinkunmi G.; Santos, Josefina S.; Pujatti, Priscilla B.; Couto, Renata M.; Massicano, Felipe; Araujo, Elaine Bortoleti de

2009-01-01

Lymphomas are cancers of the lymphatic system, being the most common the non-Hodgkin lymphoma (NHL). The Radioimmunotherapy (RIT), that increase the cytotoxic effect of monoclonal antibodies (mAb), therefore labeling these Mab with different radioisotopes. RIT combines the specificity of the antibody and the toxicity of the radionuclides. The mAb anti-CD20 is used for treatment of relapse or refractory NHL. The labeling of anti- CD20 with 177 Lu, requires a bifunctional chelating agent that is designed to make a 'connect bridge' between the mAb and the radionuclide. The incorporation of the chelating group in mAb structure is called derivatization. The aim of this work is to study the derivatization of anti-CD20 antibody with DOTA-NHS-ester chelating group and labeling parameters to produce 177 Lu-DOTA-Anti CD20. Five milligrams of anti-CD20 were purified by dialysis against phosphate buffer pH 8.0 and derivatized with DOTA-NHS-ester in 1:250, 1:500 and 1:1000 molar ratios. The reaction was conducted for 1 hour in gently mixing at room temperature and remained under refrigeration for 48 hours. The reaction mixture was purified in gel column Sephadex G-50 ; the aliquots that presented greater protein concentration, were mixed and concentrated. The purified antibody conjugated was added to 111-185MBq (3-5mCi) of 177 LuCl3 diluted in 0.4 M acetate buffer pH 5.5. Radiochemical purity was less than 95% in all the molar ratios, indicating necessity of the purification after the labeling. The mAb derivatized showed stable when stored for to 1 month to 4 deg C and 4 days at -20 deg C. (author)

Radiolabeling parameters of 177Lu-DOTA-RITUXIMAB

International Nuclear Information System (INIS)

Massicano, Adriana V.F.; Alcarde, Lais F.; Oliveira, Ricardo S.; Mengatti, Jair; Araujo, Elaine B. de

2013-01-01

Cancer treatment using radioimmunotherapy (RIT) has been the focus of much research in the last two decades. In RIT, a radioisotope is coupled to a monoclonal antibody (mAb) to form a tumor-specific target agent to improve the cytocidal effect of the mAbs. RIT allows the systemic delivery of radiation to disease target by mAbs while sparing normal tissues. Rituximab® (Mabthera - Roche) is a chimeric mouse-human monoclonal antibody; it selectively binds with high affinity to the CD20 antigen, a hydrophobic transmembrane protein, which is expressed on B-lymphocytes and in more than 90% of B cell non-Hodgkin's lymphomas (NHL). The conjugation and radiolabeling process involve special conditions of pH and temperature, long processes of manipulation and mixing. All this process can damage the antibody structure and compromise its clinical application. Therefore, these parameters must be largely studied. The aim of this work was to evaluate the best radiolabeling conditions of DOTA-rituximab. Briefly, 10 mg of antibody previously purified by ultrafiltration device was conjugated with DOTA-NHS-ester (Macrocyclics) in 50 fold molar excess. The reaction was conducted for 1 hour in phosphate buffer pH 8.0 and gently mixing at room temperature, remaining for 24 hours under refrigeration. The immunoconjugated was purified by size exclusion column and ultrafiltration device. The radiolabeled parameters studied were: immunoconjugated mass, activity of 177 LuCl 3 , reaction time, temperature and pH. The radiochemical purity of the preparations was determined using analysis by thin layer chromatography (TLC-SG plates). The best studied condition presented radiochemical purity above 95% and the integrity of antibody was preserved. (author)

Comparison of bifunctional chelates for {sup 64}Cu antibody imaging

Energy Technology Data Exchange (ETDEWEB)

Ferreira, Cara L.; Crisp, Sarah; Bensimon, Corinne [MDS Nordion, Vancouver, BC (Canada); Yapp, Donald T.T.; Ng, Sylvia S.W. [British Columbia Cancer Agency Research Centre, Vancouver, BC (Canada); University of British Columba, The Faculty of Pharmaceutical Sciences, Vancouver, BC (Canada); Sutherland, Brent W. [British Columbia Cancer Agency Research Centre, Vancouver, BC (Canada); Gleave, Martin [Prostate Centre at Vancouver General Hospital, Vancouver, BC (Canada); Jurek, Paul; Kiefer, Garry E. [Macrocyclics Inc., Dallas, TX (United States)

2010-11-15

Improved bifunctional chelates (BFCs) are needed to facilitate efficient {sup 64}Cu radiolabeling of monoclonal antibodies (mAbs) under mild conditions and to yield stable, target-specific agents. The utility of two novel BFCs, 1-Oxa-4,7,10-triazacyclododecane-5-S-(4-isothiocyanatobenzyl)-4,7,10-triacetic acid (p-SCN-Bn-Oxo-DO3A) and 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-4-S-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (p-SCN-Bn-PCTA), for mAb imaging with {sup 64}Cu were compared to the commonly used S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-tetraacetic acid (p-SCN-Bn-DOTA). The BFCs were conjugated to trastuzumab, which targets the HER2/neu receptor. {sup 64}Cu radiolabeling of the conjugates was optimized. Receptor binding was analyzed using flow cytometry and radioassays. Finally, PET imaging and biodistribution studies were done in mice bearing either HER2/neu-positive or HER2/neu-negative tumors. {sup 64}Cu-Oxo-DO3A- and PCTA-trastuzumab were prepared at room temperature in >95% radiochemical yield (RCY) in <30 min, compared to only 88% RCY after 2 h for the preparation of {sup 64}Cu-DOTA-trastuzumab under the same conditions. Cell studies confirmed that the immunoreactivity of the mAb was retained for each of the bioconjugates. In vivo studies showed that {sup 64}Cu-Oxo-DO3A- and PCTA-trastuzumab had higher uptake than the {sup 64}Cu-DOTA-trastuzumab at 24 h in HER2/neu-positive tumors, resulting in higher tumor to background ratios and better tumor images. By 40 h all three of the {sup 64}Cu-BFC-trastuzumab conjugates allowed for clear visualization of the HER2/neu-positive tumors but not the negative control tumor. The antibody conjugates of PCTA and Oxo-DO3A were shown to have superior {sup 64}Cu radiolabeling efficiency and stability compared to the analogous DOTA conjugate. In addition, {sup 64}Cu-PCTA and Oxo-DO3A antibody conjugates may facilitate earlier imaging with greater target to background ratios than

Development of new folate-based PET radiotracers: preclinical evaluation of 68Ga-DOTA-folate conjugates

International Nuclear Information System (INIS)

Fani, Melpomeni; Maecke, Helmut R.; Wang, Xuejuan; Nicolas, Guillaume; Medina, Christelle; Raynal, Isabelle; Port, Marc

2011-01-01

A number of 111 In- and 99m Tc-folate-based tracers have been evaluated as diagnostic agents for imaging folate receptor (FR)-positive tumours. A 68 Ga-folate-based radiopharmaceutical would be of great interest, combining the advantages of PET technology and the availability of 68 Ga from a generator. The aim of the study was to develop a new 68 Ga-folate-based PET radiotracer. Two new DOTA-folate conjugates, named P3026 and P1254, were synthesized using the 1,2-diaminoethane and 3-{2-[2-(3-amino-propoxy)-ethoxy]-ethoxy}-propylamine as a spacer, respectively. Both conjugates were labelled with 67/68 Ga. Binding affinity, internalization and externalization studies were performed using the FR-positive KB cell line. Biodistribution and PET/CT imaging studies were performed in nude mice, on a folate-deficient diet, bearing KB and HT1080 (FR-negative) tumours, concurrently. The new radiotracers were evaluated comparatively to the reference molecule 111 In-DTPA-folate ( 111 In-P3139). The K d values of 67/68 Ga-P3026 (4.65 ± 0.82 nM) and 67/68 Ga-P1254 (4.27 ± 0.42 nM) showed high affinity for the FR. The internalization rate followed the order 67/68 Ga-P3026 > 67/68 Ga-P1254 > 111 In-P3139, while almost double cellular retention was found for 67/68 Ga-P3026 and 67/68 Ga-P1254, compared to 111 In-P3139. The biodistribution data of 67/68 Ga-DOTA-folates showed high and receptor-mediated uptake on the FR-positive tumours and kidneys, with no significant differences compared to 111 In-P3139. PET/CT images, performed with 68 Ga-P3026, showed high uptake in the kidneys and clear visualization of the FR-positive tumours. The DOTA-folate conjugates can be efficiently labelled with 68 Ga in labelling yields and specific activities which allow clinical application. The characteristics of the 67/68 Ga-DOTA-folates are comparable to 111 In-DTPA-folate, which has already been used in clinical trials, showing that the new conjugates are promising candidates as PET radiotracers

Radiolabeling optimization and characterization of (68)Ga labeled DOTA-polyamido-amine dendrimer conjugate - Animal biodistribution and PET imaging results.

Science.gov (United States)

Ghai, Aanchal; Singh, Baljinder; Panwar Hazari, Puja; Schultz, Michael K; Parmar, Ambika; Kumar, Pardeep; Sharma, Sarika; Dhawan, Devinder; Kumar Mishra, Anil

2015-11-01

The present study describes the optimization of (68)Ga radiolabeling with PAMAM dendrimer-DOTA conjugate. A conjugate (PAMAM-DOTA) concentration of 11.69µM, provided best radiolabeling efficiency of more than 93.0% at pH 4.0, incubation time of 30.0min and reaction temperature ranging between 90 and 100°C. The decay corrected radiochemical yield was found to be 79.4±0.01%. The radiolabeled preparation ([(68)Ga]-DOTA-PAMAM-D) remained stable (radiolabeling efficiency of 96.0%) at room temperature and in serum for up to 4-h. The plasma protein binding was observed to be 21.0%. After intravenous administration, 50.0% of the tracer cleared from the blood circulation by 30-min and less than 1.0% of the injected activity remained in blood by 1.0h. The animal biodistribution studies demonstrated that the tracer excretes through the kidneys and about 0.33% of the %ID/g accumulated in the tumor at 1h post injection. The animal organ's biodistribution data was supported by animal PET imaging showing good 'non-specific' tracer uptake in tumor and excretion is primarily through kidneys. Additionally, DOTA-PAMAM-D conjugation with αVβ3 receptors targeting peptides and drug loading on the dendrimers may improve the specificity of the (68)Ga labeled product for imaging and treating angiogenesis respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bone targeting compounds for radiotherapy and imaging: *Me(III)-DOTA conjugates of bisphosphonic acid, pamidronic acid and zoledronic acid.

Science.gov (United States)

Meckel, M; Bergmann, R; Miederer, M; Roesch, F

2017-01-01

Bisphosphonates have a high adsorption on calcified tissues and are commonly used in the treatment of bone disorder diseases. Conjugates of bisphosphonates with macrocyclic chelators open new possibilities in bone targeted radionuclide imaging and therapy. Subsequent to positron emission tomography (PET) examinations utilizing 68 Ga-labelled analogues, endoradiotheraphy with 177 Lu-labelled macrocyclic bisphosphonates may have a great potential in the treatment of painful skeletal metastases. Based on the established pharmaceuticals pamidronate and zoledronate two new DOTA-α-OH-bisphosphonates, DOTA PAM and DOTA ZOL (MM1.MZ) were successfully synthesized. The ligands were labelled with the positron emitting nuclide 68 Ga and the β - emitting nuclide 177 Lu and compared in in vitro studies and in ex vivo biodistribution studies together with small animal PET and single photon emission computed tomography (SPECT) studies against [ 18 F]NaF and a known DOTA-α-H-bisphosphonate conjugate (BPAPD) in healthy Wistar rats. The new DOTA-bisphosphonates can be labelled in high yield of 80 to 95 % in 15 min with post-processed 68 Ga and >98 % with 177 Lu. The tracers showed very low uptake in soft tissue, a fast renal clearance and a high accumulation on bone. The best compound was [ 68 Ga]DOTA ZOL (SUV Femur  = 5.4 ± 0.6) followed by [ 18 F]NaF (SUV Femur  = 4.8 ± 0.2), [ 68 Ga]DOTA PAM (SUV Femur  = 4.5 ± 0.2) and [ 68 Ga]BPAPD (SUV Femur  = 3.2 ± 0.3). [ 177 Lu]DOTA ZOL showed a similar distribution as the diagnostic 68 Ga complex. The 68 Ga labelled compounds showed a promising pharmacokinetics, with similar uptake profile and distribution kinetics. Bone accumulation was highest for [ 68 Ga]DOTA ZOL , which makes this compound probably an interesting bone targeting agent for a therapeutic approach with 177 Lu. The therapeutic compound [ 177 Lu]DOTA ZOL showed a high target-to-background ratio. SPECT experiments showed concordance

Synthesis of Poly[APMA]-DOTA-64Cu Conjugates for Interventional Radionuclide Therapy of Prostate Cancer: Assessment of Intratumoral Retention by Micro–Positron Emission Tomography

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Jianchao Yuan

2007-01-01

Full Text Available To develop new radiopharmaceuticals for interventional radionuclide therapy of locally recurrent prostate cancer, poly[N-(3-aminopropylmethacrylamide] [poly(APMA] polymers were synthesized by free radical precipitation polymerization in acetonedimethylsulfoxide using N,N‘-azobis(isobutyronitrile as the initiator. The polymers were characterized with nuclear magnetic resonance, size exclusion chromatography, and dynamic light scattering (Mn 5 2.40 × 104, Mw/Mn = 1.87. Subsequently, poly[APMA] was coupled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA using 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride as an activator, followed by conjugation with 64Cu radionuclide. Prolonged retention of poly[APMA]-DOTA-64Cu conjugates within the tumor tissues was demonstrated by micro–positron emission tomography at 24 hours following intra-tumoral injection of the conjugates to human prostate xenografts in mice. The data suggest that the poly[APMA]-DOTA-64Cu conjugates might be useful for interventional radionuclide therapy of locally recurrent prostate cancer in humans.

Radiolabeling parameters of {sup 177}Lu-DOTA-RITUXIMAB

Energy Technology Data Exchange (ETDEWEB)

Massicano, Adriana V.F.; Alcarde, Lais F.; Oliveira, Ricardo S.; Mengatti, Jair; Araujo, Elaine B. de, E-mail: adriana.avfernandes@gmail.com [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

2013-07-01

Cancer treatment using radioimmunotherapy (RIT) has been the focus of much research in the last two decades. In RIT, a radioisotope is coupled to a monoclonal antibody (mAb) to form a tumor-specific target agent to improve the cytocidal effect of the mAbs. RIT allows the systemic delivery of radiation to disease target by mAbs while sparing normal tissues. Rituximab® (Mabthera - Roche) is a chimeric mouse-human monoclonal antibody; it selectively binds with high affinity to the CD20 antigen, a hydrophobic transmembrane protein, which is expressed on B-lymphocytes and in more than 90% of B cell non-Hodgkin's lymphomas (NHL). The conjugation and radiolabeling process involve special conditions of pH and temperature, long processes of manipulation and mixing. All this process can damage the antibody structure and compromise its clinical application. Therefore, these parameters must be largely studied. The aim of this work was to evaluate the best radiolabeling conditions of DOTA-rituximab. Briefly, 10 mg of antibody previously purified by ultrafiltration device was conjugated with DOTA-NHS-ester (Macrocyclics) in 50 fold molar excess. The reaction was conducted for 1 hour in phosphate buffer pH 8.0 and gently mixing at room temperature, remaining for 24 hours under refrigeration. The immunoconjugated was purified by size exclusion column and ultrafiltration device. The radiolabeled parameters studied were: immunoconjugated mass, activity of {sup 177}LuCl{sub 3}, reaction time, temperature and pH. The radiochemical purity of the preparations was determined using analysis by thin layer chromatography (TLC-SG plates). The best studied condition presented radiochemical purity above 95% and the integrity of antibody was preserved. (author)

The use of radiocobalt as a label improves imaging of EGFR using DOTA-conjugated Affibody molecule.

Science.gov (United States)

Garousi, Javad; Andersson, Ken G; Dam, Johan H; Olsen, Birgitte B; Mitran, Bogdan; Orlova, Anna; Buijs, Jos; Ståhl, Stefan; Löfblom, John; Thisgaard, Helge; Tolmachev, Vladimir

2017-07-20

Several anti-cancer therapies target the epidermal growth factor receptor (EGFR). Radionuclide imaging of EGFR expression in tumours may aid in selection of optimal cancer therapy. The 111 In-labelled DOTA-conjugated Z EGFR:2377 Affibody molecule was successfully used for imaging of EGFR-expressing xenografts in mice. An optimal combination of radionuclide, chelator and targeting protein may further improve the contrast of radionuclide imaging. The aim of this study was to evaluate the targeting properties of radiocobalt-labelled DOTA-Z EGFR:2377 . DOTA-Z EGFR:2377 was labelled with 57 Co (T 1/2  = 271.8 d), 55 Co (T 1/2  = 17.5 h), and, for comparison, with the positron-emitting radionuclide 68 Ga (T 1/2  = 67.6 min) with preserved specificity of binding to EGFR-expressing A431 cells. The long-lived cobalt radioisotope 57 Co was used in animal studies. Both 57 Co-DOTA-Z EGFR:2377 and 68 Ga-DOTA-Z EGFR:2377 demonstrated EGFR-specific accumulation in A431 xenografts and EGFR-expressing tissues in mice. Tumour-to-organ ratios for the radiocobalt-labelled DOTA-Z EGFR:2377 were significantly higher than for the gallium-labelled counterpart already at 3 h after injection. Importantly, 57 Co-DOTA-Z EGFR:2377 demonstrated a tumour-to-liver ratio of 3, which is 7-fold higher than the tumour-to-liver ratio for 68 Ga-DOTA-Z EGFR:2377 . The results of this study suggest that the positron-emitting cobalt isotope 55 Co would be an optimal label for DOTA-Z EGFR:2377 and further development should concentrate on this radionuclide as a label.

Tetrafluorophenolate of HBED-CC: a versatile conjugation agent for 68Ga-labeled small recombinant antibodies

International Nuclear Information System (INIS)

Eder, Matthias; Waengler, Bjoern; Eisenhut, Michael; Knackmuss, Stefan; LeGall, Fabrice; Little, Melvyn; Haberkorn, Uwe; Mier, Walter

2008-01-01

The success of 68 Ga-labeled peptides for positron emission tomography of neuroendocrine tumors is mainly depending on the complex chemistry of this radioisotope. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), the chelator of choice has however limitations if its application is expanded to heat-sensitive proteins. Recombinant antibodies like single chain Fv or diabodies belong to this class of proteins. They are suited to provide imaging contrast despite the short-lived 68 Ga because of their rapid blood clearances and nanomolar affinities. The heterobifunctional agent N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC) was chosen as an alternative ligand because this agent is complexing [ 68 Ga]Ga 3+ much faster than DOTA at ambient temperatures. A versatile technology for HBED-CC conjugation of proteins and 68 Ga-labeling has been developed. This included HBED-CC-tetrafluorophenol (TFP) ester synthesis, coupling to the antibody at various pH and complexation reactions performed in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer under different conditions. The synthesis of the monoreactive 2,3,5,6-tetrafluorophenolate of HBED-CC at a carboxyl group not participating in complex formation used [Fe(HBED-CC)] - for ester formation. The removal of Fe 3+ from purified (HBED-CC)TFP ester was achieved with RP 18 cartridge technology. The conjugation chemistry was performed with mAb425 which binds to the epidermal growth factor receptor (EGFR). This protein was used for optimizing purposes only. The influence of complexation parameters like temperature, pH, reaction time, and HBED-CC/antibody ratio on the biological activity of this model antibody was investigated. Furthermore, the outcome of this labeling procedure on the biological activity of a recombinant diabody (50 kDa) was studied. It is known that small HBED-CC/antibody ratios are prerequisites for minimal interference of labels with antigen

Optimized preparation and preliminary evaluation of [64Cu]-DOTA-trastuzumab for targeting ErbB2/Neu expression

International Nuclear Information System (INIS)

Behrooz Alirezapour; Mohammad Javad Rasaee

2013-01-01

Breast cancer radioimmunoscintigraphy targeting HER2/neu expression is a growing field of work in nuclear medicine research. Trastuzumab is a monoclonal antibody that binds with high affinity to HER2/neu, which is over expressed on breast and other tumors. Developing new tracers for the detection of this cancer is of great interest. In this study, trastuzumab was successively labeled with [ 64 Cu]CuCl 2 after conjugation with DOTA-NHS-ester. The conjugate was purified by molecular filtration, the average number of DOTA conjugated per mAb was calculated and total concentration was determined by spectrophotometric method. DOTA-trastuzumab was labeled with 64 Cu produced by 68 Zn(p,αn) 64 Cu nuclear reaction (30 MeV protons at 180 μA). Radiochemical purity, integrity of protein after radiolabeling and immunoreactivity of radiolabeled mAb trastuzumab with HER2/neu antigen and SkBr3 cell line were performed by RIA. In vitro stability of radiolabeled mAb in human serum was determined by thin layer chromatography. In vitro internalization studies were performed with the SkBr3 cell line and the tissue biodistribution of the 64 Cu-DOTA-trastuzumab was evaluated in wild-type rat (90 ± 5.5 μCi, 2, 6, 12, 24 h p.i.). The radioimmunoconjugate was prepared with a radiochemical purity of higher than 96 ± 0.5 % (ITLC) and specific activity as high as 5.3 μCi/μg. The average number of chelators per antibody for the conjugate used in this study was 5.8/1. The sample was showed to have similar patterns of migration in the gel electrophoresis. The 64 Cu-DOTA-trastuzumab showed high immunoreactivity towards HER2/neu antigen and SkBr3 cell line. In vitro stability of the labeled product was found to be more than 94 % in PBS and 82 ± 0.5 % in human serum over 48 h. In vitro internalization studies of the 64 Cu-DOTA-trastuzumab showed that up to 11.5 % of the radioimmunoconjugate internalized after 10 h. The accumulation of the radiolabeled mAb in liver, skin, intestine, lung

Synthesis, radiolabeling and quality control of {sup 111}In-DOTA-bevacizumab for radioimmunoscintigraphy of VEGF receptors

Energy Technology Data Exchange (ETDEWEB)

Khorami-Moghadam, A.; Jalilian, A.R.; Yavari, K.; Alirezapour, B.; Mazidi, M.; Mirzaii, M. [Nuclear Science and Technology Research Institute (NSTRI), Tehran (Iran, Islamic Republic of)

2013-11-01

In this study, bevacizumab was successively labeled with {sup 111}In-InCl{sub 3} after conjugation with DOTA-NHS-ester followed by molecular filtration and determination of the average number ofDOTAconjugated per mAb (6:1) by spectrophotometric method. Radiochemical purity (> 97%, measured by ITLC and HPLC), integrity of protein after radiolabeling (gel electrophoresis) and stability of {sup 111}In-DOTA-Bevacizumab (in final formulation, human serum, liver/kidney homogenates) were determined in 24-72 h as well as biodistribution studies in wild-type rats and human colon cancer (SW-480) bearing mice. The accumulation of the radiolabeled antibody was consistent with the former reported Bevacizumab conjugates. Significant tumor uptake (8%) was observed at 72 h p.i. Tumor/muscle uptake ratios were 2.6 (24 h), 9.74 (48 h) and 25 (72 h). {sup 111}In-DOTA-Bevacizumab was prepared as a SPECT molecular imaging agent for diagnosis and follow-up of vascular endothelial growth factor A (VEGF-A) expression in oncology. (orig.)

Development of [{sup 62}Zn/{sup 62}Cu]-DOTA-rituximab as a possible novel in vivo PET generator for anti-CD20 antigen imaging

Energy Technology Data Exchange (ETDEWEB)

Gholipour, Nazila [Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of). Dept. of Radiopharmacy; Jalilian, Amir R.; Fazaeli, Yousef; Moradkhani, Sedigheh; Bolourinovin, Fateme [Nuclear Science and Technology Research Institute (NSTRI), Tehran (Iran, Islamic Republic of); Sabzevari, Omid [Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of). Dept. of Toxicology and Pharmacology; Khalaj, Ali [Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of). Dept. of Medical Chemistry

2014-07-01

In this study, zinc-62 was prepared at radiopharmaceutical grade (for {sup 62}Zn/{sup 62}Cu generator production) using {sup nat}Cu(p, xn) reaction with the production yield of 5.9 mCi/μAh at 30 MeV proton energy (radiochemical separation yield >95%, radionuclidic purity >99% and radiochemical purity >99%). In the next step, rituximab was successively labeled with [{sup 62}Zn]-ZnCl{sub 2} after conjugation with p-SCN-Bz-DOTA followed by molecular filtration and determination of the average number of DOTA conjugated per mAb (6:1) by spectrophotometric method. Radiochemical purity (>97%, measured by ITLC and HPLC), integrity of protein after radiolabeling (gel electrophoresis) and stability of [{sup 62}Zn]-DOTA-rituximab (in final formulation, and human serum) were determined 1-8 h as well as biodistribution studies in wild-type rats followed by coincidence imaging for 6 h. However, the accumulation of the radiolabeled antibody was not consistent with the former reported rituximab conjugates. [{sup 62}Zn]-labeled monoclonal antibodies and fragments can be prepared as potential in vivo PET generators for molecular imaging however, the search for application of stable zinc complexes must be continued.

Complexes of DOTA-bisphosphonate conjugates: probes for determination of adsorption capacity and affinity constants of hydroxyapatite.

Science.gov (United States)

Vitha, Tomas; Kubícek, Vojtech; Hermann, Petr; Kolar, Zvonimir I; Wolterbeek, Hubert Th; Peters, Joop A; Lukes, Ivan

2008-03-04

The adsorption on hydroxyapatite of three conjugates of a bisphosphonate and a macrocycle having C1, C2, and C3 spacers and their terbium complexes was studied by the radiotracer method using 160Tb as the label. The radiotracer-containing complex of the conjugate with the C3 spacer was used as a probe for the determination of the adsorption parameters of other bisphosphonates that lack a DOTA unit. A physicochemical model describing the competitive adsorption was successfully applied in the fitting of the obtained data. The maximum adsorption capacity of bisphosphonates containing bulky substituents is determined mainly by their size. For bisphosphonates having no DOTA moiety, the maximum adsorption capacity is determined by the electrostatic repulsion between negatively charged bisphosphonate groups. Compounds with a hydroxy or amino group attached to the alpha-carbon atom show higher affinities. Macrocyclic compounds containing a short spacer between the different bisphosphonic acid groups and the macrocyclic unit exhibit high affinities, indicating a synergic effect of the bisphosphonic and the macrocyclic groups during adsorption. The competition method described uses a well-characterized complex and allows a simple evaluation of the adsorption behavior of bisphosphonates. The application of the macrocycle-bisphosphonate conjugates allows easy radiolabeling via complexation of a suitable metal isotope.

Development of radioinmunoconjugate 90Y-DOTA-nimotuzumab-Fab for therapy of EGFR over expressing tumors

International Nuclear Information System (INIS)

Rodriguez, A. M.; Alonso, L. M.; Gongora, M.; Leyva, R.; Solana, A.

2015-01-01

Many monoclonal antibodies conjugated with 1,4,7,10-tetraaza cyclododecane-N, N', N'', N'''-tetraacetic acid (DOTA) and radiolabeled with 90 Y, have been used for radioimmunotherapy. As know IgG molecules are heavy proteins with a molecular weight of approximately 150 kDa. Accordingly, intact IgG antibodies may have significant slow kinetics biodistribution and severely limited properties of tissue penetration. Antibody fragments labeled with radio metals could be promising radiopharmaceuticals for imaging and non-invasive therapy due to its high affinity to the tumor, the lack of effector function and rapid pharmacokinetic. In this work, the nimotuzumab Fab fragment was obtained by cleavage with papain in molar excess. After separating the reaction mixture in three steps using affinity, size exclusion and ion exchange chromatography; the Fab fragment showed high values of purity, integrity and identity. The Fab fragment was derivatized with DOTA and labeled with 90 Y. The radioimmunoconjugate with high radiochemical yield was assessed by in vitro stability with an excess of 50mM DTPA. The development of 90 Y-DOTA-Nimotuzumab-Fab radioimmunoconjugate allows to count on as a potential agent for radioimmunotherapy. (Author)

The study of conjugation of anti-CD20 monoclonal antibody for labeling with metallic or lanthanides radionuclides; Estudo de conjugacao do anticorpo anti-CD20 para marcacao com radionuclideos metalicos ou lantanideos

Energy Technology Data Exchange (ETDEWEB)

Akanji, Akinkunmi Ganiyu

2012-07-01

Lymphomas are malignancies or cancers that start from the malign transformation of a lymphocyte in the lymphatic system. Generally, lymphomas start from the lymph nodes or from the agglomeration of the lymphatic tissues, organs like stomach, intestines, in some cases it can involve the bone marrow and the blood, it can also disseminate to other organs. Lymphomas are divided in two major categories: Hodgkin lymphoma and non-Hodgkin lymphoma (NHL). Patient with NHL are generally treated with radiotherapy alone or combined with immunotherapy using monoclonal antibody rituximab (MabThera Registered-Sign ). Currently, monoclonal antibodies (Acm) conjugated with bifunctional chelate agents and radiolabeled with metallic or lanthanides radionuclides are a treatment reality for patients with NHL by the principle of radioimmunotherapy (RIT). This study focused on the conditions of conjugation of Acm rituximab (MabThera Registered-Sign ) with bifunctional chelating agents DOTA and DTPA. Various parameters were studied: method of Acm purification, conditions of Acm conjugation, the method for determination of number of chelate agent coupled to the Acm, method for purification of the conjugated antibody Acm, conditions of labeling of the conjugated antibody with lutetium-177, method of purification of the radiolabeled immuno conjugate, method of radiochemical purity (RP), specific binding in vitro Raji cells (Human Burkitt) and biological distribution performed in normal Balb-c mouse. The three methodologies employed in pre-purification of Acm (dialysis, size exclusion chromatograph and dial filtration) demonstrated to be efficient; they provided sample recovery exceeding 90%. However, the methodology of dial filtration presents minimal sample loss, and gave the final recovery of the sample in micro liters; thereby facilitating sample use in subsequent experiments. Numbers of chelators attached to the Acm molecule was proportional to the molar ratio studied. When we evaluated

Antibody-radioisotope conjugates for tumor localization and treatment

International Nuclear Information System (INIS)

Larson, S.M.; Carrasquillo, J.A.

1985-01-01

In principle, anti-tumor antibodies can be used to carry radioactivity to tumors for in-vivo diagnosis and treatment of cancer. First, for diagnostic purposes, an antibody that targets a specific antigen (for example, the p97 antigen of human melanoma tumor), is labeled with a tracer amount of radioactivity. When this antibody-radioisotope conjugate is injected into the blood stream, the antibody carries the radioactivity throughout the body and in time, percolates through all the tissues of the body. Because the tumor has specific antigens to which the antibody can bind, the antibody conjugate progressively accumulates in the tumor. Using conventional nuclear medicine imaging equipment, the body of the patient is scanned for radioactivity content, and a map of the distribution of the radioactivity is displayed on photographic film. The tumor shows up as a dense area of radio-activity. These same antibody-radioisotope conjugates may be used for therapy of tumors, except that in this case large amounts of radioactivity are loaded on the antibody. After localization of the conjugate there is sufficient radiation deposited in the tumor of radiotherapy. The success of this approach in the clinic is determined in large measure by the concentration gradient that can be achieved between tissue antibody conjugate in tumor versus normal tissue

Development of DOTA-Rituximab to be Labeled with 90Y for Radioimmunotherapy of B-cell Non-Hodgkin Lymphoma

Science.gov (United States)

Johari doha, Fariba; Rahmani, Siyavash; Rikhtechi, Pedram; Rasaneh, Samira; Sheikholislam, Zahra; Shahhosseini, Soraya

2017-01-01

NHL is the most common hematologic cancer in adults. Rituximab is the FDA approved treatment of relapsed or refractory low grade B-cell Non-Hodgkin Lymphoma (NHL). But patients eventually become resistant to rituximab. Since lymphocytes and lymphoma cells are highly radiosensitive, low grade NHL that has relapsed or refractory to standard therapy is treated by RIT in which a beta-emitting radionuclide coupled to anti-CD20 antibody. The association of beta emitter radionuclide to rituximab enhances its therapeutic efficacy. The cells which lack antigen or cells which cannot be reached due to poor vascularization and intratumoral pressure in a bulky tumor would be irradiated and killed by cross fire effect of beta emitter. 90Y, a pure high energy β-emitter with a half-life of 64 h, a maximum energy of 2.28 MeV, and maximum board of 11.3 mm in tissue is radionuclide of choice for radioimmunotherapy of outpatient administration. In this study, rituximab was conjugated to DOTA and radiolabeled with 90YCl3. The stability, affinity, and immunoreactivity of radiolabeled antibody was determined in vitro and the conditions were optimized. Biodistribution studies were done in normal mice. The optimum conditions of conjugation and radiolabeling was 1-2 h at 37 °C and 1 h at 45 °C, respectively. Results showed approximately 4 DOTA molecules conjugated per antibody molecule. The purified antibody was stable and intact over 6 months stored at -20 °C. The result of immunoreactivity (≈70%), affinity (≈3 nM) and biodistribution in normal mice are acceptable. PMID:28979315

Optimized conditions for chelation of yttrium-90-DOTA immunoconjugates.

Science.gov (United States)

Kukis, D L; DeNardo, S J; DeNardo, G L; O'Donnell, R T; Meares, C F

1998-12-01

Radioimmunotherapy (RIT) with 90Y-labeled immunoconjugates has shown promise in clinical trials. The macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) binds 90Y with extraordinary stability, minimizing the toxicity of 90Y-DOTA immunoconjugates arising from loss of 90Y to bone. However, reported 90Y-DOTA immunoconjugate product yields have been typically only BAD) was conjugated to the monoclonal antibody Lym-1 via 2-iminothiolane (2IT). The immunoconjugate product, 2IT-BAD-Lym-1, was labeled in excess yttrium in various buffers over a range of concentrations and pH. Kinetic studies were performed in selected buffers to estimate radiolabeling reaction times under prospective radiopharmacy labeling conditions. The effect of temperature on reaction kinetics was examined. Optimal radiolabeling conditions were identified and used in eight radiolabeling experiments with 2IT-BAD-Lym-1 and a second immunoconjugate, DOTA-peptide-chimeric L6, with 248-492 MBq (6.7-13.3 mCi) of 90Y. Ammonium acetate buffer (0.5 M) was associated with the highest uptake of yttrium. On the basis of kinetic data, the time required to chelate 94% of 90Y (four half-times) under prospective radiopharmacy labeling conditions in 0.5 M ammonium acetate was 17-148 min at pH 6.5, but it was only 1-10 min at pH 7.5. Raising the reaction temperature from 25 degrees C to 37 degrees C markedly increased the chelation rate. Optimal radiolabeling conditions were identified as: 30-min reaction time, 0.5 M ammonium acetate buffer, pH 7-7.5 and 37 degrees C. In eight labeling experiments under optimal conditions, a mean product yield (+/- s.d.) of 91%+/-8% was achieved, comparable to iodination yields. The specific activity of final products was 74-130 MBq (2.0-3.5 mCi) of 90Y per mg of monoclonal antibody. The immunoreactivity of 90Y-labeled immunoconjugates was 100%+/-11%. The optimization of 90Y-DOTA chelation conditions represents an important advance in 90Y RIT

Country report: Italy (Chinol). Pre-clinical evaluation of a new biotin-DOTA conjugate labeled with 90Y for application in pretargeting clinical protocols

International Nuclear Information System (INIS)

Chinol, Marco

2010-01-01

In the attempt to improve the therapeutic efficacy of radiolabeled mAbs in cancer radioimmunotherapy, various studies have examined the concept of tumor pretargeting. The so called three-step pretargeting technique, employing the avidin–biotin system, was applied in phase I-II clinical trials showing low toxicity and therapeutic efficacy. The final step of the pretargeting protocols consists in the systemic injection of radiolabeled biotin. The worldwide recognized “successful association” is between 90 Y and the tetraazamacrocycle DOTA chelator chemically bound to biotin. Improvements in the structure of the biotin-DOTA conjugate have been reported by our group following a novel approach which simplified the synthetic pattern by reducing the amide group to a methylene group, thus transforming the amide into a secondary amine without affecting the length of the biotin side arm. Preliminary in-vitro experiments, previously published by our group, indicated the potential of the new conjugate. Based on our previous experience with avidin-based pre-targeting followed 90 Y-DOTA-biotin in the locoregional treatment of peritoneal carcinomatosis and malignant glioma suggested that similar radionuclide therapy might be worth investigating as a partial replacement of external beam radiotherapy in breast cancer. We have developed IART® the Intra-operative Avidination for Radionuclide Therapy that relies on the avidin-biotin binding system. In fact, the “avidination” of the anatomical area of the tumor with native avidin, directly injected by the surgeon into and around the tumor bed, provides a target for the radiolabeled biotin intravenously (iv) injected one day later. In order to optimize the overall strategy, further efforts were needed to optimize the use of the labeled new biotin conjugate and to elucidate its chemical and biological properties. In the first 18 months of this CRP, the pre-clinical evaluation of this new reduced biotinamidohexylamine-DOTA

Mono(pyridine-N-oxide) DOTA analog and its G1/G4-PAMAM dendrimer conjugates labeled with 177Lu: Radiolabeling and biodistribution studies

International Nuclear Information System (INIS)

Laznickova, A.; Biricova, V.; Laznicek, M.; Hermann, P.

2014-01-01

177 Lu radiolabeling of the first (G1-) or fourth (G4-) generation polyaminoamide (PAMAM) dendrimer conjugates with DOTA-like bifunctional chelator with one methylenepyridine-N-oxide pendant arm (DO3A-py NO-C ) stability of the radiolabeled species and their pharmacokinetic characteristics were evaluated in preclinical experiments. The results showed that the G1- and G4-dendrimer conjugates, modified in average with 7.5 or 57 DO3A-py NO-C chelating units, respectively, can also be labeled with 177 Lu with a high specific activity and radiochemical purity even at 37 °C. The radiolabeled species were stable for at least 24 h. Distribution profile of G1-dendrimer conjugate in organs and tissues of rats was more favorable than that of G4 one. On the other hand, the later dendrimer conjugate bears a substantially higher number of metal chelators per molecule enabling binding of a considerably larger number of radiometals. Our results indicate that an employment of dendrimer-chelate conjugates with bound radiometals might represent a prospective way for radiolabeling of biologically active target-specific macromolecules to obtain markedly high specific activity. - Highlights: • Chelation of DOTA-like ligands suitable for biomacromolecules modification. • Radiolabeling of modified PAMAM-dendrimers with 177 Lu. • Determination of stability of the labeled conjugates. • Pharmacokinetic characteristics evaluated in preclinical experiments

Evaluation of iodovinyl antibody conjugates: Comparison with a p-iodobenzoyl conjugate and direct radioiodination

International Nuclear Information System (INIS)

Hadley, S.W.; Wilbur, D.S.

1990-01-01

The preparations and conjugations of 2,3,5,6-tetrafluorophenyl 5-[125I/131I]iodo-4-pentenoate (7a) and 2,3,5,6-tetrafluorophenyl 3,3-dimethyl-5-[125I/131I]iodo-4-pentenoate (7b) to monoclonal antibodies are reported. Reagents 7a and 7b were prepared in high radiochemical yield by iododestannylation of their corresponding 5-tri-n-butylstannyl precursors. Radioiodinated antibody conjugates were prepared by reaction of 7a or 7b with the protein at basic pH. Evaluation of these conjugates by several in vitro procedures demonstrated that the radiolabel was attached to the antibody in a stable manner and that the conjugates maintained immunoreactivity. Comparative dual-isotope biodistribution studies of a monoclonal antibody Fab fragment conjugate of 7a and 7b with the same Fab fragment labeled with N-succinimidyl p-[131I]iodobenzoate (PIB, p-iodobenzoate, 2) or directly radioiodinated have been carried out in tumor-bearing nude mice. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 2 demonstrated that the biodistributions were similar in most organs, except the neck tissue (thyroid-containing) and the stomach, which contained substantially increased levels of the 7a label. Coinjection of the Fab conjugate of 7a with the Fab fragment radioiodinated by using the chloramine-T method demonstrated that the biodistributions were remarkably similar, suggesting roughly equivalent in vivo deiodination of these labeled antibody fragments. Coinjection of the Fab conjugate of 7a with the Fab conjugate of 7b indicated that there was ∼ a 2-fold reduction in the amount of in vivo deiodination of the 7b conjugate as compared to the 7a conjugate

Country report: Italy (Chinol). Pre-clinical evaluation of a new biotin-DOTA conjugate labeled with {sup 90}Y for application in pretargeting clinical protocols

Energy Technology Data Exchange (ETDEWEB)

Chinol, Marco

2010-07-01

In the attempt to improve the therapeutic efficacy of radiolabeled mAbs in cancer radioimmunotherapy, various studies have examined the concept of tumor pretargeting. The so called three-step pretargeting technique, employing the avidin–biotin system, was applied in phase I-II clinical trials showing low toxicity and therapeutic efficacy. The final step of the pretargeting protocols consists in the systemic injection of radiolabeled biotin. The worldwide recognized “successful association” is between {sup 90}Y and the tetraazamacrocycle DOTA chelator chemically bound to biotin. Improvements in the structure of the biotin-DOTA conjugate have been reported by our group following a novel approach which simplified the synthetic pattern by reducing the amide group to a methylene group, thus transforming the amide into a secondary amine without affecting the length of the biotin side arm. Preliminary in-vitro experiments, previously published by our group, indicated the potential of the new conjugate. Based on our previous experience with avidin-based pre-targeting followed {sup 90}Y-DOTA-biotin in the locoregional treatment of peritoneal carcinomatosis and malignant glioma suggested that similar radionuclide therapy might be worth investigating as a partial replacement of external beam radiotherapy in breast cancer. We have developed IART® the Intra-operative Avidination for Radionuclide Therapy that relies on the avidin-biotin binding system. In fact, the “avidination” of the anatomical area of the tumor with native avidin, directly injected by the surgeon into and around the tumor bed, provides a target for the radiolabeled biotin intravenously (iv) injected one day later. In order to optimize the overall strategy, further efforts were needed to optimize the use of the labeled new biotin conjugate and to elucidate its chemical and biological properties. In the first 18 months of this CRP, the pre-clinical evaluation of this new reduced biotinamidohexylamine-DOTA

Development of [⁶⁴Cu]-DOTA-PR81 radioimmunoconjugate for MUC-1 positive PET imaging.

Science.gov (United States)

Alirezapour, Behrouz; Rasaee, Mohammad Javad; Jalilian, Amir Reza; Rajabifar, Saeed; Mohammadnejad, Javad; Paknejad, Malihe; Maadi, Ehsan; Moradkhani, Sedigheh

2016-01-01

Breast cancer radioimmunoscintigraphy targeting MUC1 expression is a growing field of work in nuclear medicine research. PR81 is a monoclonal antibody that binds with high affinity to MUC1, which is over expressed on breast tumors. In this study, we report production, quality control and preclinical qualifications of a copper-64 labeled PR81 for PET imaging of breast cancer. PR81 was conjugated with DOTA-NHS-ester and purified by molecular filtration followed by chelate:mAb ratio determination by spectrophotometric method. DOTA-PR81 was labeled with (64)Cu followed by radiochemical purity, in vitro stability, in vitro internalization and immunoreactivity determination. The tissue biodistribution of the (64)Cu-DOTA-PR81 and (64)Cu-DOTA-hIgG was evaluated in BALB/c mice with breast carcinoma tumors using tissue counting and imaging. The radiochemical purity of radioimmunoconjugate was >95±1.9% (ITLC) (specific activity; 4.6 μCi/μg). The average number of chelators per antibody was 3.4±0.3:1. The (64)Cu-DOTA-PR81 showed immunoreactivity towards MUC1 antigen and MCF7 cell line with significant in vitro stability (>89% in PBS and 78±0.5% in human serum) over 48 h. Maximum internalized activity of radiolabeled PR81 in 4-8 h was 81.5%. The biodistribution and scintigraphy studies showed the accumulation of the complex at the site of tumors with high sensitivity and specificity compared to control probes. The results showed that (64)Cu-DOTA-PR81 may be considered as a potential PET tracer for diagnosis and follow-up of MUC1 expression in oncology. Copyright © 2015 Elsevier Inc. All rights reserved.

Remote-loading of liposomes with manganese-52 and in vivo evaluation of the stabilities of 52Mn-DOTA and 64Cu-DOTA using radiolabelled liposomes and PET imaging

DEFF Research Database (Denmark)

Jensen, Andreas I.; Severin, Gregory W.; Hansen, Anders Elias

2018-01-01

to 8days. Liposomes with surface-conjugated 52Mn-DOTA exhibited a significantly shorter plasma half-life (T½=14.4h) when compared to the remote-loaded counterpart (T½=21.3h), whereas surface-conjugated 64Cu-DOTA cleared only slightly faster and non-significantly, when compared to remote-loaded (17.2±2.......9h versus 20.3±1.2h). From our data, we conclude the successful remote-loading of liposomes with 52Mn, and furthermore that 52Mn-DOTA may be unstable in vivo whereas 64Cu-DOTA appears suitable for quantitative imaging....... protocols for radiolabeling liposomes with 52Mn, through both remote-loading and surface labeling. For comparison, liposomes were also remote-loaded and surface labeled with copper-64 (64Cu, T½=12.7h) through conventional means. The chelator DOTA was used in all cases. The in vivo stability of radiometal...

Preparation and biological studies of 68Ga-DOTA-alendronate

Directory of Open Access Journals (Sweden)

Ashraf Fakhari

2016-07-01

Full Text Available Objective(s: In line with previous research on the development of conjugated bisphosphonate ligands as new bone-avid agents, in this study, DOTA conjugated alendronate (DOTA-ALN was synthesized and evaluated after labeling with gallium-68 (68Ga.Methods: DOTA-ALN was synthesized and characterized, followed by 68Ga-DOTA-ALN preparation, using DOTA-ALN and 68GaCl3 (pH: 4-5 at 92-95°C for 10 min. Stability tests, hydroxyapatite assay, partition coefficient calculation,biodistribution studies, and imaging were performed on the developed agent in normal rats.Results: The complex was prepared with high radiochemical purity (>99% as depicted by radio thin-layer chromatography; specific activity: 310-320GBq/mmol after solid phase purification and was stabilized for up to 90 min with a logP value of -2.91. Maximum ligand binding (65% was observed in the presence of 50 mg of hydroxyapatite; a major portion of the activity was excreted through the kidneys. With the exception of excretory organs, gastrointestinal tract organs, including the liver, intestine, and colon, showed significant uptake; however, the bone uptake was low (

Antibody conjugate radioimmunotherapy of superficial bladder cancer

International Nuclear Information System (INIS)

Perkins, Alan; Hopper, Melanie; Murray, Andrea; Frier, Malcolm; Bishop, Mike

2002-01-01

The administration of antibody conjugates for cancer therapy is now proving to be of clinical value. We are currently undertaking a programme of clinical studies using the monoclonal antibody C 595 (gG3) which reacts with the MUC1 glycoprotein antigen that is aberrantly expressed in a high proportion of bladder tumours. Radio immuno conjugates of the C 595 antibody have been produced with high radiolabelling efficiency and immuno reactivity using Tc-99 m and In-111 for diagnostic imaging, and disease staging and the cytotoxic radionuclides Cu-67 and Re-188 for therapy of superficial bladder cancer. A Phase I/II therapeutic trail involving the intravesical administration of antibody directly into the bladder has now begun. (author)

Synthesis and stability test of radiogadolinium(III-DOTA-PAMAM G3.0-trastuzumab as SPECT-MRI molecular imaging agent for diagnosis of HER-2 positive breast cancer

Directory of Open Access Journals (Sweden)

Hardiani Rahmania

2015-01-01

Full Text Available Nonivasive diagnosis of cancer can be provided by molecular imaging using hybrid modality to obtain better sensitivity, specificity and depiction localization of the disease. In this study, we developed a new molecular imaging agent, radiogadolinium(III-DOTA-PAMAM G3.0-trastuzumab in the form of 147Gd-DOTA-PAMAM G3.0-trastuzumab, that can be both target-specific radiopharmaceutical in SPECT as well as targeted contrast agent in MRI for the purpose of diagnosis of HER-2 positive breast cancer. 147Gd radionuclide emits γ-rays that can be used in SPECT modality, but because of technical constraint, 147Gd radionuclide was simulated by its radioisotope, 153Gd. Gd-DOTA complex has also been known as good MRI contrast agent. PAMAM G3.0 is useful to concentrate Gd-DOTA compelexes in large quantities, thus minimizing the number of trastuzumab molecules used. Trastuzumab is human monoclonal antibody that can spesifically interact with HER-2. Synthesis of radiogadolinium(III-DOTA-PAMAM G3.0-trastuzumab was initiated by conjugating DOTA NHS ester ligand with PAMAM G3.0 dendrimer. The DOTA-PAMAM G3.0 produced was conjugated to trastuzumab molecule and labeled with 153Gd. Characterization DOTA-PAMAM G3.0-trastuzumab immunoconjugate was performed using HPLC system equipped with SEC. The formation of immunoconjugate was indicated by the shorter retention time (6.82 min compared to that of trastuzumab (7.06 min. Radiochemical purity of radiogadolinium(III-DOTA-PAMAM G3.0-trastuzumab was >99% after purification process by PD-10 desalting column. Radiogadolinium(III-DOTA-PAMAM G3.0-trastuzumab compound was stable at room temperature and at 2–8 0C as indicated by its radiochemical purity 97.6 ± 0.5%–99.1 ± 0.5% after 144 h storage.

Less is More: A Comparison of Antibody-Gold Nanoparticle Conjugates of Different Ratios.

Science.gov (United States)

Byzova, Nadezhda A; Safenkova, Irina V; Slutskaya, Elvira S; Zherdev, Anatoly V; Dzantiev, Boris B

2017-11-15

This comprehensive study is related to gold nanoparticles (GNPs) conjugated with antibodies. The goal of the study is to determine the minimal concentration of antibodies for conjugate synthesis when the conjugates have high antigen-capturing activity. Two systems were studied: gold nanoparticles conjugated with monoclonal antibodies (mAb-GNP) specific to Helicobacter pylori and gold nanoparticles conjugated with polyclonal antibodies (pAb-GNP) specific to mouse immunoglobulins. Several conjugates were synthesized with different GNP-to-antibody molar ratios (from 1:1 to 1:245) through nondirectional and noncovalent immobilization on a surface of GNPs with a diameter of 25.3 ± 4.6 nm. The maximal antigen-capturing activities and equilibrium constants of the conjugates correlate with the formation of a constant hydrodynamic radius of the conjugates for mAb-GNP (GNP to antibody molar ratio 1:58) and with the stabilizing concentration by flocculation curves for pAb-GNP (GNP to antibody molar ratio 1:116). The application of the conjugates to the lateral flow immunoassay shows that the antibody concentrations used for the conjugation can be reduced (below the stabilizing concentration) without losing activity for the mAb-GNP conjugates. The findings highlight that the optimal concentration of antibodies immobilized on the surface of GNPs is not always equal to the stabilizing concentration determined by the flocculation curve.

Preparation and Biological Study of (68)Ga-DOTA-alendronate.

Science.gov (United States)

Fakhari, Ashraf; Jalilian, Amir R; Johari-Daha, Fariba; Shafiee-Ardestani, Mehdi; Khalaj, Ali

2016-01-01

In line with previous research on the development of conjugated bisphosphonate ligands as new bone-avid agents, in this study, DOTA-conjugated alendronate (DOTA-ALN) was synthesized and evaluated after labeling with gallium-68 ((68)Ga). DOTA-ALN was synthesized and characterized, followed by (68)Ga-DOTA-ALN preparation, using DOTA-ALN and (68)GaCl3 (pH: 4-5) at 92-95° C for 10 min. Stability tests, hydroxyapatite assay, partition coefficient calculation, biodistribution studies, and imaging were performed on the developed agent in normal rats. The complex was prepared with high radiochemical purity (>99% as depicted by radio thin-layer chromatography; specific activity: 310-320 GBq/mmol) after solid phase purification and was stabilized for up to 90 min with a log P value of -2.91. Maximum ligand binding (65%) was observed in the presence of 50 mg of hydroxyapatite; a major portion of the activity was excreted through the kidneys. With the exception of excretory organs, gastrointestinal tract organs, including the liver, intestine, and colon, showed significant uptake; however, the bone uptake was low (<1%) at 30 min after the injection. The data were also confirmed by sequential imaging at 30-90 min following the intravenous injection. The high solubility and anionic properties of the complex led to major renal excretion and low hydroxyapatite uptake; therefore, the complex failed to demonstrate bone imaging behaviors.

Anti-transferrin receptor antibody and antibody-drug conjugates cross the blood-brain barrier

International Nuclear Information System (INIS)

Friden, P.M.; Walus, L.R.; Musso, G.F.; Taylor, M.A.; Malfroy, B.; Starzyk, R.M.

1991-01-01

Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. The authors have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration, labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling of brain capillaries becomes extremely punctate ∼ 4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a time-dependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier. Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabeled methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26

Dosimetric analysis of 177Lu-DOTA-rituximab in patients with relapsed/refractory non-Hodgkin's lymphoma.

Science.gov (United States)

Yadav, Madhav P; Singla, Suhas; Thakral, Parul; Ballal, Sanjana; Bal, Chandrasekhar

2016-07-01

Radioimmunotherapy targeting CD20 receptors in lymphoma using radiolabeled chimeric antibodies may lead to better therapeutic responses than cold anti-CD20 antibodies. This study aimed to assess the biodistribution and present reasonable estimates of normal organ doses, including red marrow using Lu-DOTA-rituximab. Patients with relapsed/refractory CD20+ B-cell non-Hodgkin's lymphoma were recruited into this prospective study. In-house labeling of Lu-DOTA-rituximab was performed and administered after quality assurance. Rituximab (375 mg/m), followed by 50 mCi (1850 MBq) of Lu-DOTA-rituximab was administered as a slow intravenous infusion and emission images were acquired. Regions of interest were drawn for kidney, liver, heart, bladder, spleen, and tumor lesions on both anterior and posterior images. Internal dose estimation was performed using OLINDA v1.0 software. The mean age of the 10 patients (eight men and two women) was 52±13 years. The uptake of radiolabeled antibody was visualized within 30 min of administration in the liver, kidneys, heart, spleen, and bladder. The coefficient of determination (R) was greater than 0.95 for organs and the whole body in all patients. The effective half-life of radioimmunoconjugate was 100±28 h (42-126 h). The critical organ in our study was the red marrow. The average total body dose, effective dose, and effective dose equivalent calculated in all 10 patients were 0.13±0.02, 0.15±0.03, and 0.22±0.04 mGy/MBq, respectively. There may be considerable interindividual differences in absorbed doses of organs and generalization or extrapolation of doses in the clinical setting at present is not feasible with Lu-DOTA-rituximab in non-Hodgkin's lymphoma patients. Patient-specific dosimetry is thus recommended to eliminate the variations and reduce the possibility of dose-limiting toxicity.

Antibody-Conjugated Nanoparticles for Biomedical Applications

Directory of Open Access Journals (Sweden)

Manuel Arruebo

2009-01-01

Full Text Available Nanoscience and Nanotechnology have found their way into the fields of Biotechnology and Medicine. Nanoparticles by themselves offer specific physicochemical properties that they do not exhibit in bulk form, where materials show constant physical properties regardless of size. Antibodies are nanosize biological products that are part of the specific immune system. In addition to their own properties as pathogens or toxin neutralizers, as well as in the recruitment of immune elements (complement, improving phagocytosis, cytotoxicity antibody dependent by natural killer cells, etc., they could carry several elements (toxins, drugs, fluorochroms, or even nanoparticles, etc. and be used in several diagnostic procedures, or even in therapy to destroy a specific target. The conjugation of antibodies to nanoparticles can generate a product that combines the properties of both. For example, they can combine the small size of nanoparticles and their special thermal, imaging, drug carrier, or magnetic characteristics with the abilities of antibodies, such as specific and selective recognition. The hybrid product will show versatility and specificity. In this review, we analyse both antibodies and nanoparticles, focusing especially on the recent developments for antibody-conjugated nanoparticles, offering the researcher an overview of the different applications and possibilities of these hybrid carriers.

In vitro Evaluation of a Bombesin Antagonistic Analogue Conjugated with DOTA-Ala(SO3H)-Aminooctanoyl for Targeting of the Gastrin-releasing Peptide Receptor

International Nuclear Information System (INIS)

Lim, Jae Cheong; Cho, Eun Ha; Kim, Jin Joo; Lee, So Young; Choi, Sang Mu

2014-01-01

As Bombesin (BBS) binds with high affinity to GRPR, BBS derivatives have been labeled with various radionuclides such as 99 mTc, 111 In, 90 Y, 64 Cu, 177 Lu, 68 Ga, or 18 F and have proved to be successful candidates for peptide receptor radiotherapy (PRRT). In this study, we employed Ala(SO 3 H)-Aminooctanoyl as a linker of BBS antagonistic peptide sequence, Gln-Trp-Ala-Val-N methyl Gly-His-Statine-Leu-NH 2 , with DOTA to prepare radiolabeled candidates for GRPR targeting. A DOTA-conjugated BBS antagonistic analogue was synthesized and radiolabeled with 177 Lu, and in vitro characteristics on GRPR-overexpressing human prostate tumor cells were evaluated. In conclusion, a novel BBS antagonistic analogue, 177 Lu-DOTA-sBBNA, is a promising candidate for the targeting of GRPR-over-expressing tumors. Further investigations to evaluate its in vivo characteristics and therapeutic efficacy are needed

DOTA-derivatives of octreotide dicarba-analogues with high affinity for somatostatin sst2,5 receptors

Science.gov (United States)

Pratesi, Alessandro; Ginanneschi, Mauro; Lumini, Marco; Papini, Anna M.; Novellino, Ettore; Brancaccio, Diego; Carotenuto, Alfonso

2017-02-01

In vivo somatostatin receptor scintigraphy is a valuable method for the visualization of human endocrine tumours and their metastases. In fact, peptide ligands of somatostatin receptors (sst’s) conjugated with chelating agents are in clinical use. We have recently developed octreotide dicarba-analogues, which show interesting binding profiles at sst’s. In this context, it was mandatory to explore the possibility that our analogues could maintain their activity also upon conjugation with DOTA. In this paper, we report and discuss the synthesis, binding affinity and conformational preferences of three DOTA-conjugated dicarba-analogues of octreotide. Interestingly, two conjugated analogues exhibited nanomolar affinities on sst2 and sst5 somatostatin receptor subtypes.

Recent advances in the construction of antibody-drug conjugates

Science.gov (United States)

Chudasama, Vijay; Maruani, Antoine; Caddick, Stephen

2016-02-01

Antibody-drug conjugates (ADCs) comprise antibodies covalently attached to highly potent drugs using a variety of conjugation technologies. As therapeutics, they combine the exquisite specificity of antibodies, enabling discrimination between healthy and diseased tissue, with the cell-killing ability of cytotoxic drugs. This powerful and exciting class of targeted therapy has shown considerable promise in the treatment of various cancers with two US Food and Drug Administration approved ADCs currently on the market (Adcetris and Kadcyla) and approximately 40 currently undergoing clinical evaluation. However, most of these ADCs exist as heterogeneous mixtures, which can result in a narrow therapeutic window and have major pharmacokinetic implications. In order for ADCs to deliver their full potential, sophisticated site-specific conjugation technologies to connect the drug to the antibody are vital. This Perspective discusses the strategies currently used for the site-specific construction of ADCs and appraises their merits and disadvantages.

Tubulin Inhibitor-Based Antibody-Drug Conjugates for Cancer Therapy

Directory of Open Access Journals (Sweden)

Hao Chen

2017-08-01

Full Text Available Antibody-drug conjugates (ADCs are a class of highly potent biopharmaceutical drugs generated by conjugating cytotoxic drugs with specific monoclonal antibodies through appropriate linkers. Specific antibodies used to guide potent warheads to tumor tissues can effectively reduce undesired side effects of the cytotoxic drugs. An in-depth understanding of antibodies, linkers, conjugation strategies, cytotoxic drugs, and their molecular targets has led to the successful development of several approved ADCs. These ADCs are powerful therapeutics for cancer treatment, enabling wider therapeutic windows, improved pharmacokinetic/pharmacodynamic properties, and enhanced efficacy. Since tubulin inhibitors are one of the most successful cytotoxic drugs in the ADC armamentarium, this review focuses on the progress in tubulin inhibitor-based ADCs, as well as lessons learned from the unsuccessful ADCs containing tubulin inhibitors. This review should be helpful to facilitate future development of new generations of tubulin inhibitor-based ADCs for cancer therapy.

Mechanism and energetics for complexation of 90Y with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a model for cancer radioimmunotherapy

International Nuclear Information System (INIS)

Jang, Y.H.; Blanco, M.; Dasgupta, S.; Keire, D.A.; Shively, J.E.; Goddard, W.A. III

1999-01-01

A promising cancer therapy involves the use of the macrocyclic polyaminoacetate DOTA (1,4,6,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) attached to a tumor-targeting antibody complexed with the β emitter 90 Y 3+ . However, incorporation of the 90 Y into the DOTA conjugate is too slow. To identify the origins of this problem, ab initio quantum chemistry methods (B3LYP/:ACVP* and HF/LACVP*) were used to predict structures and energetics. The authors find that the initial complex YH 2 (DOTA) + is 4-coordinate (the four equivalent carboxylate oxygens), which transforms to YH(DOTA) (5-coordinate with one ring N and four carboxylate oxygens), and finally to Y(DOTA) - , which is 8-coordinate (four oxygens and four nitrogens). The rate-determining step is the conversion of YH(DOTA) to Y(DOTA) - , which was calculated to have an activation free energy (aqueous phase) of 8.4 kcal/mol, in agreement with experimental results (8.1--9.3 kcal/mol) for various metals to DOTA [Kumar, K.; Tweedle, M.F. Inorg. Chem. 1993, 32, 4193--4199; Wu, S.L.; Horrocks, W.D., Jr., Inorg. Chem. 1995, 34, 3724--2732]. On the basis of this mechanism the authors propose a modified chelate, DO3AlPr, which has calculated at a much faster rate of incorporation

A rapid approach for characterization of thiol-conjugated antibody-drug conjugates and calculation of drug-antibody ratio by liquid chromatography mass spectrometry.

Science.gov (United States)

Firth, David; Bell, Leonard; Squires, Martin; Estdale, Sian; McKee, Colin

2015-09-15

We present the demonstration of a rapid "middle-up" liquid chromatography mass spectrometry (LC-MS)-based workflow for use in the characterization of thiol-conjugated maleimidocaproyl-monomethyl auristatin F (mcMMAF) and valine-citrulline-monomethyl auristatin E (vcMMAE) antibody-drug conjugates. Deconvoluted spectra were generated following a combination of deglycosylation, IdeS (immunoglobulin-degrading enzyme from Streptococcus pyogenes) digestion, and reduction steps that provide a visual representation of the product for rapid lot-to-lot comparison-a means to quickly assess the integrity of the antibody structure and the applied conjugation chemistry by mass. The relative abundance of the detected ions also offer information regarding differences in drug conjugation levels between samples, and the average drug-antibody ratio can be calculated. The approach requires little material (<100 μg) and, thus, is amenable to small-scale process development testing or as an early component of a complete characterization project facilitating informed decision making regarding which aspects of a molecule might need to be examined in more detail by orthogonal methodologies. Copyright © 2015 Elsevier Inc. All rights reserved.

Intracellular trafficking of new anticancer therapeutics: antibody-drug conjugates.

Science.gov (United States)

Kalim, Muhammad; Chen, Jie; Wang, Shenghao; Lin, Caiyao; Ullah, Saif; Liang, Keying; Ding, Qian; Chen, Shuqing; Zhan, Jinbiao

2017-01-01

Antibody-drug conjugate (ADC) is a milestone in targeted cancer therapy that comprises of monoclonal antibodies chemically linked to cytotoxic drugs. Internalization of ADC takes place via clathrin-mediated endocytosis, caveolae-mediated endocytosis, and pinocytosis. Conjugation strategies, endocytosis and intracellular trafficking optimization, linkers, and drugs chemistry present a great challenge for researchers to eradicate tumor cells successfully. This inventiveness of endocytosis and intracellular trafficking has given considerable momentum recently to develop specific antibodies and ADCs to treat cancer cells. It is significantly advantageous to emphasize the endocytosis and intracellular trafficking pathways efficiently and to design potent engineered conjugates and biological entities to boost efficient therapies enormously for cancer treatment. Current studies illustrate endocytosis and intracellular trafficking of ADC, protein, and linker strategies in unloading and also concisely evaluate practically applicable ADCs.

78 FR 18999 - Prospective Grant of Start-Up Exclusive License: Photosensitizing Antibody-Fluorophore Conjugates...

Science.gov (United States)

2013-03-28

...-Up Exclusive License: Photosensitizing Antibody-Fluorophore Conjugates for Photoimmunotherapy AGENCY...-205-2010/0-US-01), and entitled ``Photosensitizing Antibody- Fluorophore Conjugates,'' to Aspyrian... invention. The field of use may be limited to ``use of photosensitizing antibody-fluorophore conjugate by...

DNA-templated antibody conjugation for targeted drug delivery to cancer cells

DEFF Research Database (Denmark)

Liu, Tianqiang

2016-01-01

-templated organic synthesis due to the wide existence of the 3-histidine cluster in most wild-type proteins. In this thesis, three projects that relate to targeted drug delivery to cancer cells based on the DTPC method is described. The first project was a delivery system which uses transferrin as the targeting....... The study shows that DNA is a highly useful tool for the assembly of proteins with potential therapeutic applications. The DNA-templated protein conjugation shows a promising application in constructing antibody-toxin conjugates or antibody-drug conjugates. In addition, DNA strands used for antibody...... either antibody engineering or special expression systems and are both time and labor consuming. To avoid the drawbacks caused by conventional chemical modification and recombinant methodologies, an ideal site specific conjugation technique would use natural amino acid residues to the protein by a new...

In vitro characterization of 177Lu-radiolabelled chimeric anti-CD20 monoclonal antibody and a preliminary dosimetry study

International Nuclear Information System (INIS)

Forrer, Flavio; Mueller-Brand, Jan; Chen, Jianhua; Fani, Melpomeni; Powell, Pia; Maecke, Helmut R.; Lohri, Andreas; Moldenhauer, Gerhard

2009-01-01

131 I- and 90 Y-labelled anti-CD20 antibodies have been shown to be effective in the treatment of low-grade, B-cell non-Hodgkin's lymphoma (NHL). However, the most appropriate radionuclide in terms of high efficiency and low toxicity has not yet been established. In this study we evaluated an immunoconjugate formed by the anti-CD20 antibody rituximab and the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). DOTA-rituximab was prepared as a kit formulation and can be labelled in a short time ( 177 Lu or 90 Y. Immunoconjugates with different numbers of DOTA molecules per rituximab were prepared using p-SCN-Bz-DOTA. In vitro immunoreactivity and stability were tested and preliminary dosimetric results were acquired in two patients. The immunological binding properties of DOTA-rituximab to the CD20 antigen were found to be retained after conjugation with up to four chelators. The labelled product was stable against a 10 5 times excess of diethylenetriaminepentaacetic acid (DTPA, 37 C, 7 days). Two patients with relapsed NHL were treated with 740 MBq/m 2 body surface 177 Lu-DOTA-rituximab. Scintigraphic images showed specific uptake at tumour sites and acceptable dosimetric results. The mean whole-body dose was found to be 314 mGy. The administration of 177 Lu-DOTA-rituximab was tolerated well. Our results show that DOTA-rituximab (4:1) can be labelled with 177 Lu with sufficient stability while the immunoconjugate retains its immunoreactivity. 177 Lu-DOTA-rituximab is an interesting, well-tolerated radiolabelled antibody with clinical activity in a low dose range, and provides an approach to the efficient treatment with few side effects for patients with relapsed NHL. (orig.)

Electrochemical separation of 90-yttrium in the electrochemical 90Sr/90Y generator and its use for radiolabelling of DOTA-conjugated somatostatin analog [DOTA0, Tyr3] octreotate

Directory of Open Access Journals (Sweden)

Petrović Đorđe Ž.

2012-01-01

Full Text Available Radiopharmaceuticals based on 90Y are widely used in the treatment of malignant deseases. In order to meet the requirements for their future application, a 90Sr/90Y generator was developed and 90Y eluted from this locally produced generator was used for the radiolabelling of the DOTA-conjugated somatostatin analog [DOTA0,Tyr3] octreotate and the preparation of [90Y-DOTA0,Tyr3] octreotate (90Y-DOTATATE for peptide receptore radionuclide therapy. 90Sr/90Y generator was based on the electrochemical separation of 90Y from 90Sr in a two-cycle electrolysis procedure. Three electrode cells were used to perform both electrolyses. In both cycles, working electrodes were kept on constant potential. The pH of the solution was adjusted to 2.7 of the value before the electrolyses. The radionuclidic purity of the 90Y solution was analysed by ITLC and extraction paper chromatography. The labelling of peptide (100 mg DOTATATE with 90YCl3 was performed at 95°C for 30 minutes. Radiochemical purity was determined by HPLC and chromatographic separation, using a solid SepPak C-18 column. Results obtained confirmed the efficiency of our electrochemical separation technique and quality control methods for 90Y. The achieved efficiency of the 90Sr/90Y generator above 96% of the theoretical value represents a good basis for the further development of this generator. The labelling of the DOTATATE with 90Y exhibited a high efficiency, too: there was less than 1% of 90Y3+in the 90Y-DOTATATE.

In vitro Evaluation of a Bombesin Antagonistic Analogue Conjugated with DOTA-Ala(SO{sub 3}H)-Aminooctanoyl for Targeting of the Gastrin-releasing Peptide Receptor

Energy Technology Data Exchange (ETDEWEB)

Lim, Jae Cheong; Cho, Eun Ha; Kim, Jin Joo; Lee, So Young; Choi, Sang Mu [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2014-05-15

As Bombesin (BBS) binds with high affinity to GRPR, BBS derivatives have been labeled with various radionuclides such as {sup 99}mTc, {sup 111}In, {sup 90}Y, {sup 64}Cu, {sup 177}Lu, {sup 68}Ga, or {sup 18}F and have proved to be successful candidates for peptide receptor radiotherapy (PRRT). In this study, we employed Ala(SO{sub 3}H)-Aminooctanoyl as a linker of BBS antagonistic peptide sequence, Gln-Trp-Ala-Val-N methyl Gly-His-Statine-Leu-NH{sub 2}, with DOTA to prepare radiolabeled candidates for GRPR targeting. A DOTA-conjugated BBS antagonistic analogue was synthesized and radiolabeled with {sup 177}Lu, and in vitro characteristics on GRPR-overexpressing human prostate tumor cells were evaluated. In conclusion, a novel BBS antagonistic analogue, {sup 177}Lu-DOTA-sBBNA, is a promising candidate for the targeting of GRPR-over-expressing tumors. Further investigations to evaluate its in vivo characteristics and therapeutic efficacy are needed.

In vitro characterization of {sup 177}Lu-radiolabelled chimeric anti-CD20 monoclonal antibody and a preliminary dosimetry study

Energy Technology Data Exchange (ETDEWEB)

Forrer, Flavio; Mueller-Brand, Jan [University Hospital Basel, Institute of Nuclear Medicine, Basel (Switzerland); Chen, Jianhua; Fani, Melpomeni; Powell, Pia; Maecke, Helmut R. [University Hospital Basel, Division of Radiological Chemistry, Basel (Switzerland); Lohri, Andreas [Basel University Medical Clinic, Liestal (Switzerland); Moldenhauer, Gerhard [German Cancer Research Center, Division of Molecular Immunology, Heidelberg (Germany)

2009-09-15

{sup 131}I- and {sup 90}Y-labelled anti-CD20 antibodies have been shown to be effective in the treatment of low-grade, B-cell non-Hodgkin's lymphoma (NHL). However, the most appropriate radionuclide in terms of high efficiency and low toxicity has not yet been established. In this study we evaluated an immunoconjugate formed by the anti-CD20 antibody rituximab and the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). DOTA-rituximab was prepared as a kit formulation and can be labelled in a short time (<20 min) with either {sup 177}Lu or {sup 90}Y. Immunoconjugates with different numbers of DOTA molecules per rituximab were prepared using p-SCN-Bz-DOTA. In vitro immunoreactivity and stability were tested and preliminary dosimetric results were acquired in two patients. The immunological binding properties of DOTA-rituximab to the CD20 antigen were found to be retained after conjugation with up to four chelators. The labelled product was stable against a 10{sup 5} times excess of diethylenetriaminepentaacetic acid (DTPA, 37 C, 7 days). Two patients with relapsed NHL were treated with 740 MBq/m{sup 2} body surface {sup 177}Lu-DOTA-rituximab. Scintigraphic images showed specific uptake at tumour sites and acceptable dosimetric results. The mean whole-body dose was found to be 314 mGy. The administration of {sup 177}Lu-DOTA-rituximab was tolerated well. Our results show that DOTA-rituximab (4:1) can be labelled with {sup 177}Lu with sufficient stability while the immunoconjugate retains its immunoreactivity. {sup 177}Lu-DOTA-rituximab is an interesting, well-tolerated radiolabelled antibody with clinical activity in a low dose range, and provides an approach to the efficient treatment with few side effects for patients with relapsed NHL. (orig.)

Guideline for PET/CT imaging of neuroendocrine neoplasms withGa-DOTA-conjugated somatostatin receptor targeting peptides andF-DOPA

DEFF Research Database (Denmark)

Bozkurt, Murat Fani; Virgolini, Irene; Balogova, Sona

2017-01-01

PURPOSE & METHODS: Neuroendocrine neoplasms are a heterogenous group of tumours, for which nuclear medicine plays an important role in the diagnostic work-up as well as in the targeted therapeutic options. This guideline is aimed to assist nuclear medicine physicians in recommending, performing......, reporting and interpreting the results of somatostatin receptor (SSTR) PET/CT imaging using68Ga-DOTA-conjugated peptides, as well as18F-DOPA imaging for various neuroendocrine neoplasms. RESULTS & CONCLUSION: The previous procedural guideline by EANM regarding the use PET/CT tumour imaging with68Ga...

In vivo evaluation of a radiogallium-labeled bifunctional radiopharmaceutical, Ga-DOTA-MN2, for hypoxic tumor imaging.

Science.gov (United States)

Sano, Kohei; Okada, Mayumi; Hisada, Hayato; Shimokawa, Kenta; Saji, Hideo; Maeda, Minoru; Mukai, Takahiro

2013-01-01

On the basis of the findings obtained by X-ray crystallography of Ga-DOTA chelates and the drug design concept of bifunctional radiopharmaceuticals, we previously designed and synthesized a radiogallium-labeled DOTA chelate containing two metronidazole moieties, (67)Ga-DOTA-MN2, for hypoxic tumor imaging. As expected, (67)Ga-DOTA-MN2 exhibited high in vivo stability, although two carboxyl groups in the DOTA skeleton were conjugated with metronidazole moieties. In this study, we evaluated (67/68)Ga-DOTA-MN2 as a nuclear imaging agent for hypoxic tumors. (67)Ga-labeling of DOTA-MN2 with (67)GaCl(3) was achieved with high radiochemical yield (>85%) by 1-min of microwave irradiation (50 W). The pharmacokinetics of (67)Ga-DOTA-MN2 were examined in FM3A tumor-bearing mice, and compared with those of (67)Ga-DOTA-MN1 containing one metronidazole unit and (67)Ga-DOTA. Upon administration, (67)Ga-DOTA-MN2 exhibited higher accumulation in the implanted tumors than (67)Ga-DOTA. Tumor-to-blood ratios of (67)Ga-DOTA-MN2 were about two-fold higher than those of (67)Ga-DOTA-MN1. Autoradiographic analysis showed the heterogeneous localization of (67)Ga-DOTA-MN2 in the tumors, which corresponds to hypoxic regions suggested by well-established hypoxia marker drug, pimonidazole. Furthermore, in positron emission tomography (PET) study, the tumors of mice administered (68)Ga-labeled DOTA-MN2 were clearly imaged by small-animal PET at 1 h after administration. This study demonstrates the potential usefulness of (67/68)Ga-DOTA-MN2 as a nuclear imaging agent for hypoxic tumors and suggests that two functional moieties, such as metronidazole, can be conjugated to radiogallium-DOTA chelate without reducing the complex stability. The present findings provide useful information about the chemical design of radiogallium-labeled radiopharmaceuticals for PET and single photon emission computed tomography (SPECT) studies.

Syntheses and evaluation of 68 Ga- and 153 Sm-labeled DOTA-conjugated bisphosphonate ligand for potential use in detection of skeletal metastases and management of pain arising from skeletal metastases.

Science.gov (United States)

Chakraborty, Sudipta; Goswami, Dibakar; Chakravarty, Rubel; Mohammed, Sahiralam Khan; Sarma, Haladhar Deb; Dash, Ashutosh

2018-05-05

This article reports the syntheses and evaluation of 68 Ga- and 153 Sm-complexes of a new DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid)-conjugated geminal bisphosphonate, DOTA-Bn-SCN-BP, for their potential uses in the early detection of skeletal metastases by imaging and palliation of pain arising from skeletal metastases, respectively. The conjugate was synthesized in high purity following an easily adaptable three-step reaction scheme. Gallium-68- and 153 Sm-complexes were prepared in high yield (>98%) and showed excellent in vitro stability in phosphate-buffered saline (PBS) and human serum. Both the complexes showed high affinity for hydroxyapatite particles in in vitro binding study. In biodistribution studies carried out in normal Wistar rats, both the complexes exhibited rapid skeletal accumulation with almost no retention in any other major organ. The newly synthesized molecule DOTA-Bn-SCN-BP would therefore be a promising targeting ligand for the development of radiopharmaceuticals for both imaging skeletal metastases and palliation of pain arising out of it in patients with cancer when radiolabeled with 68 Ga and 153 Sm, respectively. A systematic comparative evaluation, however, showed that there was no significant improvement of skeletal accumulation of the 153 Sm-DOTA-Bn-SCN-BP complex over 153 Sm-DOTMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethylenephosphonic acid) as the later itself demonstrated optimal properties required for an agent for bone pain palliation. © 2018 John Wiley & Sons A/S.

Guideline for PET/CT imaging of neuroendocrine neoplasms with {sup 68}Ga-DOTA-conjugated somatostatin receptor targeting peptides and {sup 18}F-DOPA

Energy Technology Data Exchange (ETDEWEB)

Bozkurt, Murat Fani [Hacettepe University Faculty of Medicine Department of Nuclear Medicine, Ankara (Turkey); Virgolini, Irene; Decristoforo, Clemens [Medical University Innsbruck, Department of Nuclear Medicine, Innsbruck (Austria); Balogova, Sona [Comenius University and St. Elisabeth Oncology Institute, Department of Nuclear Medicine, Bratislava (Slovakia); Tenon Hospital AP-HP and Universite Pierre et Marie Curie, Department of Nuclear Medicine, Paris (France); Beheshti, Mohsen [St. Vincent' s Hospital, PET-CT Center, Department of Nuclear Medicine and Endocrinology, Linz (Austria); Paracelsus Medical University, Department of Nuclear Medicine, Salzburg (Austria); Rubello, Domenico [Santa Maria della Misericordia Hospital, Department of Nuclear Medicine, PET Center and Medical Physics and Radiology, Rovigo (Italy); Ambrosini, Valentina; Fanti, Stefano [University of Bologna, Department of Experimental, Diagnostic and Specialty Medicine-DIMES, Bologna (Italy); Kjaer, Andreas [National University Hospital and University of Copenhagen, Department of Clinical Physiology, Nuclear Medicine and PET, Rigshospitalet, Copenhagen (Denmark); Delgado-Bolton, Roberto [San Pedro Hospital and Centre for Biomedical Research of La Rioja (CIBIR), Department of Diagnostic Imaging (Radiology) and Nuclear Medicine, Logrono (Spain); Kunikowska, Jolanta [Medical University of Warsaw, Nuclear Medicine, Warsaw (Poland); Oyen, Wim J.G. [Institute of Cancer Research and Royal Marsden NHS Foundation Trust, London (United Kingdom); Chiti, Arturo [Humanitas University, Nuclear Medicine Department, Rozzano, MI (Italy); Giammarile, Francesco [University of Lyon, Nuclear Medicine, Lyon (France)

2017-08-15

Neuroendocrine neoplasms are a heterogenous group of tumours, for which nuclear medicine plays an important role in the diagnostic work-up as well as in the targeted therapeutic options. This guideline is aimed to assist nuclear medicine physicians in recommending, performing, reporting and interpreting the results of somatostatin receptor (SSTR) PET/CT imaging using {sup 68}Ga-DOTA-conjugated peptides, as well as {sup 18}F-DOPA imaging for various neuroendocrine neoplasms. The previous procedural guideline by EANM regarding the use PET/CT tumour imaging with {sup 68}Ga-conjugated peptides has been revised and updated with the relevant and recent literature in the field with contribution of distinguished experts. (orig.)

Development of 90Y-DOTA-nimotuzumab Fab fragment for radioimmunotherapy

International Nuclear Information System (INIS)

Alonso Martinez, L.M.; Marylaine Perez-Malo Cruz; Rene Leyva Montana; Calzada Falcon, V.N.; Minely Zamora Barrabi; Alejandro Arbesu Valdivia; Ignacio Hernandez Gonzalez; Mariela Leon Perez

2014-01-01

Yttrium-90-( 90 Y) labeled monoclonal antibodies prepared with a chelating agent, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), have been used for radioimmunotherapy of cancer. In the present work, the Fab fragment of anti-EGFR monoclonal antibody nimotuzumab was prepared with high purity, integrity and biological activity. The Fab fragment with high specific recognition of EGFR in NCI-H125 human lung adenocarcinoma cells was derivatized with DOTA-NHS applying a simple procedure. DOTA-nimotuzumab Fab fragment was successfully radiolabeled with 90 Y with high radiochemical yield. The in vitro stability of labeled product was optimal over 24 h in buffered solution at 37 deg C. Biodistribution and pharmacokinetic studies correctly evaluated the in vivo non-tumor uptake, dosage regimen and excretion pathway in normal Wistar rats. (author)

Bispecific small molecule-antibody conjugate targeting prostate cancer.

Science.gov (United States)

Kim, Chan Hyuk; Axup, Jun Y; Lawson, Brian R; Yun, Hwayoung; Tardif, Virginie; Choi, Sei Hyun; Zhou, Quan; Dubrovska, Anna; Biroc, Sandra L; Marsden, Robin; Pinstaff, Jason; Smider, Vaughn V; Schultz, Peter G

2013-10-29

Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant αCD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility. The efficacy of the conjugate was optimized by modifying the linker structure, relative binding orientation, and stoichiometry of the ligand. The optimized conjugate showed potent and selective in vitro activity (EC50 ~ 100 pM), good serum half-life, and potent in vivo activity in prophylactic and treatment xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors.

Bispecific small molecule–antibody conjugate targeting prostate cancer

Science.gov (United States)

Kim, Chan Hyuk; Axup, Jun Y.; Lawson, Brian R.; Yun, Hwayoung; Tardif, Virginie; Choi, Sei Hyun; Zhou, Quan; Dubrovska, Anna; Biroc, Sandra L.; Marsden, Robin; Pinstaff, Jason; Smider, Vaughn V.; Schultz, Peter G.

2013-01-01

Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant αCD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility. The efficacy of the conjugate was optimized by modifying the linker structure, relative binding orientation, and stoichiometry of the ligand. The optimized conjugate showed potent and selective in vitro activity (EC50 ∼100 pM), good serum half-life, and potent in vivo activity in prophylactic and treatment xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors. PMID:24127589

Remote-loading of liposomes with manganese-52 and in vivo evaluation of the stabilities of 52Mn-DOTA and 64Cu-DOTA using radiolabelled liposomes and PET imaging.

Science.gov (United States)

Jensen, Andreas I; Severin, Gregory W; Hansen, Anders E; Fliedner, Frederikke P; Eliasen, Rasmus; Parhamifar, Ladan; Kjær, Andreas; Andresen, Thomas L; Henriksen, Jonas R

2018-01-10

Liposomes are nanoparticles used in drug delivery that distribute over several days in humans and larger animals. Radiolabeling with long-lived positron emission tomography (PET) radionuclides, such as manganese-52 ( 52 Mn, T½=5.6days), allow the imaging of this biodistribution. We report optimized protocols for radiolabeling liposomes with 52 Mn, through both remote-loading and surface labeling. For comparison, liposomes were also remote-loaded and surface labeled with copper-64 ( 64 Cu, T½=12.7h) through conventional means. The chelator DOTA was used in all cases. The in vivo stability of radiometal chelates is widely debated but studies that mimic a realistic in vivo setting are lacking. Therefore, we employed these four radiolabeled liposome types as platforms to demonstrate a new concept for such in vivo evaluation, here of the chelates 52 Mn-DOTA and 64 Cu-DOTA. This was done by comparing "shielded" remote-loaded with "exposed" surface labeled variants in a CT26 tumor-bearing mouse model. Remote loading (90min at 55°C) and surface labeling (55°C for 2h) of 52 Mn gave excellent radiolabeling efficiencies of 97-100% and 98-100% respectively, and the liposome biodistribution was imaged by PET for up to 8days. Liposomes with surface-conjugated 52 Mn-DOTA exhibited a significantly shorter plasma half-life (T ½ =14.4h) when compared to the remote-loaded counterpart (T ½ =21.3h), whereas surface-conjugated 64 Cu-DOTA cleared only slightly faster and non-significantly, when compared to remote-loaded (17.2±2.9h versus 20.3±1.2h). From our data, we conclude the successful remote-loading of liposomes with 52 Mn, and furthermore that 52 Mn-DOTA may be unstable in vivo whereas 64 Cu-DOTA appears suitable for quantitative imaging. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioanalysis of antibody-drug conjugates: American Association of Pharmaceutical Scientists Antibody-Drug Conjugate Working Group position paper.

Science.gov (United States)

Gorovits, Boris; Alley, Stephen C; Bilic, Sanela; Booth, Brian; Kaur, Surinder; Oldfield, Phillip; Purushothama, Shobha; Rao, Chetana; Shord, Stacy; Siguenza, Patricia

2013-05-01

Antibody-drug conjugates (ADCs) typically consist of a cytotoxic drug covalently bound to an antibody by a linker. These conjugates have the potential to substantially improve efficacy and reduce toxicity compared with cytotoxic small-molecule drugs. Since ADCs are generally complex heterogeneous mixtures of multiple species, these novel therapeutic products present unique bioanalytical challenges. The growing number of ADCs being developed across the industry suggests the need for alignment of the bioanalytical methods or approaches used to assess the multiple species and facilitate consistent interpretation of the bioanalytical data. With limited clinical data, the current strategies that can be used to provide insight into the relationship between the multiple species and the observed clinical safety and efficacy are still evolving. Considerations of the bioanalytical strategies for ADCs based on the current industry practices that take into account the complexity and heterogeneity of ADCs are discussed.

Preclinical evaluation of [ 111 In]-DOTA-trastuzumab for clinical trials

Directory of Open Access Journals (Sweden)

Behrouz Alirezapour

2014-01-01

Full Text Available Context: Herceptin and its fragments have been radiolabeled and used in the imaging of human epidermal growth factor receptor 2 (HER2/neu-positive tumors and development of diagnostic kits is of great importance in radiopharmacy. Aims: In this study, 111 In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-trastuzumab ( 111 In-DOTA-trastuzumab was successively prepared and evaluated for ultimate use in the HER2 antigen imaging in oncology. Settings and Design: The conjugate was prepared, labeled and evaluated using in vitro (radioimmunoassay [RIA], enzyme-linked immunosorbent assay (ELISA, stability, binding, internalization/in vivo (bio-distribution, single-photon emission computed tomography [SPECT] experiments. Materials and Methods: 111 In-DOTA-trastuzumab was prepared followed by determination of radiochemical purity (RCP, integrity of protein, immunoreactivity of radiolabeled antibody with HER2/neu antigen (by SkBr3 cell line binding and RIA methods were determined followed by stability tests, internalization studies and the tissue bio-distribution determination in wild-type rats as well as SPECT imaging in SkBr3-bearing mice. Statistical Analysis Used: All values were expressed as mean ± standard deviation (mean ± SD and the data were compared using Student′s t-test. Statistical significance was defined as P 95 ± 0.5%, S.A. 5.3 μCi/μg with the average number of chelators per antibody of 6:1 showing significant immune-reactivity retention using ELISA. In vitro stability was >90% in phosphate buffered saline and 80 ± 0.5% in serum over 48 h. Cell binding was significant (>0.79. In vitro internalization reached up to %12-13 in 10 h. Significant tumor uptake was observed. Conclusions: In vitro and in vivo/SPECT imaging in SkBr3-bearing mice demonstrated that 111 In-DOTA-trastuzumab is a potential compound for molecular imaging of SPECT for diagnosis and follow-up of HER2 expression in oncology.

CD133 antibody conjugation to decellularized human heart valves intended for circulating cell capture.

Science.gov (United States)

Vossler, John D; Min Ju, Young; Williams, J Koudy; Goldstein, Steven; Hamlin, James; Lee, Sang Jin; Yoo, James J; Atala, Anthony

2015-09-03

The long term efficacy of tissue based heart valve grafts may be limited by progressive degeneration characterized by immune mediated inflammation and calcification. To avoid this degeneration, decellularized heart valves with functionalized surfaces capable of rapid in vivo endothelialization have been developed. The aim of this study is to examine the capacity of CD133 antibody-conjugated valve tissue to capture circulating endothelial progenitor cells (EPCs). Decellularized human pulmonary valve tissue was conjugated with CD133 antibody at varying concentrations and exposed to CD133 expressing NTERA-2 cl.D1 (NT2) cells in a microflow chamber. The amount of CD133 antibody conjugated on the valve tissue surface and the number of NT2 cells captured in the presence of shear stress was measured. Both the amount of CD133 antibody conjugated to the valve leaflet surface and the number of adherent NT2 cells increased as the concentration of CD133 antibody present in the surface immobilization procedure increased. The data presented in this study support the hypothesis that the rate of CD133(+) cell adhesion in the presence of shear stress to decellularized heart valve tissue functionalized by CD133 antibody conjugation increases as the quantity of CD133 antibody conjugated to the tissue surface increases.

Somatostatin receptor PET in neuroendocrine tumours: 68Ga-DOTA0,Tyr3-octreotide versus 68Ga-DOTA0-lanreotide.

Science.gov (United States)

Putzer, Daniel; Kroiss, Alexander; Waitz, Dietmar; Gabriel, Michael; Traub-Weidinger, Tatjana; Uprimny, Christian; von Guggenberg, Elisabeth; Decristoforo, Clemens; Warwitz, Boris; Widmann, Gerlig; Virgolini, Irene Johanna

2013-02-01

The aim of this study was to evaluate the impact of (68)Ga-labelled DOTA(0)-lanreotide ((68)Ga-DOTA-LAN) on the diagnostic assessment of neuroendocrine tumour (NET) patients with low to moderate uptake on planar somatostatin receptor (SSTR) scintigraphy or (68)Ga-labelled DOTA(0),Tyr(3)-octreotide ((68)Ga-DOTA-TOC) positron emission tomography (PET). Fifty-three patients with histologically confirmed NET and clinical signs of progressive disease, who had not qualified for peptide receptor radionuclide therapy (PRRT) on planar SSTR scintigraphy or (68)Ga-DOTA-TOC PET (n = 38) due to lack of tracer uptake, underwent (68)Ga-DOTA-LAN PET to evaluate a treatment option with (90)Y-labelled lanreotide according to the MAURITIUS trial. The included patients received 150 ± 30 MBq of each radiopharmaceutical intravenously. PET scans were acquired 60-90 min after intravenous bolus injection. Image results from both PET scans were compared head to head, focusing on the intensity of tracer uptake in terms of treatment decision. CT was used for morphologic correlation of tumour lesions. To further evaluate the binding affinities of each tracer, quantitative and qualitative values were calculated for target lesions. (68)Ga-DOTA-LAN and (68)Ga-DOTA-TOC both showed equivalent findings in 24/38 patients when fused PET/CT images were interpreted. The sensitivity, specificity and accuracy of (68)Ga-DOTA-LAN in comparison to CT were 0.63, 0.5 and 0.62 (n = 53; p < 0.0001) and for (68)Ga-DOTA-TOC in comparison to CT 0.78, 0.5 and 0.76 (n = 38; p < 0.013), respectively. (68)Ga-DOTA-TOC showed a significantly higher maximum standardized uptake value (SUV(max)) regarding the primary tumour in 25 patients (p < 0.003) and regarding the liver in 30 patients (p < 0.009) compared to (68)Ga-DOTA-LAN. Corresponding values of both PET scans for tumour and liver did not show any significant correlation. (68)Ga-DOTA-TOC revealed more tumour sites than (68)Ga-DOTA

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

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Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F

2015-08-01

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab')2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro. © 2015 The Authors. American Journal of Transplantation Published by Wiley Periodicals, Inc.

Cathepsin B Cleavage of vcMMAE-Based Antibody-Drug Conjugate Is Not Drug Location or Monoclonal Antibody Carrier Specific.

Science.gov (United States)

Gikanga, Benson; Adeniji, Nia S; Patapoff, Thomas W; Chih, Hung-Wei; Yi, Li

2016-04-20

Antibody-drug conjugates (ADCs) require thorough characterization and understanding of product quality attributes. The framework of many ADCs comprises one molecule of antibody that is usually conjugated with multiple drug molecules at various locations. It is unknown whether the drug release rate from the ADC is dependent on drug location, and/or local environment, dictated by the sequence and structure of the antibody carrier. This study addresses these issues with valine-citrulline-monomethylauristatin E (vc-MMAE)-based ADC molecules conjugated at reduced disulfide bonds, by evaluating the cathepsin B catalyzed drug release rate of ADC molecules with different drug distributions or antibody carriers. MMAE drug release rates at different locations on ADC I were compared to evaluate the impact of drug location. No difference in rates was observed for drug released from the V(H), V(L), or C(H)2 domains of ADC I. Furthermore, four vc-MMAE ADC molecules were chosen as substrates for cathepsin B for evaluation of Michaelis-Menten parameters. There was no significant difference in K(M) or k(cat) values, suggesting that different sequences of the antibody carrier do not result in different drug release rates. Comparison between ADCs and small molecules containing vc-MMAE moieties as substrates for cathepsin B suggests that the presence of IgG1 antibody carrier, regardless of its bulkiness, does not impact drug release rate. Finally, a molecular dynamics simulation on ADC II revealed that the val-cit moiety at each of the eight possible conjugation sites was, on average, solvent accessible over 50% of its maximum solvent accessible surface area (SASA) during a 500 ns trajectory. Combined, these results suggest that the cathepsin cleavage sites for conjugated drugs are exposed enough for the enzyme to access and that the drug release rate is rather independent of drug location or monoclonal antibody carrier. Therefore, the distribution of drug conjugation at different

[64Cu]-Labelled Trastuzumab: Optimisation of Labelling by DOTA and NODAGA Conjugation and Initial Evaluation in Mice

DEFF Research Database (Denmark)

Schjøth-Eskesen, Christina; Nielsen, Carsten Haagen; Heissel, Søren

2015-01-01

The human epidermal growth factor receptor-2 (HER2) is overexpressed in 20-30% of all breast cancer cases, leading to increased cell proliferation, growth and migration. The monoclonal antibody, trastuzumab, binds to HER2 and is used for treatment of HER2-positive breast cancer. Trastuzumab has...... [(64) Cu]DOTA-trastuzumab and [(64) Cu]NODAGA-trastuzumab were produced after purification with radiochemical purities of >97%. The tracers were injected into mice with HER2 expressing tumours. The mice were imaged by positron emission tomography and showed high tumour uptake of 3-9% ID/g for both...

Preparation and evaluation of a radiogallium complex-conjugated bisphosphonate as a bone scintigraphy agent

International Nuclear Information System (INIS)

Ogawa, Kazuma; Takai, Kenichiro; Kanbara, Hiroya; Kiwada, Tatsuto; Kitamura, Yoji; Shiba, Kazuhiro; Odani, Akira

2011-01-01

Introduction: 68 Ga is a radionuclide of great interest as a positron emitter for positron emission tomography (PET). To develop a new bone-imaging agent with radiogallium, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was chosen as a chelating site and Ga-DOTA complex-conjugated bisphosphonate, which has a high affinity for bone, was prepared and evaluated. Although we are interested in developing 68 Ga-labeled bone imaging agents for PET, in these initial studies 67 Ga was used because of its longer half-life. Methods: DOTA-conjugated bisphosphonate (DOTA-Bn-SCN-HBP) was synthesized by conjugation of 2-(4-isothiocyanatebenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid to 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate). 67 Ga-DOTA-Bn-SCN-HBP was prepared by coordination with 67 Ga, and its in vitro and in vivo evaluations were performed. Results: 67 Ga-DOTA-Bn-SCN-HBP was prepared with a radiochemical purity of over 95% without purification. 67 Ga-DOTA-Bn-SCN-HBP had great affinity for hydroxyapatite in binding assay. In biodistribution experiments, 67 Ga-DOTA-Bn-SCN-HBP accumulated in bone rapidly but was hardly observed in tissues other than bone. Pretreatment of an excess amount of alendronate inhibited the bone accumulation of 67 Ga-DOTA-Bn-SCN-HBP. Conclusions: 67 Ga-DOTA-Bn-SCN-HBP showed ideal biodistribution characteristics as a bone-imaging agent. These findings should provide useful information on the drug design of bone imaging agents for PET with 68 Ga.

177Lu-DOTA-Bevacizumab: Radioimmunotherapy Agent for Melanoma.

Science.gov (United States)

Camacho, Ximena; Calzada, Victoria; Fernandez, Marcelo; Alonso, Omar; Chammas, Roger; Riva, Eloisa; Gambini, Juan Pablo; Cabral, Pablo

2017-01-01

Vascular endothelial growth factor (VEGF) is one of the classic factors to tumor-induced angiogenesis in several types, including melanoma. Bevacizumab is a humanized monoclonal antibody directed against VEGF. To radiolabel Bevacizumab with 177-Lutetium as a potential radioimmunotherapy agent for melanoma. Bevacizumab was derivatized with DOTA-NHS-ester at 4 ºC for 18 h. DOTABevacizumab was radiolabeled with 177LuCl3 (15 MBq/mg) at 37 ºC for 1 h. The studies were performed in healthy and B16F1 tumor-bearing C57BL/6J mice at 24 and 48 h (n = 5). Scinthigraphic imaging studies were performed at 24 h to determine the radiochemical stability, targeting specificity and pharmacokinetics of the 177Lutetium-labeled antibody. DOTA-Bevacizumab was efficiently labeled with 177LuCl3 at 37 °C. The in-vitro stability of labeled product was optimal over 72 h. In-vivo biodistribution studies showed a high liver and tumor uptake of 177Lu-DOTA-Bevacizumab, with tumor-to-muscle ratios of 11.58 and 6.37 at 24 and 48 h p.i. Scintigraphic imaging of melanoma tumor-bearing C57BL/6J mice showed liver and a high tumor selective uptake of 177Lu-DOTA-Bevacizumab at 24 h. Our results support the potential role of 177Lu-DOTA-Bevacizumab as a novel radioimmunotherapy agent for melanoma. We hope that these novel molecular imaging agents will open the path to new diagnostic and therapeutic strategies for Melanoma disease. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Synthesis of DOTA-conjugated multimeric [Tyr3]octreotide peptides via a combination of Cu(I)-catalyzed "click" cycloaddition and thio acid/sulfonyl azide "sulfo-click" amidation and their in vivo evaluation.

NARCIS (Netherlands)

Yim, C.B.; Dijkgraaf, I.; Merkx, R.; Versluis, C.; Eek, A.; Mulder, G.E.; Rijkers, D.T.; Boerman, O.C.; Liskamp, R.M.

2010-01-01

Herein, we describe the design, synthesis, and biological evaluation of a series of DOTA-conjugated monomeric, dimeric, and tetrameric [Tyr(3)]octreotide-based analogues as a tool for tumor imaging and/or radionuclide therapy. These compounds were synthesized using a Cu(I)-catalyzed 1,3-dipolar

68Ga-labeling and in vivo evaluation of a uPAR binding DOTA- and NODAGA-conjugated peptide for PET imaging of invasive cancers

DEFF Research Database (Denmark)

Persson, Morten; Madsen, Jacob; Østergaard, Søren

2012-01-01

, uPAR binding affinity and cell uptake were determined. To characterize the in vivo properties, dynamic microPET imaging was carried out in nude mice bearing human glioma U87MG tumor xenograft. RESULTS: In vitro experiments revealed uPAR binding affinities in the lower nM range for both conjugated......-AE105-NH(2) was observed. Good stability in phosphate-buffered saline and mouse plasma was observed. High cell uptake was found for both tracers in U87MG tumor cells. Dynamic microPET imaging demonstrated good tumor-to-background ratio for both tracers. Tumor uptake was 2.1% ID/g and 1.3% ID/g 30 min...... positron emission tomography (PET) in human cancer xenograft mice models. Here we introduce (68)Ga-DOTA-AE105-NH(2) and (68)Ga-NODAGA-AE105-NH(2) and evaluate their imaging properties using small-animal PET. METHODS: Synthesis of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) was based on solid-phase peptide...

Antibody-nanoparticle conjugates to enhance the sensitivity of ELISA-based detection methods.

Directory of Open Access Journals (Sweden)

Margaret M Billingsley

Full Text Available Accurate antigen detection is imperative for clinicians to diagnose disease, assess treatment success, and predict patient prognosis. The most common technique used for the detection of disease-associated biomarkers is the enzyme linked immunosorbent assay (ELISA. In an ELISA, primary antibodies are incubated with biological samples containing the biomarker of interest. Then, detectible secondary antibodies conjugated with horseradish peroxidase (HRP bind the primary antibodies. Upon addition of a color-changing substrate, the samples provide a colorimetric signal that directly correlates to the targeted biomarker concentration. While ELISAs are effective for analyzing samples with high biomarker content, they lack the sensitivity required to analyze samples with low antigen levels. We hypothesized that the sensitivity of ELISAs could be enhanced by replacing freely delivered primary antibodies with antibody-nanoparticle conjugates that provide excess binding sites for detectible secondary antibodies, ultimately leading to increased signal. Here, we investigated the use of nanoshells (NS decorated with antibodies specific to epidermal growth factor receptor (EGFR as a model system (EGFR-NS. We incubated one healthy and two breast cancer cell lines, each expressing different levels of EGFR, with EGFR-NS, untargeted NS, or unconjugated EGFR antibodies, as well as detectable secondary antibodies. We found that EGFR-NS consistently increased signal intensity relative to unconjugated EGFR antibodies, with a substantial 13-fold enhancement from cells expressing high levels of EGFR. Additionally, 40x more unconjugated antibodies were required to detect EGFR compared to those conjugated to NS. Our results demonstrate that antibody-nanoparticle conjugates lower the detection limit of traditional ELISAs and support further investigation of this strategy with other antibodies and nanoparticles. Owing to their enhanced sensitivity, we anticipate that

Preparation and evaluation of a radiogallium complex-conjugated bisphosphonate as a bone scintigraphy agent

Energy Technology Data Exchange (ETDEWEB)

Ogawa, Kazuma, E-mail: kogawa@p.kanazawa-u.ac.jp [Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192 (Japan); Takai, Kenichiro; Kanbara, Hiroya; Kiwada, Tatsuto [Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192 (Japan); Kitamura, Yoji; Shiba, Kazuhiro [Advanced Science Research Center, Kanazawa University, Kanazawa 920-8640 (Japan); Odani, Akira [Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-1192 (Japan)

2011-07-15

Introduction: {sup 68}Ga is a radionuclide of great interest as a positron emitter for positron emission tomography (PET). To develop a new bone-imaging agent with radiogallium, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was chosen as a chelating site and Ga-DOTA complex-conjugated bisphosphonate, which has a high affinity for bone, was prepared and evaluated. Although we are interested in developing {sup 68}Ga-labeled bone imaging agents for PET, in these initial studies {sup 67}Ga was used because of its longer half-life. Methods: DOTA-conjugated bisphosphonate (DOTA-Bn-SCN-HBP) was synthesized by conjugation of 2-(4-isothiocyanatebenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid to 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate). {sup 67}Ga-DOTA-Bn-SCN-HBP was prepared by coordination with {sup 67}Ga, and its in vitro and in vivo evaluations were performed. Results: {sup 67}Ga-DOTA-Bn-SCN-HBP was prepared with a radiochemical purity of over 95% without purification. {sup 67}Ga-DOTA-Bn-SCN-HBP had great affinity for hydroxyapatite in binding assay. In biodistribution experiments, {sup 67}Ga-DOTA-Bn-SCN-HBP accumulated in bone rapidly but was hardly observed in tissues other than bone. Pretreatment of an excess amount of alendronate inhibited the bone accumulation of {sup 67}Ga-DOTA-Bn-SCN-HBP. Conclusions: {sup 67}Ga-DOTA-Bn-SCN-HBP showed ideal biodistribution characteristics as a bone-imaging agent. These findings should provide useful information on the drug design of bone imaging agents for PET with {sup 68}Ga.

Chemical de-conjugation for investigating the stability of small molecule drugs in antibody-drug conjugates.

Science.gov (United States)

Chen, Tao; Su, Dian; Gruenhagen, Jason; Gu, Christine; Li, Yi; Yehl, Peter; Chetwyn, Nik P; Medley, Colin D

2016-01-05

Antibody-drug conjugates (ADCs) offer new therapeutic options for advanced cancer patients through precision killing with fewer side effects. The stability and efficacy of ADCs are closely related, emphasizing the urgency and importance of gaining a comprehensive understanding of ADC stability. In this work, a chemical de-conjugation approach was developed to investigate the in-situ stability of the small molecule drug while it is conjugated to the antibody. This method involves chemical-mediated release of the small molecule drug from the ADC and subsequent characterization of the released small molecule drug by HPLC. The feasibility of this technique was demonstrated utilizing a model ADC containing a disulfide linker that is sensitive to the reducing environment within cancer cells. Five reducing agents were screened for use in de-conjugation; tris(2-carboxyethyl) phosphine (TCEP) was selected for further optimization due to its high efficiency and clean impurity profile. The optimized de-conjugation assay was shown to have excellent specificity and precision. More importantly, it was shown to be stability indicating, enabling the identification and quantification of the small molecule drug and its degradation products under different formulation pHs and storage temperatures. In summary, the chemical de-conjugation strategy demonstrated here offers a powerful tool to assess the in-situ stability of small molecule drugs on ADCs and the resulting information will shed light on ADC formulation/process development and storage condition selection. Copyright © 2015 Elsevier B.V. All rights reserved.

Characterization of hapten-protein conjugates: antibody generation and immunoassay development for chlorophenoxyacetic acid pesticides.

Science.gov (United States)

Boro, Robin C; Singh, K Vikas; Suri, C Raman

2009-01-01

The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten-protein conjugates. In this study, we report a new fluorescence-based method for the characterization of hapten-protein conjugates. The method is based on an effect promoted by hapten-protein conjugation density upon the fluorescence intensity of the intrinsic tryptophan chromophore molecules of the protein. The proposed methodology is applied to quantify the hapten-protein conjugation density for two different chlorophenoxyacetic acid pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenoxybutyric acid (2,4-DB), coupled to carrier protein. Highly sensitive anti-2,4-D and anti-2,4-DB antibodies were obtained using these well-characterized hapten-protein conjugates. The generated antibodies were used in an immunoassay format demonstrating inhibitory concentration (IC50) values equal to 30 and 7 ng/mL for 2,4-D and 2,4-DB, respectively. Linearity was observed in the concentration range between 0.1-500 nglmL with LODs around 4 and 3 ng/mL for 2,4-D and 2,4-DB, respectively, in standard water samples. The proposed method was successfully applied for the determination of the extent of hapten-protein conjugation to produce specific antibodies for immunoassay development against pesticides.

Effect of different hapten-carrier conjugation ratios and molecular orientations on antibody affinity against a peptide antigen

DEFF Research Database (Denmark)

Pedersen, M. K.; Sørensen, Nanna Skall; Heegaard, Peter M. H.

2006-01-01

-based assay systems and in deciding whether a vaccine-induced antibody response will be protective. With ovalbumin as a carrier protein and a peptide (7.2NY) representing a 19 ammo acid sequence from the E. coli-derived Verotoxin 2e as a model hapten we investigated whether it was possible to influence...... ten dines at two-weeks intervals with low doses of the eight conjugates, Blood samples collected between each immunisation were analysed by ELISA for specific antibody titres and relative affinities. With both types of conjugations, the anti-peptide antibody titres increased in response to increasing...... for terminal conjugation. Thus, it appears that the molar ratio of a peptide and its carrier may affect the resulting antibody affinities, and that a conjugation ratio between a terminally Conjugated peptide and its carrier approaching one will result in relatively high antibody affinities. Furthermore...

Metal chelate conjugated monoclonal antibodies, wherein the metal is an α emitter

International Nuclear Information System (INIS)

Gansow, O.A.; Strand, M.

1984-01-01

Methods of manufacturing and purifying metal chelate conjugated monoclonal antibodies are described, wherein the chelated metal emits alpha radiation. The conjugates are suited for therapeutic uses being substantially free of nonchelated radiometal. (author)

BRCAA1 antibody- and Her2 antibody-conjugated amphiphilic polymer engineered CdSe/ZnS quantum dots for targeted imaging of gastric cancer

Science.gov (United States)

Li, Chao; Ji, Yang; Wang, Can; Liang, Shujing; Pan, Fei; Zhang, Chunlei; Chen, Feng; Fu, Hualin; Wang, Kan; Cui, Daxiang

2014-05-01

Successful development of safe and highly effective nanoprobes for targeted imaging of in vivo early gastric cancer is a great challenge. Herein, we choose the CdSe/ZnS (core-shell) quantum dots (QDs) as prototypical materials, synthesized one kind of a new amphiphilic polymer including dentate-like alkyl chains and multiple carboxyl groups, and then used the prepared amphiphilic polymer to modify QDs. The resultant amphiphilic polymer engineered QDs (PQDs) were conjugated with BRCAA1 and Her2 monoclonal antibody, and prepared BRCAA1 antibody- and Her2 antibody-conjugated QDs were used for in vitro MGC803 cell labeling and in vivo targeted imaging of gastric cancer cells. Results showed that the PQDs exhibited good water solubility, strong photoluminescence (PL) intensity, and good biocompatibility. BRCAA1 antibody- and Her2 antibody-conjugated QD nanoprobes successfully realized targeted imaging of in vivo gastric cancer MGC803 cells. In conclusion, BRCAA1 antibody- and Her2 antibody-conjugated PQDs have great potential in applications such as single cell labeling and in vivo tracking, and targeted imaging and therapeutic effects' evaluation of in vivo early gastric cancer cells in the near future.

An optimized antibody-chelator conjugate for imaging of carcinoembryonic antigen with indium-111

International Nuclear Information System (INIS)

Sumerdon, G.A.; Rogers, P.E.; Lombardo, C.M.; Schnobrich, K.E.; Melvin, S.L.; Tribby, I.I.E.; Stroupe, S.D.; Johnson, D.K.; Hobart, E.D.

1990-01-01

A monoclonal antibody to carcinoembryonic antigen showing minimal cross-reactivity with blood cells and normal tissues was derivatized with benzylisothiocyanate derivatives of EDTA and DTPA. Seven chelators per immunoglobulin could be incorporated without loss of immunoreactivity. The resulting conjugates, labeled with indium-111, showed low liver uptake in animals. A cold kit, comprising the DTPA conjugate at a molarity of antibody bound chelator exceeding 1 x 10 -4 M, gave radiochemical yields of indium labeled antibody of ≥ 95% and was stable for 1 yr. (author)

A VAR2CSA:CSP conjugate capable of inducing dual specificity antibody responses

DEFF Research Database (Denmark)

Matondo, Sungwa; Thrane, Susan; Janitzek, Christoph Mikkel

2017-01-01

Catcher peptide. The covalent interaction between SpyTag/SpyCatcher enables the formation of DBL1x-DBL2x-ID2a:CSP conjugate vaccine. Immunogenicity and quality of antibody responses induced by the conjugate vaccine, as well as a control CSP-SpyCatcher vaccine, was tested in BALB/c mice.  Results: Serum samples...... obtained from mice immunized with the conjugate vaccine were able to recognize both untagged DBL1x-DBL2x-ID2a as well as CSP antigen. Moreover, the geometric mean anti-CSP antibody titer was 1.9-fold higher in serum (at day 35 and 55 post-first immunization) from mice immunized with the conjugate vaccine......, as compared to mice receiving the control vaccine.  Conclusion: The data obtained in this study serves as proof-of-concept for the simultaneous induction of antibodies directed against individual antigen components in a dual stage anti-malaria vaccine....

[Effect of Photodynamic Inactivation (PDI) using Riboflavin-Conjugated Antibody against Staphylococcus aureus].

Science.gov (United States)

Song, X; Stachon, T; Seitz, B; Wang, J; Bischoff, M; Langenbucher, A; Janunts, E; Szentmáry, N

2015-08-01

Crosslinking/riboflavin-UVA photodynamic therapy is a potential treatment alternative in antibiotic resistant infectious keratitis. For photodynamic therapy a specific (against bacteria) conjugated antibody may be used in order to increase the effect of the treatment. In our present study we analysed the impact of photodynamic inactivation using riboflavin-conjugated antibody or riboflavin alone on Staphylococcus aureus, in vitro. Staphylococcus aureus (S. aureus) was incubated in 1 : 100 diluted riboflavin-conjugated antibody (R-AB) for 30 minutes in darkness. Following UVA-light illumination (375 nm) with an energy dose of 2, 3, 4 and 8 J/cm(2), bacteria were brought to blood agar Plates for 24 hours before colony-forming unit (CFU) counting. In an additional group, we incubated bacteria to 0, 0.05 or 0.1 % riboflavin 5-phosphate as described above followed by illumination using UVA light (375 nm) with an energy dose of 2 J/cm(2), before CFU counting. The number of CFU decreased significantly (inactivation of 36 %, p = 0.022) using 1 : 100 diluted riboflavin-conjugated antibody and 2 J/cm(2) UVA-light illumination, compared to untreated controls. The use of 3, 4 und 8 J/cm(2) energy dose and R-AB in 1 : 100 dilution did not further change the decrease of CFU (inactivation of 39, 39 and 40 %; p = 0.016; p = 0.016; p = 0.015). The use of 0.05 % or 0.1 % riboflavin 5-phosphate alone and UVA-light illumination reduced the CFU count significantly (inactivation of 73 and 55 %; p = 0.002; p = 0.005), compared to untreated controls. The use of riboflavin-conjugated antibody or 0.05 % or 0.1 % riboflavin 5-phosphate and UVA-light illumination reduces the number of CFU of S. aureus. However, none of these photodynamic therapies reached the necessary 99 % killing rate of these bacteria. Further work is needed to increase the efficacy of riboflavin-conjugated antibodies against antibiotic resistant bacteria. Georg

Somatostatin receptor PET in neuroendocrine tumours: {sup 68}Ga-DOTA{sup 0},Tyr{sup 3}-octreotide versus {sup 68}Ga-DOTA{sup 0}-lanreotide

Energy Technology Data Exchange (ETDEWEB)

Putzer, Daniel; Kroiss, Alexander; Waitz, Dietmar; Gabriel, Michael; Uprimny, Christian; Guggenberg, Elisabeth von; Decristoforo, Clemens; Warwitz, Boris; Virgolini, Irene Johanna [Innsbruck Medical University, Department of Nuclear Medicine, Innsbruck (Austria); Traub-Weidinger, Tatjana [Vienna Medical University, Department of Nuclear Medicine, Vienna (Austria); Widmann, Gerlig [Innsbruck Medical University, Department of Radiology, Innsbruck (Austria)

2013-03-15

The aim of this study was to evaluate the impact of {sup 68}Ga-labelled DOTA{sup 0}-lanreotide ({sup 68}Ga-DOTA-LAN) on the diagnostic assessment of neuroendocrine tumour (NET) patients with low to moderate uptake on planar somatostatin receptor (SSTR) scintigraphy or {sup 68}Ga-labelled DOTA{sup 0},Tyr{sup 3}-octreotide ({sup 68}Ga-DOTA-TOC) positron emission tomography (PET). Fifty-three patients with histologically confirmed NET and clinical signs of progressive disease, who had not qualified for peptide receptor radionuclide therapy (PRRT) on planar SSTR scintigraphy or {sup 68}Ga-DOTA-TOC PET (n = 38) due to lack of tracer uptake, underwent {sup 68}Ga-DOTA-LAN PET to evaluate a treatment option with {sup 90}Y-labelled lanreotide according to the MAURITIUS trial. The included patients received 150 {+-} 30 MBq of each radiopharmaceutical intravenously. PET scans were acquired 60-90 min after intravenous bolus injection. Image results from both PET scans were compared head to head, focusing on the intensity of tracer uptake in terms of treatment decision. CT was used for morphologic correlation of tumour lesions. To further evaluate the binding affinities of each tracer, quantitative and qualitative values were calculated for target lesions. {sup 68}Ga-DOTA-LAN and {sup 68}Ga-DOTA-TOC both showed equivalent findings in 24/38 patients when fused PET/CT images were interpreted. The sensitivity, specificity and accuracy of {sup 68}Ga-DOTA-LAN in comparison to CT were 0.63, 0.5 and 0.62 (n = 53; p < 0.0001) and for {sup 68}Ga-DOTA-TOC in comparison to CT 0.78, 0.5 and 0.76 (n = 38; p < 0.013), respectively. {sup 68}Ga-DOTA-TOC showed a significantly higher maximum standardized uptake value (SUV{sub max}) regarding the primary tumour in 25 patients (p < 0.003) and regarding the liver in 30 patients (p < 0.009) compared to {sup 68}Ga-DOTA-LAN. Corresponding values of both PET scans for tumour and liver did not show any significant correlation. {sup 68}Ga-DOTA

177Lu-DOTA-HH1, a novel anti-CD37 radio-immunoconjugate: a study of toxicity in nude mice.

Directory of Open Access Journals (Sweden)

Ada H V Repetto-Llamazares

Full Text Available CD37 is an internalizing B-cell antigen expressed on Non-Hodgkin lymphoma (NHL and chronic lymphocytic leukemia cells (CLL. The anti-CD37 monoclonal antibody HH1 was conjugated to the bifunctional chelator p-SCN-Bn-DOTA and labelled with the beta-particle emitting radionuclide 177Lu creating the radio-immunoconjugate (RIC 177Lu-DOTA-HH1 (177Lu-HH1, trade name Betalutin. The present toxicity study was performed prior to initiation of clinical studies with 177Lu-HH1.Nude mice with or without tumor xenografts were treated with 50 to 1000 MBq/kg 177Lu- HH1 and followed for clinical signs of toxicity up to ten months. Acute, life threatening bone marrow toxicity was observed in animals receiving 800 and 1000 MBq/kg 177Lu-HH1. Significant changes in serum concentrations of liver enzymes were evident for treatment with 1000 MBq/kg 177Lu-HH1. Lymphoid depletion, liver necrosis and atrophy, and interstitial cell hyperplasia of the ovaries were also observed for mice in this dose group.177Lu-DOTA-HH1 was well tolerated at dosages about 10 times above those considered relevant for radioimmunotherapy in patients with B-cell derived malignancies.The toxicity profile was as expected for RICs. Our experimental results have paved the way for clinical evaluation of 177Lu-HH1 in NHL patients.

Towards tumour targeting with copper-radiolabelled macrocycle-antibody conjugates

International Nuclear Information System (INIS)

Morphy, J.R.; Parker, David; Kataky, Ritu

1989-01-01

Tetraaza-macrocycles covalently attached to a monoclonal antibody may be efficiently radiolabelled with 64 Cu or 67 Cu at pH4, minimising non-specific binding to the protein, giving a kinetically stable conjugate in vivo. (author)

Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

Science.gov (United States)

Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

Towards tumour targeting with copper-radiolabelled macrocycle-antibody conjugates

Energy Technology Data Exchange (ETDEWEB)

Morphy, J.R.; Parker, David; Kataky, Ritu; Harrison, Alice; Walker, Carole; Eaton, M.A.W.; Millican, Andrew; Phipps, Alison

1989-06-15

Tetraaza-macrocycles covalently attached to a monoclonal antibody may be efficiently radiolabelled with /sup 64/Cu or /sup 67/Cu at pH4, minimising non-specific binding to the protein, giving a kinetically stable conjugate in vivo. (author).

of radioconjugated DOTA-1-Nal3-octreotide labeled with gallium-68 using non-aqueous solvents

International Nuclear Information System (INIS)

Pérez-Malo Cruz, Marylaine; Leyva Montaña, René

2016-01-01

Neuroendocrine tumors specifically over-expressing somatostatin receptors. Diagnosis has expanded due to radiolabelling of DOTA-peptides such as somatostatin analogue DOTA-1-Nal 3 -Octreotide (DOTA-NOC) conjugated to β+ emitting radionuclides such as 68 Ga, which has very favorable physics-nuclear properties. This paper describes the radiolabeling procedures of DOTA-NOC with 68 Ga, in pure aqueous medium and in presence of non-aqueous solvents as well as the methods used for quality control where a formulation is obtained with a radiochemical yield exceeding 95%. The addition of ethanol (30% - v / v) to reaction mixture allowed to increase the specific activity of 68 Ga-DOTA-NOC radioconjugate, reaching a value of 182 MBq / nmol, higher than reported in the literature (50 MBq / nmol ) for labeling in pure aqueous medium. Stability studies are also presented (in presence of saline solution and saline phosphate buffer, transmetallation studies in Fe 3+ , Ca 2+ , Mg 2+ and Zn 2+ solutions, challenges competition against EDTA and DTPA chelators and in vitro stability in human transferrin) performed to 68Ga-DOTA-NOC radioconjugated, showing its high stability (> 95%). (author)

Hacking into the granuloma: could antibody antibiotic conjugates be developed for TB?

Science.gov (United States)

Ekins, Sean

2014-12-01

Alternatives to small molecule or vaccine approaches to treating tuberculosis are rarely discussed. Attacking Mycobacterium tuberculosis in the granuloma represents a challenge. It is proposed that the conjugation of small molecules onto a monoclonal antibody that recognizes macrophage or lymphocytes cell surface receptors, might be a way to target the bacteria in the granuloma. This antibody drug conjugate approach is currently being used in 2 FDA approved targeted cancer therapies. The pros and cons of this proposal for further research are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

Drug-to-antibody determination for an antibody-drug-conjugate utilizing cathepsin B digestion coupled with reversed-phase high-pressure liquid chromatography analysis.

Science.gov (United States)

Adamo, Michael; Sun, Guoyong; Qiu, Difei; Valente, Joseph; Lan, Wenkui; Song, Hangtian; Bolgar, Mark; Katiyar, Amit; Krishnamurthy, Girija

2017-01-20

Antibody drug conjugates or ADCs are currently being evaluated for their effectiveness as targeted chemotherapeutic agents across the pharmaceutical industry. Due to the complexity arising from the choice of antibody, drug and linker; analytical methods for release and stability testing are required to provide a detailed understanding of both the antibody and the drug during manufacturing and storage. The ADC analyzed in this work consists of a tubulysin drug analogue that is randomly conjugated to lysine residues in a human IgG1 antibody. The drug is attached to the lysine residue through a peptidic, hydrolytically stable, cathepsin B cleavable linker. The random lysine conjugation produces a heterogeneous mixture of conjugated species with a variable drug-to-antibody ratio (DAR), therefore, the average amount of drug attached to the antibody is a critical parameter that needs to be monitored. In this work we have developed a universal method for determining DAR in ADCs that employ a cathepsin B cleavable linker. The ADC is first cleaved at the hinge region and then mildly reduced prior to treatment with the cathepsin B enzyme to release the drug from the antibody fragments. This pre-treatment allows the cathepsin B enzyme unrestricted access to the cleavage sites and ensures optimal conditions for the cathepsin B to cleave all the drug from the ADC molecule. The cleaved drug is then separated from the protein components by reversed phase high performance liquid chromatography (RP-HPLC) and quantitated using UV absorbance. This method affords superior cleavage efficiency to other methods that only employ a cathepsin digestion step as confirmed by mass spectrometry analysis. This method was shown to be accurate and precise for the quantitation of the DAR for two different random lysine conjugated ADC molecules. Copyright © 2016 Elsevier B.V. All rights reserved.

Method to conjugate polysaccharide antigens to surfaces for the detection of antibodies.

Science.gov (United States)

Boas, Ulrik; Lind, Peter; Riber, Ulla

2014-11-15

A new generic method for the conjugation of lipopolysaccharide (LPS)-derived polysaccharide antigens from gram-negative bacteria has been developed using Salmonella as a model. After removal of lipid A from the LPS by mild acidolysis, the polysaccharide antigen was conjugated to polystyrene microbeads modified with N-alkyl hydroxylamine and N-alkyl-O-methyl hydroxylamine surface groups by incubation of antigen and beads for 16 h at 40 °C without the need for coupling agents. The efficiency of the new method was evaluated by flow cytometry in model samples and serum samples containing antibodies against Salmonella typhimurium and Salmonella dublin. The presented method was compared with a similar method for conjugation of Salmonella polysaccharide antigens to surfaces. Here, the new method showed higher antigen coupling efficiency by detecting low concentrations of antibodies. Furthermore, the polysaccharide-conjugated beads showed preserved bioactivity after 1 year of use. Copyright © 2014 Elsevier Inc. All rights reserved.

Are radiogallium-labelled DOTA-conjugated somatostatin analogues superior to those labelled with other radiometals?

Energy Technology Data Exchange (ETDEWEB)

Antunes, P.; Ginj, M.; Zhang, H.; Maecke, H. [University Hospital Basel, Division of Radiological Chemistry, Basel (Switzerland); Waser, B.; Reubi, J.C. [University of Bern, Institute of Pathology, Bern (Switzerland); Baum, R.P. [Zentralklinik Bad Berka, Department of Nuclear Medicine/PETCT-Center, Bad Berka (Germany)

2007-07-15

Gallium-68 is a metallic positron emitter with a half-life of 68 min that is ideal for the in vivo use of small molecules, such as [{sup 68}Ga-DOTA,Tyr{sup 3}]octreotide, in the diagnostic imaging of somatostatin receptor-positive tumours. In preclinical studies it has shown a striking superiority over its {sup 111}In-labelled congener. The purpose of this study was to evaluate whether third-generation somatostatin-based, radiogallium-labelled peptides show the same superiority. Peptides were synthesised on solid phase. The receptor affinity was determined by in vitro receptor autoradiography. The internalisation rate was studied in AR4-2J and hsst-HEK-transfected cell lines. The pharmacokinetics was studied in a rat xenograft tumour model, AR4-2J. All peptides showed high affinities on hsst2, with the highest affinity for the Ga{sup III}-complexed peptides. On hsst3 the situation was reversed, with a trend towards lower affinity of the Ga{sup III} peptides. A significantly increased internalisation rate was found in sst2-expressing cells for all {sup 67}Ga-labelled peptides. Internalisation into HEK-sst3 was usually faster for the {sup 111}In-labelled peptides. No internalisation was found into sst5. Biodistribution studies employing [{sup 67}Ga-DOTA,1-Nal{sup 3}]octreotide in comparison to [{sup 111}In-DOTA,1-Nal{sup 3}]octreotide and [{sup 67}Ga-DOTA,Tyr{sup 3}]octreotide showed a significantly higher and receptor-mediated uptake of the two{sup 67}Ga-labelled peptides in the tumour and somatostatin receptor-positive tissues. A patient study illustrated the potential advantage of a broad receptor subtype profile radiopeptide over a high-affinity sst2-selective radiopeptide. This study demonstrates that {sup 67/68}Ga-DOTA-octapeptides show distinctly better preclinical, pharmacological performances than the {sup 111}In-labelled peptides, especially on sst2-expressing cells and the corresponding animal models. They may be excellent candidates for further

Are radiogallium-labelled DOTA-conjugated somatostatin analogues superior to those labelled with other radiometals?

International Nuclear Information System (INIS)

Antunes, P.; Ginj, M.; Zhang, H.; Maecke, H.; Waser, B.; Reubi, J.C.; Baum, R.P.

2007-01-01

Gallium-68 is a metallic positron emitter with a half-life of 68 min that is ideal for the in vivo use of small molecules, such as [ 68 Ga-DOTA,Tyr 3 ]octreotide, in the diagnostic imaging of somatostatin receptor-positive tumours. In preclinical studies it has shown a striking superiority over its 111 In-labelled congener. The purpose of this study was to evaluate whether third-generation somatostatin-based, radiogallium-labelled peptides show the same superiority. Peptides were synthesised on solid phase. The receptor affinity was determined by in vitro receptor autoradiography. The internalisation rate was studied in AR4-2J and hsst-HEK-transfected cell lines. The pharmacokinetics was studied in a rat xenograft tumour model, AR4-2J. All peptides showed high affinities on hsst2, with the highest affinity for the Ga III -complexed peptides. On hsst3 the situation was reversed, with a trend towards lower affinity of the Ga III peptides. A significantly increased internalisation rate was found in sst2-expressing cells for all 67 Ga-labelled peptides. Internalisation into HEK-sst3 was usually faster for the 111 In-labelled peptides. No internalisation was found into sst5. Biodistribution studies employing [ 67 Ga-DOTA,1-Nal 3 ]octreotide in comparison to [ 111 In-DOTA,1-Nal 3 ]octreotide and [ 67 Ga-DOTA,Tyr 3 ]octreotide showed a significantly higher and receptor-mediated uptake of the two 67 Ga-labelled peptides in the tumour and somatostatin receptor-positive tissues. A patient study illustrated the potential advantage of a broad receptor subtype profile radiopeptide over a high-affinity sst2-selective radiopeptide. This study demonstrates that 67/68 Ga-DOTA-octapeptides show distinctly better preclinical, pharmacological performances than the 111 In-labelled peptides, especially on sst2-expressing cells and the corresponding animal models. They may be excellent candidates for further development for clinical studies. (orig.)

Data on atherosclerosis specific antibody conjugation to nanoemulsions

Directory of Open Access Journals (Sweden)

Geoffrey Prévot

2017-12-01

Full Text Available This article present data related to the publication entitled “Iron oxide core oil-in-water nanoemulsion as tracer for atherosclerosis MPI and MRI imaging” (Prévot et al., 2017 [1]. Herein we describe the engineering in the baculovirus-insect cell system and purification processes of the human scFv-Fc TEG4-2C antibody, specific of platelets within the atheroma plaque. For molecular targeting purpose, atheroma specific antibody was conjugated to nanoemulsions (NEs using a heterobifunctional linker (DSPE-PEG-maleimide. Atheroma labelling was assayed by immunochemistry on arterial sections from rabbits.

Robust labeling and comparative preclinical characterization of DOTA-TOC and DOTA-TATE

International Nuclear Information System (INIS)

Velikyan, Irina; Xu Hui; Nair, Manoj; Hall, Håkan

2012-01-01

Objectives: Various radionuclide-labeled somatostatin analogues are used currently for diagnosis and therapy of neuroendocrine tumors. In particular, [ 68 Ga]Ga-DOTA-TOC is commonly used for diagnosis, while [ 177 Lu]Lu-DOTA-TATE is used for therapy. With the development of theranostics and personalized medicine where the imaging diagnosis is tailored to the subsequent radiotherapy, it is of paramount importance to investigate the relevance of the ligand exchange. The aim of this study was to compare binding capacity of [ 67/68 Ga]Ga-DOTA-TOC ([ 67/68 Ga]Ga-N-(4,7,10-(tris(carboxymethyl)-1,4,7, 10-tetraazacyclododecan-1-yl) acetyl-D-Phe-c[Cys-D-Tyr-Trp-Lys-Thr-Cys]-Thr(ol)) and [ 67/68 Ga]Ga-DOTA-TATE ([ 67/68 Ga]Ga-N-(4,7,10-(tris(carboxymethyl)-1,4,7, 10-tetraazacyclododecan-1-yl)acetyl-D-Phe-c[Cys-D-Tyr-Trp-Lys-Thr-Cys]-Thr) in vitro in monkey brain cryosections and in vivo in the rat, where, in contrast to transfected cell lines, there is a heterogeneous distribution of somatostatin receptor (SSTR) subtypes. The influence of various production methods of [ 68 Ga]Ga-DOTA-TOC and [ 68 Ga]Ga-DOTA-TATE on the biological performance of the tracers was also studied. Material and Methods: [ 67 Ga]Ga-DOTA-TOC, [ 68 Ga]Ga-DOTA-TOC, [ 67 Ga]Ga-DOTA-TATE and [ 68 Ga]Ga-DOTA-TATE were synthesized including preconcentration and purification of the generator eluate. The binding of the radioligands was assessed in vitro using autoradiography on cryosections of Rhesus monkey brains and in vivo/ex vivo using organ distribution studies in rats. Results and Discussion: The tracer production method was improved in terms of higher robustness, simplification and good manufacturing practice (GMP) relevance. The synthesis variation did not influence the biological performance of the tracers. There was no statistically significant difference observed in the binding of [ 67/68 Ga]Ga-DOTA-TOC and [ 67/68 Ga]Ga-DOTA-TATE either in brain cortex in vitro or in rat biodistribution and uptake

Robust labeling and comparative preclinical characterization of DOTA-TOC and DOTA-TATE.

Science.gov (United States)

Velikyan, Irina; Xu, Hui; Nair, Manoj; Hall, Håkan

2012-07-01

Various radionuclide-labeled somatostatin analogues are used currently for diagnosis and therapy of neuroendocrine tumors. In particular, [68Ga]Ga-DOTA-TOC is commonly used for diagnosis, while [177Lu]Lu-DOTA-TATE is used for therapy. With the development of theranostics and personalized medicine where the imaging diagnosis is tailored to the subsequent radiotherapy, it is of paramount importance to investigate the relevance of the ligand exchange. The aim of this study was to compare binding capacity of [67/68Ga]Ga-DOTA-TOC ([67/68Ga]Ga-N-(4,7,10-(tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetyl-D-Phe-c[Cys-D-Tyr-Trp-Lys-Thr-Cys]-Thr(ol)) and [67/68Ga]Ga-DOTA-TATE ([67/68Ga]Ga-N-(4,7,10-(tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetyl-D-Phe-c[Cys-D-Tyr-Trp-Lys-Thr-Cys]-Thr) in vitro in monkey brain cryosections and in vivo in the rat, where, in contrast to transfected cell lines, there is a heterogeneous distribution of somatostatin receptor (SSTR) subtypes. The influence of various production methods of [68Ga]Ga-DOTA-TOC and [68Ga]Ga-DOTA-TATE on the biological performance of the tracers was also studied. [67Ga]Ga-DOTA-TOC, [68Ga]Ga-DOTA-TOC, [67Ga]Ga-DOTA-TATE and [68Ga]Ga-DOTA-TATE were synthesized including preconcentration and purification of the generator eluate. The binding of the radioligands was assessed in vitro using autoradiography on cryosections of Rhesus monkey brains and in vivo/ex vivo using organ distribution studies in rats. The tracer production method was improved in terms of higher robustness, simplification and good manufacturing practice (GMP) relevance. The synthesis variation did not influence the biological performance of the tracers. There was no statistically significant difference observed in the binding of [67/68Ga]Ga-DOTA-TOC and [67/68Ga]Ga-DOTA-TATE either in brain cortex in vitro or in rat biodistribution and uptake in SSTR-positive tissues such as pancreas, adrenals and pituitary. The uptake in these

Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using a IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex

Science.gov (United States)

Cheal, Sarah M.; Xu, Hong; Guo, Hong-fen; Zanzonico, Pat B.; Larson, Steven M.; Cheung, Nai-Kong

2014-01-01

Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and immunogenicity (e.g. of streptavidin-antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive (GD2(+)) tumors. For this purpose, a IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 (1) and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with beta-particle emitting radiometals such as 177Lu and 90Y (2, 3). A three-step regimen including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with 177Lu (as 177Lu-DOTA-Bn; t1/2 = 6.71 days (d)) was optimized in immunocompromised mice carrying subcutaneous (s.c.) human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were ∼85 cGy/MBq and ≤3.7 cGy/MBq, respectively, with therapeutic indicies (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5 per group; tumor volume: 240 ± 160 mm3) with three successive PRIT cycles (total 177Lu: ∼33 MBq; tumor dose ∼3400 cGy), revealed complete tumor response in 5/5 animals, with no recurrence up to 28 d post-treatment. Tumor ablation was confirmed histologically in 4/5 mice, and normal organs showed minimal overall toxicities. All non-treated mice required sacrifice within 12 d (>1.0 cm3 tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate s.c. GD2(+)–NB in mice while sparing kidney and bone marrow. PMID:24944121

Site-specific antibody-drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development.

Science.gov (United States)

Agarwal, Paresh; Bertozzi, Carolyn R

2015-02-18

Antibody-drug conjugates (ADCs) combine the specificity of antibodies with the potency of small molecules to create targeted drugs. Despite the simplicity of this concept, generation of clinically successful ADCs has been very difficult. Over the past several decades, scientists have learned a great deal about the constraints on antibodies, linkers, and drugs as they relate to successful construction of ADCs. Once these components are in hand, most ADCs are prepared by nonspecific modification of antibody lysine or cysteine residues with drug-linker reagents, which results in heterogeneous product mixtures that cannot be further purified. With advances in the fields of bioorthogonal chemistry and protein engineering, there is growing interest in producing ADCs by site-specific conjugation to the antibody, yielding more homogeneous products that have demonstrated benefits over their heterogeneous counterparts in vivo. Here, we chronicle the development of a multitude of site-specific conjugation strategies for assembly of ADCs and provide a comprehensive account of key advances and their roots in the fields of bioorthogonal chemistry and protein engineering.

Synthesis and stability test of radioimmunoconjugate 177Lu-DOTA-F(ab′2-trastuzumab for theranostic agent of HER2 positive breast cancer

Directory of Open Access Journals (Sweden)

Sandra Hermanto

2016-10-01

Full Text Available The use of trastuzumab as intact IgG labeling radionuclide for HER2 positive breast cancer theranostic agent is not ideal because it is slowly eliminated from the blood and normal tissues resulting in low tumor/blood (T/B and tumor/normal tissue (T/NT ratios. To overcome this limitation, we developed the trastuzumab F(ab′2 fragments and radiolabeling of the fragments by β and γ-particle of Lutetium-177. F(ab2 fragments were produced by digestion of trastuzumab IgG (Herceptin with pepsin for 18 h at 37 °C. The F(ab′2 fragment fractionated in PD-10 column, followed by the conjugation with 2-(4-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA as a metal chelator and radiolabeling with 177LuCl3. Molecular weight of fragments was calculated by LCMS (Liquid Chromatography Mass Spectroscopy and the radiochemical purity was evaluated by ITLC-SG (Instan Thin Layer Chromatography. Our study showed that the purity of F(ab′2 fragment generated by PD-10 fractions was >98% and the molecular weight of F(ab′2 was 98.35 kDa. The average numbers of pSCN-Bn-DOTA chelates per antibody fragment were 5.03 ± 1.5 and the optimum conjugation reactions was performed at molar ratio 20:1 (chelator to antibody. The stability test of the radioimmunoconjugate in the human serum albumin (HSA at 37 °C showed the radiochemical purity was 91.96 ± 0.26% after 96 h storage. This indicated that the radioimmunoconjugate is relatively stable when applied to the human body's physiological condition.

Selective cytotoxicity of an oxygen-radical-generating enzyme conjugated to a monoclonal antibody.

Science.gov (United States)

Battelli, M G; Abbondanza, A; Tazzari, P L; Dinota, A; Rizzi, S; Grassi, G; Gobbi, M; Stirpe, F

1988-07-01

The monoclonal antibody 8A, which recognizes a human plasma cell-associated antigen, was covalently linked to xanthine oxidase in a conjugate maintaining both immunological and enzymatic properties. A significant degree of target cell lysis was obtained at an enzyme concentration that was ineffective on non-target cells and on myeloid staminal cells (CFU-GM). The cytotoxic activity was abolished by an excess of antibody, by allopurinol and by superoxide dismutase and catalase. A possible use of the conjugate for bone marrow purging in multiple myeloma patients is suggested.

Antibody derivatization and conjugation strategies: application in preparation of stealth immunoliposome to target chemotherapeutics to tumor.

Science.gov (United States)

Manjappa, Arehalli S; Chaudhari, Kiran R; Venkataraju, Makam P; Dantuluri, Prudhviraju; Nanda, Biswarup; Sidda, Chennakesavulu; Sawant, Krutika K; Murthy, Rayasa S Ramachandra

2011-02-28

A great deal of effort has been made over the years to develop liposomes that have targeting vectors (oligosaccharides, peptides, proteins and vitamins) attached to the bilayer surface. Most studies have focused on antibody conjugates since procedures for producing highly specific monoclonal antibodies are well established. Antibody conjugated liposomes have recently attracted a great deal of interest, principally because of their potential use as targeted drug delivery systems and in diagnostic applications. A number of methods have been reported for coupling antibodies to the surface of stealth liposomes. The objective of this review is to enumerate various strategies which are employed in the modification and conjugation of antibodies to the surface of stealth liposomes. This review also describes various derivatization techniques of lipids prior and after their use in the preparation of liposomes. The use of single chain variable fragments and affibodies as targeting ligands in the preparation of immunoliposomes is also discussed. Copyright © 2010 Elsevier B.V. All rights reserved.

Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity.

Directory of Open Access Journals (Sweden)

Martin Kreutz

Full Text Available Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA and CpG oligodeoxynucleotides (ODN. We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

Gallium-68-DOTA-albumin as a PET blood-pool marker: experimental evaluation in vivo

International Nuclear Information System (INIS)

Hoffend, Johannes; Mier, Walter; Schuhmacher, Jochen; Schmidt, Kerstin; Dimitrakopoulou-Strauss, Antonia; Strauss, Ludwig G.; Eisenhut, Michael; Kinscherf, Ralf; Haberkorn, Uwe

2005-01-01

Investigations into tumor angiogenesis and antiangiogenic treatment have renewed interest in tumor perfusion. To image tumor blood-pool by PET, suitable tracers are not generally available. In this experimental study, we characterized a 68 Ga-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugate of rat serum albumin ( 68 Ga-DOTA-RSA) in vivo using a generator-produced isotope. Biodistribution was determined in ACI rats after intravenous administration of 3-6 MBq of 68 Ga-DOTA-RSA. Three ACI rats were imaged over 1 h by dynamic PET after intravenous administration of 15-25 MBq of 68 Ga-DOTA-RSA while the blood-pool activity was recorded simultaneously in a closed extracorporeal loop (ECL) between the carotid artery and the jugular vein. Time-activity curves (TACs) were obtained from volume of interest (VOI) analysis and from the ECL data. Stability and metabolites in plasma and urine were analyzed by size exclusion HPLC (SE-HPLC) 1 h after intravenous injection of 67 Ga-DOTA-RSA. Blood radioactivity decreased by 10% and 18% from 10 to 60 min p.i. by biodistribution and PET or ECL, respectively. Tissue sampling between 10 and 60 min p.i. showed slight increases in the uptake of spleen, myocardium, kidney and skeletal muscle while hepatic accretion remained unchanged. Total urinary excretion after 60 min amounted to 9% of the injected dose. HPLC demonstrated a single urinary metabolite corresponding in size to gallium-labeled DOTA. 68 Ga-DOTA-RSA is a blood-pool tracer whose physical and biological half-life is well suited for PET. Our findings support clinical imaging using 68 Ga-DOTA-labeled human serum albumin (HSA). The generator-produced label makes 68 Ga-DOTA-labeled albumin continuously available even to centers lacking an in-house cyclotron

Labeling of monoclonal antibody conjugates with 90Y

International Nuclear Information System (INIS)

Motta-Hennessy, Cecilia; Sharkey, R.M.; Goldenberg, D.M.

1991-01-01

An anti-carcinoembryonic antigen (CEA) antibody, NP-4, was labeled with 90 Y using p-isothiocyanatobenzyl DTPA (SCN-Bz-DTPA) and its derivatives 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1B3M), 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1M3B), 1-(2)-methyl-4-isothiocyanatobenzyl-DTPA (MX-DTPA) as the chelating agents. The 90 Y conjugates were purified from unbound 90 Y by two different methods, HPLC or acrylamide size exclusion gel chromatography, in order to evaluate the best purification method. Labeling efficiency, reaction kinetics and immunoreactivity were compared to the same antibodies labeled with [ 111 In]citrate. Labeling efficiency, as determined by either HPLC or ITLC (instant thin layer chromatography), was consistently higher by ITLC than HPLC for 90 Y-labeled MAb, but equal for 111 In-labeled MAbs. Discrepancies between the 2 methods were linked to impurities in the 90 Y that remained at the origin of ITLC plates. After purification by acrylamide gel filtration, recovery was 50-60% of loaded 90 Y activity, but was more than 87% for the 111 In compounds. Using HPLC, the recovery measured 85% for 90 Y-labeled MAb and more than 93% for 111 In-labeled conjugates. Immunoreactivity of the [ 90 Y]MAb was comparable to the 111 In-labeled conjugates. These studies indicate that HPLC purification of the [ 90 Y] MAbs improves recovery of activity, and suggests that impurities found in the 90 Y and metal-binding properties of acrylamide may have contributed to the poor recoveries from acrylamide gels. (author)

Novel Phospholipid-Protein Conjugates Allow Improved Detection of Antibodies in Patients with Autoimmune Diseases

DEFF Research Database (Denmark)

Samuelsen, Simone V; Maity, Arindam; Nybo, Mads

2016-01-01

Reliable measurement of clinically relevant autoimmune antibodies toward phospholipid-protein conjugates is highly desirable in research and clinical assays. To date, the development in this field has been limited to the use of natural heterogeneous antigens. However, this approach does not take ...... on the correlation of detected autoantibodies with disease activity and manifestations. This confirms the crucial importance of antigens' composition on research and diagnostic assays, and opens up exciting perspectives for synthetic antigens in future studies of autoimmunity.......Reliable measurement of clinically relevant autoimmune antibodies toward phospholipid-protein conjugates is highly desirable in research and clinical assays. To date, the development in this field has been limited to the use of natural heterogeneous antigens. However, this approach does not take...... structural features of biologically active antigens into account and leads to low reliability and poor scientific test value. Here we describe novel phospholipid-protein conjugates for specific detection of human autoimmune antibodies. Our synthetic approach includes mild oxidation of synthetic phospholipid...

Imaging of primary and metastatic colorectal carcinoma with monoclonal antibody 791T/36 and the therapeutic potential of antibody-drug conjugates

International Nuclear Information System (INIS)

Pimm, M.V.; Armitage, N.C.; Ballantyne, K.; Baldwin, R.W.; Perkins, A.C.; Durrant, L.G.; Garnett, M.C.; Hardcastle, J.D.

1987-01-01

Monoclonal antibody 791T/36, prepared against a tumor-associated 72,000 dalton glycoprotein, reacted with cells from primary and metastatic colorectal carcinomas. I-131 or In-111-labelled antibody localized in xenografts of colorectal carcinomas established from in vitro clonogenic populations. Clinically, with I-131-labelled antibody, 8/11 colonic tumors imaged positively. Imaging was negative in four patients with benign colon disease. 5/11 rectal tumors were positively imaged, but excreted I-131 in the bladder obscured tumors in several studies. In-111-labelled antibody gave superior images and positively imaged primary and metastatic sites in 13/14 patients. Prospectively in the detection of recurrent disease, I-131 or In-111-antibody detected 29/33 separate sites in 24 patients. Seven negative patients remain disease free. There were 3 false positives; overall sensitivity was 88%, with 70% specificity. Specific localization of radiolabel was confirmed immunochemically and by counting radioactivity in resected specimens. Antibody conjugates with methotrexate, vindesine and daunomycin retained drug activity and antibody function, including xenograft localization and conjugates were therapeutically effective against xenografts. 791T/36 antibody has potential for immunodetection of primary and recurrent colorectal carcinoma and for targeting of therapeutic agents

Characterization of antibody-chelator conjugates: Determination of chelator content by terbium fluorescence titration

Energy Technology Data Exchange (ETDEWEB)

Brandt, K.D.; Schnobrich, K.E.; Johnson, D.K. (Abbott Laboratories, Department 90M, Abbott Park, IL (United States))

1991-01-01

Fluorescence titrations were performed by adding varying mole ratios of terbium(III) to antibody conjugates formed by benzyl isothiocyanate derivatives of three different polyaminopolycarboxylate chelators (NTA, EDTA, and DTPA) and the results compared to values for average chelator content obtained by cobalt-57 binding assays. For two different murine monoclonal antibodies, the average chelator content obtained by terbium fluorescence titration correlated closely with that measured by the cobalt-57 binding assay. It is concluded that lanthanide fluorescence titrations provide a useful alternative to radiometal binding assays for the determination of chelator content in protein-chelator conjugates.

Characterization of antibody-chelator conjugates: Determination of chelator content by terbium fluorescence titration

International Nuclear Information System (INIS)

Brandt, K.D.; Schnobrich, K.E.; Johnson, D.K.

1991-01-01

Fluorescence titrations were performed by adding varying mole ratios of terbium(III) to antibody conjugates formed by benzyl isothiocyanate derivatives of three different polyaminopolycarboxylate chelators (NTA, EDTA, and DTPA) and the results compared to values for average chelator content obtained by cobalt-57 binding assays. For two different murine monoclonal antibodies, the average chelator content obtained by terbium fluorescence titration correlated closely with that measured by the cobalt-57 binding assay. It is concluded that lanthanide fluorescence titrations provide a useful alternative to radiometal binding assays for the determination of chelator content in protein-chelator conjugates

Internalization, Trafficking, Intracellular Processing and Actions of Antibody-Drug Conjugates.

Science.gov (United States)

Xu, Shi

2015-11-01

This review discusses the molecular mechanism involved in the targeting and delivery of antibody-drug conjugates (ADCs), the new class of biopharmaceuticals mainly designed for targeted cancer therapy. this review goes over major progress in preclinical and clinical studies of ADCs, in the past 5 years. The pharmacokinetics and pharmacodynamics of ADCs involve multiple mechanisms, including internalization of ADCs by target cells, intracellular trafficking, release of conjugated drugs, and payload. These mechanisms actually jointly determine the efficacy of ADCs. Therefore, the optimization of ADCs should take them as necessary rationales.

Cohort study of somatostatin-based radiopeptide therapy with [(90)Y-DOTA]-TOC versus [(90)Y-DOTA]-TOC plus [(177)Lu-DOTA]-TOC in neuroendocrine cancers.

Science.gov (United States)

Villard, Linda; Romer, Anna; Marincek, Nicolas; Brunner, Philippe; Koller, Michael T; Schindler, Christian; Ng, Quinn K T; Mäcke, Helmut R; Müller-Brand, Jan; Rochlitz, Christoph; Briel, Matthias; Walter, Martin A

2012-04-01

Radiopeptide therapy is commonly performed with a single radioisotope. We aimed to compare the effectiveness of somatostatin-based radiopeptide therapy with a single versus a combination of radioisotopes. In a cohort study, patients with metastasized neuroendocrine cancer were treated with repeated cycles of (90)yttrium-labeled tetraazacyclododecane-tetraacetic acid modified Tyr-octreotide ([(90)Y-DOTA]-TOC) or with cycles alternating between [(90)Y-DOTA]-TOC and (177)lutetium-labeled DOTA-TOC ([(177)Lu-DOTA]-TOC) until tumor progression or permanent toxicity. Multivariable Cox regression and competing risk regression were used to study predictors of survival and renal toxicity in patients completing three or more treatment cycles. A total of 486 patients completed three or more treatment cycles; 237 patients received [(90)Y-DOTA]-TOC and 249 patients received [(90)Y-DOTA]-TOC + [(177)Lu-DOTA]-TOC. Patients receiving [(90)Y-DOTA]-TOC + [(177)Lu-DOTA]-TOC had a significantly longer survival than patients receiving [(90)Y-DOTA]-TOC alone (5.51 v 3.96 years; hazard ratio, 0.64; 95% CI, 0.47 to 0.88; P = .006). The rates of severe hematologic toxicities (6.3% v 4.4%; P = .25) and severe renal toxicity (8.9% v 11.2%; P = .47) were comparable in both groups. [(90)Y-DOTA]-TOC + [(177)Lu-DOTA]-TOC was associated with improved overall survival compared with [(90)Y-DOTA]-TOC alone in patients completing three or more cycles of treatment. Contrary to the current practice in radiopeptide therapy, our results suggest an advantage of using a combination of radioisotopes.

Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate.

Science.gov (United States)

Marcoux, Julien; Champion, Thierry; Colas, Olivier; Wagner-Rousset, Elsa; Corvaïa, Nathalie; Van Dorsselaer, Alain; Beck, Alain; Cianférani, Sarah

2015-08-01

Antibody-drug conjugates (ADCs) are biochemotherapeutics consisting of a cytotoxic chemical drug linked covalently to a monoclonal antibody. Two main classes of ADCs, namely cysteine and lysine conjugates, are currently available on the market or involved in clinical trials. The complex structure and heterogeneity of ADCs makes their biophysical characterization challenging. For cysteine conjugates, hydrophobic interaction chromatography is the gold standard technique for studying drug distribution, the naked antibody content, and the average drug to antibody ratio (DAR). For lysine ADC conjugates on the other hand, which are not amenable to hydrophobic interaction chromatography because of their higher heterogeneity, denaturing mass spectrometry (MS) and UV/Vis spectroscopy are the most powerful approaches. We report here the use of native MS and ion mobility (IM-MS) for the characterization of trastuzumab emtansine (T-DM1, Kadcyla(®)). This lysine conjugate is currently being considered for the treatment of human epidermal growth factor receptor 2 (HER2)-positive breast cancer, and combines the anti-HER2 antibody trastuzumab (Herceptin(®)), with the cytotoxic microtubule-inhibiting maytansine derivative, DM1. We show that native MS combined with high-resolution measurements and/or charge reduction is beneficial in terms of the accurate values it provides of the average DAR and the drug load profiles. The use of spectral deconvolution is discussed in detail. We report furthermore the use of native IM-MS to directly determine DAR distribution profiles and average DAR values, as well as a molecular modeling investigation of positional isomers in T-DM1. © 2015 The Protein Society.

Preclinical Assessment of a 68Ga-DOTA-Functionalized Depsipeptide as a Radiodiagnostic Infection Imaging Agent.

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Ebenhan, Thomas; Mokaleng, Botshelo Brenda; Venter, Jacobus Daniel; Kruger, Hendrik Gert; Zeevaart, Jan Rijn; Sathekge, Mike

2017-08-24

The study assessed a radiolabeled depsipeptide conjugate ( 68 Ga-DOTA-TBIA101) for its potential as an imaging agent targeting infection or infection-associated inflammation. 68 Ga-labeled DOTA-TBIA101 imaging was performed in (NZR1) healthy rabbits; (NZR2) rabbits bearing muscular sterile inflammation and Staphylococcus aureus (SA) infection; and (NZR3) rabbits infected with Mycobacterium tuberculosis (MTB) combined with a subcutaneous scruff infection of SA in the same animal. All animals were imaged using a PET/CT scanner at 5 and 60 min post injection. Images showed elevated accumulation of 68 Ga-DOTA-TBIA101 in the sterile muscular inflammation site (T/NT ratio = 2.6 ± 0.37 (5 min) and 2.8 ± 2.3 (60 min)) and muscles infected with MTB (T/NT ratio = 2.6 ± 0.35 (5 min) and 2.8 ± 0.16 (60 min)). The findings suggest that 68 Ga-DOTA-TBIA101-PET/CT may detect MTB-associated inflammation, although more foundational studies need to be performed to rationalize the diagnostic value of this technique.

Compact quantum dot-antibody conjugates for FRET immunoassays with subnanomolar detection limits

Science.gov (United States)

Mattera, Lucia; Bhuckory, Shashi; Wegner, K. David; Qiu, Xue; Agnese, Fabio; Lincheneau, Christophe; Senden, Tim; Djurado, David; Charbonnière, Loïc J.; Hildebrandt, Niko; Reiss, Peter

2016-05-01

A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post-functionalization with a heterobifunctional crosslinker, containing a lipoic acid group and a maleimide function, enabled the subsequent coupling to sulfhydryl groups of proteins. This was demonstrated by QD conjugation with fragmented antibodies (F(ab)). The obtained F(ab)-QD conjugates range among the smallest antibody-functionalized nanoprobes ever reported, with a hydrodynamic diameter coating for FRET could be demonstrated by an 6.2 and 2.5 fold improvement of the limit of detection (LOD) for PSA compared to commercially available hydrophilic QDs emitting at 605 and 705 nm, respectively. While the commercial QDs contain identical inorganic cores responsible for their fluorescence, they are coated with a comparably thick amphiphilic polymer layer leading to much larger hydrodynamic diameters (>26 nm without biomolecules). The LODs of 0.8 and 3.7 ng mL-1 obtained in 50 μL serum samples are below the clinical cut-off level of PSA (4 ng mL-1) and demonstrate their direct applicability in clinical diagnostics.A novel two-step approach for quantum dot (QD) functionalization and bioconjugation is presented, which yields ultra-compact, stable, and highly luminescent antibody-QD conjugates suitable for use in FRET immunoassays. Hydrophobic InPZnS/ZnSe/ZnS (emission wavelength: 530 nm), CdSe/ZnS (605 nm), and CdSeTe/ZnS (705 nm) QDs were surface functionalized with zwitterionic penicillamine, enabling aqueous phase transfer under conservation of the photoluminescence properties. Post

Position for Site-Specific Attachment of a DOTA Chelator to Synthetic Affibody Molecules Has a Different Influence on the Targeting Properties of 68Ga-Compared to 111In-Labeled Conjugates

Directory of Open Access Journals (Sweden)

Hadis Honarvar

2014-12-01

Full Text Available Affibody molecules, small (7 kDa scaffold proteins, are a promising class of probes for radionuclide molecular imaging. Radiolabeling of Affibody molecules with the positron-emitting nuclide 68Ga would permit the use of positron emission tomography (PET, providing better resolution, sensitivity, and quantification accuracy than single-photon emission computed tomography (SPECT. The synthetic anti-HER2 ZHER2:S1 Affibody molecule was conjugated with DOTA at the N-terminus, in the middle of helix 3, or at the C-terminus. The biodistribution of 68Ga- and 111In-labeled Affibody molecules was directly compared in NMRI nu/nu mice bearing SKOV3 xenografts. The position of the chelator strongly influenced the biodistribution of the tracers, and the influence was more pronounced for 68Ga-labeled Affibody molecules than for the 111In-labeled counterparts. The best 68Ga-labeled variant was 68Ga-[DOTA-A1]-ZHER2:S1 which provided a tumor uptake of 13 ± 1 %ID/g and a tumor to blood ratio of 39 ± 12 at 2 hours after injection. 111In-[DOTA-A1]-ZHER2:S1 and 111In-[DOTA-K58]-ZHER2:S1 were equally good at this time point, providing a tumor uptake of 15 to 16 %ID/g and a tumor to blood ratio in the range of 60 to 80. In conclusion, the selection of the best position for a chelator in Affibody molecules can be used for optimization of their imaging properties. This may be important for the development of Affibody-based and other protein-based imaging probes.

Development and Characterization of a Camelid Single Domain Antibody-Urease Conjugate That Targets Vascular Endothelial Growth Factor Receptor 2.

Science.gov (United States)

Tian, Baomin; Wong, Wah Yau; Uger, Marni D; Wisniewski, Pawel; Chao, Heman

2017-01-01

Angiogenesis is the process of new blood vessel formation and is essential for a tumor to grow beyond a certain size. Tumors secrete the pro-angiogenic factor vascular endothelial growth factor, which acts upon local endothelial cells by binding to vascular endothelial growth factor receptors (VEGFRs). In this study, we describe the development and characterization of V21-DOS47, an immunoconjugate that targets VEGFR2. V21-DOS47 is composed of a camelid single domain anti-VEGFR2 antibody (V21) and the enzyme urease. The conjugate specifically binds to VEGFR2 and urease converts endogenous urea into ammonia, which is toxic to tumor cells. Previously, we developed a similar antibody-urease conjugate, L-DOS47, which is currently in clinical trials for non-small cell lung cancer. Although V21-DOS47 was designed from parameters learned from the generation of L-DOS47, additional optimization was required to produce V21-DOS47. In this study, we describe the expression and purification of two versions of the V21 antibody: V21H1 and V21H4. Each was conjugated to urease using a different chemical cross-linker. The conjugates were characterized by a panel of analytical techniques, including SDS-PAGE, size exclusion chromatography, Western blotting, and LC-MS E peptide mapping. Binding characteristics were determined by ELISA and flow cytometry assays. To improve the stability of the conjugates at physiologic pH, the pIs of the V21 antibodies were adjusted by adding several amino acid residues to the C-terminus. For V21H4, a terminal cysteine was also added for use in the conjugation chemistry. The modified V21 antibodies were expressed in the E. coli BL21 (DE3) pT7 system. V21H1 was conjugated to urease using the heterobifunctional cross-linker succinimidyl-[( N -maleimidopropionamido)-diethyleneglycol] ester (SM(PEG) 2 ), which targets lysine resides in the antibody. V21H4 was conjugated to urease using the homobifunctional cross-linker, 1,8-bis(maleimido)diethylene glycol

HAI-178 antibody-conjugated fluorescent magnetic nanoparticles for targeted imaging and simultaneous therapy of gastric cancer

Science.gov (United States)

Wang, Can; Bao, Chenchen; Liang, Shujing; Zhang, Lingxia; Fu, Hualin; Wang, Yutian; Wang, Kan; Li, Chao; Deng, Min; Liao, Qiande; Ni, Jian; Cui, Daxiang

2014-05-01

The successful development of safe and highly effective nanoprobes for targeted imaging and simultaneous therapy of in vivo gastric cancer is a great challenge. Herein we reported for the first time that anti-α-subunit of ATP synthase antibody, HAI-178 monoclonal antibody-conjugated fluorescent magnetic nanoparticles, was successfully used for targeted imaging and simultaneous therapy of in vivo gastric cancer. A total of 172 specimens of gastric cancer tissues were collected, and the expression of α-subunit of ATP synthase in gastric cancer tissues was investigated by immunohistochemistry method. Fluorescent magnetic nanoparticles were prepared and conjugated with HAI-178 monoclonal antibody, and the resultant HAI-178 antibody-conjugated fluorescent magnetic nanoparticles (HAI-178-FMNPs) were co-incubated with gastric cancer MGC803 cells and gastric mucous GES-1 cells. Gastric cancer-bearing nude mice models were established, were injected with prepared HAI-178-FMNPs via tail vein, and were imaged by magnetic resonance imaging and small animal fluorescent imaging system. The results showed that the α-subunit of ATP synthase exhibited high expression in 94.7% of the gastric cancer tissues. The prepared HAI-178-FMNPs could target actively MGC803 cells, realized fluorescent imaging and magnetic resonance imaging of in vivo gastric cancer, and actively inhibited growth of gastric cancer cells. In conclusion, HAI-178 antibody-conjugated fluorescent magnetic nanoparticles have a great potential in applications such as targeted imaging and simultaneous therapy of in vivo early gastric cancer cells in the near future.

The target invites a foe: antibody-drug conjugates in gynecologic oncology.

Science.gov (United States)

Campos, Maira P; Konecny, Gottfried E

2018-02-01

Antibody-drug conjugates (ADCs) represent a promising new class of cancer therapeutics. Currently more than 60 ADCs are in clinical development, however, only very few trials focus on gynecologic malignancies. In this review, we summarize the most recent advances in ADC drug development with an emphasis on how this progress relates to patients diagnosed with gynecologic malignancies and breast cancer. The cytotoxic payloads of the majority of the ADCs that are currently in clinical trials for gynecologic malignancies or breast cancer are auristatins (MMAE, MMAF), maytansinoids (DM1, DM4), calicheamicin, pyrrolobenzodiazepines and SN-38. Both cleavable and noncleavable linkers are currently being investigated in clinical trials. A number of novel target antigens are currently being validated in ongoing clinical trials including folate receptor alpha, mesothelin, CA-125, NaPi2b, NOTCH3, protein tyrosine kinase-like 7, ephrin-A4, TROP2, CEACAM5, and LAMP1. For most ADCs currently in clinical development, dose-limiting toxicities appear to be unrelated to the targeted antigen but more tightly associated with the payload. Rational drug design involving optimization of the antibody, the linker and the conjugation chemistry is aimed at improving the therapeutic index of new ADCs. Antibody-drug conjugates can increase the efficacy and decrease the toxicity of their payloads in comparison with traditional cyctotoxic agents. A better and quicker translation of recent scientific advances in the field of ADCs into rational clinical trials for patients diagnosed with ovarian, endometrial or cervical cancer could create real improvements in tumor response, survival and quality of life for our patients.

Somatostatin-based radiopeptide therapy with [177Lu-DOTA]-TOC versus [90Y-DOTA]-TOC in neuroendocrine tumours

International Nuclear Information System (INIS)

Romer, A.; Seiler, D.; Brunner, P.; Ng, Q.K.T.; Mueller-Brand, J.; Marincek, N.; Walter, M.A.; Koller, M.T.; Maecke, H.R.; Rochlitz, C.; Briel, M.; Schindler, C.

2014-01-01

Somatostatin-based radiopeptide treatment is generally performed using the β-emitting radionuclides 90 Y or 177 Lu. The present study aimed at comparing benefits and harms of both therapeutic approaches. In a comparative cohort study, patients with advanced neuroendocrine tumours underwent repeated cycles of [ 90 Y-DOTA]-TOC or [ 177 Lu-DOTA]-TOC until progression of disease or permanent adverse events. Multivariable Cox regression and competing risks regression were employed to examine predictors of survival and adverse events for both treatment groups. Overall, 910 patients underwent 1,804 cycles of [ 90 Y-DOTA]-TOC and 141 patients underwent 259 cycles of [ 177 Lu-DOTA]-TOC. The median survival after [ 177 Lu-DOTA]-TOC and after [ 90 Y-DOTA]-TOC was comparable (45.5 months versus 35.9 months, hazard ratio 0.91, 95 % confidence interval 0.63-1.30, p = 0.49). Subgroup analyses revealed a significantly longer survival for [ 177 Lu-DOTA]-TOC over [ 90 Y-DOTA]-TOC in patients with low tumour uptake, solitary lesions and extra-hepatic lesions. The rate of severe transient haematotoxicities was lower after [ 177 Lu-DOTA]-TOC treatment (1.4 vs 10.1 %, p = 0.001), while the rate of severe permanent renal toxicities was similar in both treatment groups (9.2 vs 7.8 %, p = 0.32). The present results revealed no difference in median overall survival after [ 177 Lu-DOTA]-TOC and [ 90 Y-DOTA]-TOC. Furthermore, [ 177 Lu-DOTA]-TOC was less haematotoxic than [ 90 Y-DOTA]-TOC. (orig.)

Somatostatin-based radiopeptide therapy with [177Lu-DOTA]-TOC versus [90Y-DOTA]-TOC in neuroendocrine tumours.

Science.gov (United States)

Romer, A; Seiler, D; Marincek, N; Brunner, P; Koller, M T; Ng, Q K T; Maecke, H R; Müller-Brand, J; Rochlitz, C; Briel, M; Schindler, C; Walter, M A

2014-02-01

Somatostatin-based radiopeptide treatment is generally performed using the β-emitting radionuclides (90)Y or (177)Lu. The present study aimed at comparing benefits and harms of both therapeutic approaches. In a comparative cohort study, patients with advanced neuroendocrine tumours underwent repeated cycles of [(90)Y-DOTA]-TOC or [(177)Lu-DOTA]-TOC until progression of disease or permanent adverse events. Multivariable Cox regression and competing risks regression were employed to examine predictors of survival and adverse events for both treatment groups. Overall, 910 patients underwent 1,804 cycles of [(90)Y-DOTA]-TOC and 141 patients underwent 259 cycles of [(177)Lu-DOTA]-TOC. The median survival after [(177)Lu-DOTA]-TOC and after [(90)Y-DOTA]-TOC was comparable (45.5 months versus 35.9 months, hazard ratio 0.91, 95% confidence interval 0.63-1.30, p = 0.49). Subgroup analyses revealed a significantly longer survival for [(177)Lu-DOTA]-TOC over [(90)Y-DOTA]-TOC in patients with low tumour uptake, solitary lesions and extra-hepatic lesions. The rate of severe transient haematotoxicities was lower after [(177)Lu-DOTA]-TOC treatment (1.4 vs 10.1%, p = 0.001), while the rate of severe permanent renal toxicities was similar in both treatment groups (9.2 vs 7.8%, p = 0.32). The present results revealed no difference in median overall survival after [(177)Lu-DOTA]-TOC and [(90)Y-DOTA]-TOC. Furthermore, [(177)Lu-DOTA]-TOC was less haematotoxic than [(90)Y-DOTA]-TOC.

Site-specific conjugation and labelling of prostate antibody 7E11C5.3 (CYT-351) with technetium-99m

International Nuclear Information System (INIS)

Stalteri, M.A.; Mather, S.J.; Belinka, B.A.; Coughlin, D.J.; Chengazi, V.U.; Britton, K.E.

1997-01-01

Attachment of chelating agents to the sugar residues of antibodies for subsequent radiolabelling is an attractive approach since it may have less effect on the immunoreactivity than attachment through lysine residues, which are distributed throughout the antibody and may be present near the antigen binding site. We have attached a new hydrazide-linked chelator CYT-395 (Cytogen Corp., Princeton, N.J.) to the sugar residues of the anti-prostate monoclonal antibody 7E11C5.3 and optimised the conditions for labelling the conjugate with technetium-99m in order to compare the conjugate to 7E11C5.3 antibody labelled directly with technetium using a mercaptoethanol reduction technique. Labelling yields of 70%-90% were obtained at specific activities up to 2000 MBq/mg antibody. The stability of the technetium-labelled conjugate in plasma or to a challenge with 0.1 or 1.0 mM cysteine was similar to that of direct-labelled antibody. In nine patients with prostate cancer, the plasma clearance of the labelled conjugate followed a two-compartment model, with an average β-phase half-life of 31.4±3.9 h. The average urinary clearance at 24 h was 15.3±5.0% of the injected dose. In this group of patients there was no significant difference between the blood and urine clearance of the labelled conjugate, and the clearances of the direct-labelled antibody. (orig.). With 5 figs

Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

Science.gov (United States)

Zhou, Zhan; Zhang, Jing; Zhang, Yan; Ma, Guanghui; Su, Zhiguo

2016-01-20

Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

PET imaging of 64Cu-DOTA-scFv-anti-PSMA lipid nanoparticles (LNPs): Enhanced tumor targeting over anti-PSMA scFv or untargeted LNPs

International Nuclear Information System (INIS)

Wong, Patty; Li, Lin; Chea, Junie; Delgado, Melissa K.; Crow, Desiree; Poku, Erasmus; Szpikowska, Barbara; Bowles, Nicole; Channappa, Divya; Colcher, David; Wong, Jeffrey Y.C.; Shively, John E.; Yazaki, Paul J.

2017-01-01

Introduction: Single chain (scFv) antibodies are ideal targeting ligands due to their modular structure, high antigen specificity and affinity. These monovalent ligands display rapid tumor targeting but have limitations due to their fast urinary clearance. Methods: An anti-prostate membrane antigen (PSMA) scFv with a site-specific cysteine was expressed and evaluated in a prostate cancer xenograft model by Cu-64 PET imaging. To enhance tumor accumulation, the scFv-cys was conjugated to the co-polymer DSPE-PEG-maleimide that spontaneously assembled into a homogeneous multivalent lipid nanoparticle (LNP). Results: The targeted LNP exhibited a 2-fold increase in tumor uptake compared to the scFv alone using two different thiol ester chemistries. The anti-PSMA scFv-LNP exhibited a 1.6 fold increase in tumor targeting over the untargeted LNP. Conclusions: The targeted anti-PSMA scFv-LNP showed enhanced tumor accumulation over the scFv alone or the untargeted DOTA-micelle providing evidence for the development of this system for drug delivery. Advances in knowledge and implications for patient care: Anti-tumor scFv antibody fragments have not achieved their therapeutic potential due to their fast blood clearance. Conjugation to an LNP enables multivalency to the tumor antigen as well as increased molecular size for chemotherapy drug delivery.

[99mTc]Demotate 2 in the detection of sst2-positive tumours: a preclinical comparison with [111In]DOTA-tate

International Nuclear Information System (INIS)

Maina, Theodosia; Nock, Berthold A.; Cordopatis, Paul; Bernard, Bert F.; Breeman, Wout A.P.; Gameren, Arthur van; Berg, Ria van den; Krenning, Eric P.; Jong, Marion de; Reubi, Jean-Claude

2006-01-01

The aim of this study was to evaluate [ 99m Tc]Demotate 2 ([ 99m Tc-N 4 0-1 ,Asp 0 ,Tyr 3 ]octreotate) as a candidate for in vivo imaging of sst 2 -positive tumours and to compare it with [ 111 In]DOTA-tate ([ 111 In-DOTA 0 ,Tyr 3 ]octreotate). Labelling of Demotate 2 with 99m Tc was performed at room temperature using SnCl 2 as reductant in the presence of citrate at alkaline pH. Radiochemical analysis involved ITLC and HPLC methods. Peptide conjugate affinities for sst 2 were determined by receptor autoradiography on rat brain cortex sections using [DOTA 0 , 125 I-Tyr 3 ]octreotate as the radioligand. The affinity profile of Demotate 2 for human sst 1 -sst 5 was studied by receptor autoradiography in cell preparations using the universal somatostatin radioligand [ 125 I][Leu 8 ,(D)Trp 22 ,Tyr 25 ]somatostatin-28. The internalisation rates of [ 99m Tc]Demotate 2 and [ 111 In]DOTA-tate were compared in sst 2 -positive and -negative control cell lines. Biodistribution of radiopeptides was studied in male Lewis rats bearing CA20948 tumours. Peptide conjugates showed selectivity and a high affinity binding for sst 2 (Demotate 2 IC 50 =3.2 nM and DOTA-tate IC 50 =5.4 nM). [ 99m Tc]Demotate 2, like [ 111 In]DOTA-tate, internalised rapidly in all sst 2 -positive cells tested, but not in sst 2 -negative control cells. After injection in CA20948 tumour-bearing rats both radiopeptides showed high and specific uptake in the sst 2 -positive organs and in the implanted tumour and rapid excretion from non-target tissues via the kidneys. [ 99m Tc]Demotate 2, similarly to the known sst 2 -targeting agent [ 111 In]DOTA-tate, showed promising biological qualities for application in the scintigraphy of sst 2 -positive tumours. (orig.)

Production and characterization of a camelid single domain antibody-urease enzyme conjugate for the treatment of cancer.

Science.gov (United States)

Tian, Baomin; Wong, Wah Yau; Hegmann, Elda; Gaspar, Kim; Kumar, Praveen; Chao, Heman

2015-06-17

A novel immunoconjugate (L-DOS47) was developed and characterized as a therapeutic agent for tumors expressing CEACAM6. The single domain antibody AFAIKL2, which targets CEACAM6, was expressed in the Escherichia coli BL21 (DE3) pT7-7 system. High purity urease (HPU) was extracted and purified from Jack bean meal. AFAIKL2 was activated using N-succinimidyl [4-iodoacetyl] aminobenzoate (SIAB) as the cross-linker and then conjugated to urease. The activation and conjugation reactions were controlled by altering pH. Under these conditions, the material ratio achieved conjugation ratios of 8-11 antibodies per urease molecule, the residual free urease content was practically negligible (95%) L-DOS47 conjugate was produced using only ultradiafiltration to remove unreacted antibody and hydrolyzed cross-linker. L-DOS47 was characterized by a panel of analytical techniques including SEC, IEC, Western blot, ELISA, and LC-MS(E) peptide mapping. As the antibody-urease conjugate ratio increased, a higher binding signal was observed. The specificity and cytotoxicity of L-DOS47 was confirmed by screening in four cell lines (BxPC-3, A549, MCF7, and CEACAM6-transfected H23). BxPC-3, a CEACAM6-expressing cell line was found to be most susceptible to L-DOS47. L-DOS47 is being investigated as a potential therapeutic agent in human phase I clinical studies for nonsmall cell lung cancer.

High-throughput oxidation screen of antibody-drug conjugates by analytical protein A chromatography following IdeS digest.

Science.gov (United States)

Buecheler, Jakob W; Winzer, Matthias; Weber, Christian; Gieseler, Henning

2018-05-01

Oxidation of protein therapeutics is a major chemical degradation pathway which may impact bioactivity, serum half-life and stability. Therefore, oxidation is a relevant parameter which has to be monitored throughout formulation development. Methods such as HIC, RPLC and LC/MS achieve a separation of oxidized and non-oxidized species by differences in hydrophobicity. Antibody-drug conjugates (ADC) although are highly more complex due to the heterogeneity in linker, drug, drug-to-antibody ratio (DAR) and conjugation site. The analytical protein A chromatography can provide a simple and fast alternative to these common methods. A miniature analytical protein A chromatography method in combination with an IdeS digest was developed to analyse ADCs. The IdeS digest efficiency of an IgG1 was monitored using SEC-HPLC and non-reducing SDS-PAGE. An antibody-fluorescent dye conjugate was conjugated at different dye-to-antibody ratios as model construct to mimic an ADC. With IdeS, an almost complete digest of a model IgG1 can be achieved (digested protein amount >98%). This enables subsequent analytical protein A chromatography, which consequently eliminates any interference of payload with the stationary phase. A novel high-throughput method for an interchain cysteine-linked ADC oxidation screens during formulation development was developed. © 2018 Royal Pharmaceutical Society.

Anti-VEGF antibody conjugated CdHgTe quantum dots as a fluorescent probe for imaging in living mouse

Energy Technology Data Exchange (ETDEWEB)

Pang, Lili; Cui, Hongjing [Department of Analytical Chemistry, China Pharmaceutical University, Nanjing (China); Liu, Yu [Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing (China); Zhong, Wenying, E-mail: wyzhong@cpu.edu.cn [Department of Analytical Chemistry, China Pharmaceutical University, Nanjing (China); Key laboratory of Biomedical Functional Materials, China Pharmaceutical University, Nanjing (China)

2016-05-15

The dual-function anti-VEGF antibody conjugated CdHgTe quantum dots with good targeting property was successfully prepared. In this system, anti-VEGF antibody is not only a target agent but also a therapeutic drug. The anti-VEGF antibody conjugated near-infrared quantum dots can achieve the purposes of detection and treatment at the same time. As-prepared dual-function fluorescent probe in this work has been successfully applied for in vivo and in vitro imaging, which is promising in rapid tumor detection.

Improved Lysosomal Trafficking Can Modulate the Potency of Antibody Drug Conjugates.

Science.gov (United States)

DeVay, Rachel M; Delaria, Kathy; Zhu, Guoyun; Holz, Charles; Foletti, Davide; Sutton, Janette; Bolton, Gary; Dushin, Russell; Bee, Christine; Pons, Jaume; Rajpal, Arvind; Liang, Hong; Shelton, David; Liu, Shu-Hui; Strop, Pavel

2017-04-19

Antibody drug conjugates (ADCs) provide an efficacious and relatively safe means by which chemotherapeutic agents can be specifically targeted to cancer cells. In addition to the selection of antibody targets, ADCs offer a modular design that allows selection of ADC characteristics through the choice of linker chemistries, toxins, and conjugation sites. Many studies have indicated that release of toxins bound to antibodies via noncleavable linker chemistries relies on the internalization and intracellular trafficking of the ADC. While this can make noncleavable ADCs more stable in the serum, it can also result in lower efficacy when their respective targets are not internalized efficiently or are recycled back to the cell surface following internalization. Here, we show that a lysosomally targeted ADC against the protein APLP2 mediates cell killing, both in vitro and in vivo, more effectively than an ADC against Trop2, a protein with less efficient lysosomal targeting. We also engineered a bispecific ADC with one arm targeting HER2 for the purpose of directing the ADC to tumors, and the other arm targeting APLP2, whose purpose is to direct the ADC to lysosomes for toxin release. This proof-of-concept bispecific ADC demonstrates that this technology can be used to shift the intracellular trafficking of a constitutively recycled target by directing one arm of the antibody against a lysosomally delivered protein. Our data also show limitations of this approach and potential future directions for development.

Labeling of monoclonal antibody conjugates with sup 90 Y

Energy Technology Data Exchange (ETDEWEB)

Motta-Hennessy, Cecilia; Sharkey, R.M.; Goldenberg, D.M. (Center for Molecular Medicine and Immunology, Newark, NJ (USA))

1991-01-01

An anti-carcinoembryonic antigen (CEA) antibody, NP-4, was labeled with {sup 90}Y using p-isothiocyanatobenzyl DTPA (SCN-Bz-DTPA) and its derivatives 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1B3M), 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1M3B), 1-(2)-methyl-4-isothiocyanatobenzyl-DTPA (MX-DTPA) as the chelating agents. The {sup 90}Y conjugates were purified from unbound {sup 90}Y by two different methods, HPLC or acrylamide size exclusion gel chromatography, in order to evaluate the best purification method. Labeling efficiency, reaction kinetics and immunoreactivity were compared to the same antibodies labeled with ({sup 111}In)citrate. Labeling efficiency, as determined by either HPLC or ITLC (instant thin layer chromatography), was consistently higher by ITLC than HPLC for {sup 90}Y-labeled MAb, but equal for {sup 111}In-labeled MAbs. Discrepancies between the 2 methods were linked to impurities in the {sup 90}Y that remained at the origin of ITLC plates. After purification by acrylamide gel filtration, recovery was 50-60% of loaded {sup 90}Y activity, but was more than 87% for the {sup 111}In compounds. Using HPLC, the recovery measured 85% for {sup 90}Y-labeled MAb and more than 93% for {sup 111}In-labeled conjugates. Immunoreactivity of the ({sup 90}Y)MAb was comparable to the {sup 111}In-labeled conjugates. These studies indicate that HPLC purification of the ({sup 90}Y) MAbs improves recovery of activity, and suggests that impurities found in the {sup 90}Y and metal-binding properties of acrylamide may have contributed to the poor recoveries from acrylamide gels. (author).

Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity.

Science.gov (United States)

Schettini, Jorge; Kidiyoor, Amritha; Besmer, Dahlia M; Tinder, Teresa L; Roy, Lopamudra Das; Lustgarten, Joseph; Gendler, Sandra J; Mukherjee, Pinku

2012-11-01

Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo.

Somatostatin-based radiotherapy with [90Y-DOTA]-TOC in neuroendocrine tumors: long-term outcome of a phase I dose escalation study.

Science.gov (United States)

Marincek, Nicolas; Jörg, Ann-Catherine; Brunner, Philippe; Schindler, Christian; Koller, Michael T; Rochlitz, Christoph; Müller-Brand, Jan; Maecke, Helmut R; Briel, Matthias; Walter, Martin A

2013-01-15

We describe the long-term outcome after clinical introduction and dose escalation of somatostatin receptor targeted therapy with [90Y-DOTA]-TOC in patients with metastasized neuroendocrine tumors. In a clinical phase I dose escalation study we treated patients with increasing [90Y-DOTA]-TOC activities. Multivariable Cox regression and competing risk regression were used to compare efficacy and toxicities of the different dosage protocols. Overall, 359 patients were recruited; 60 patients were enrolled for low dose (median: 2.4 GBq/cycle, range 0.9-7.8 GBq/cycle), 77 patients were enrolled for intermediate dose (median: 3.3 GBq/cycle, range: 2.0-7.4 GBq/cycle) and 222 patients were enrolled for high dose (median: 6.7 GBq/cycle, range: 3.7-8.1 GBq/cycle) [90Y-DOTA]-TOC treatment. The incidences of hematotoxicities grade 1-4 were 65.0%, 64.9% and 74.8%; the incidences of grade 4/5 kidney toxicities were 8.4%, 6.5% and 14.0%, and the median survival was 39 (range: 1-158) months, 34 (range: 1-118) months and 29 (range: 1-113) months. The high dose protocol was associated with an increased risk of kidney toxicity (Hazard Ratio: 3.12 (1.13-8.59) vs. intermediate dose, p = 0.03) and a shorter overall survival (Hazard Ratio: 2.50 (1.08-5.79) vs. low dose, p = 0.03). Increasing [90Y-DOTA]-TOC activities may be associated with increasing hematological toxicities. The dose related hematotoxicity profile of [90Y-DOTA]-TOC could facilitate tailoring [90Y-DOTA]-TOC in patients with preexisting hematotoxicities. The results of the long-term outcome suggest that fractionated [90Y-DOTA]-TOC treatment might allow to reduce renal toxicity and to improve overall survival. (ClinicalTrials.gov number NCT00978211).

Investigational Antibody-Drug Conjugates for Treatment of B-lineage Malignancies.

Science.gov (United States)

Herrera, Alex F; Molina, Arturo

2018-05-10

Antibody-drug conjugates (ADCs) are tripartite molecules consisting of a monoclonal antibody, a covalent linker, and a cytotoxic payload. ADC development has aimed to target the specificity inherent in antigen-antibody interactions to deliver potent cytotoxins preferentially to tumor cells and maximize antitumor activity and simultaneously minimize off-target toxicity. The earliest ADCs provided disappointing results in the clinic; however, the lessons learned regarding the need for human or humanized antibodies, more stable linkers, and greater potency payloads led to improved ADCs. Three ADCs, gemtuzumab ozogamicin, brentuximab vedotin (BV), and inotuzumab ozogamicin, have been approved for hematologic malignancies. Site-specific conjugation methods have now resulted in a new generation of more uniform, molecularly defined ADCs. These are expected to display improved in vivo properties and have recently entered the clinic. We reviewed investigational ADCs currently in clinical testing for the treatment of B-cell lineage malignancies, including leukemias, lymphomas, and multiple myeloma. The rationales for antigen targeting, data reported to date, current trial status, and preclinical results for several newer ADCs expected to enter first-in-human studies are presented. Owing to the large number of ongoing and reported BV clinical studies, only the studies of BV for diffuse large B-cell lymphoma and those combining BV with checkpoint inhibitors in B-lineage malignancies have been reviewed. With > 40 ongoing clinical trials and 7 investigational ADCs already having advanced to phase II studies, the role of ADCs in the armamentarium for the treatment of B-lineage malignancies continues to be elucidated. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The chemical fate of 212Bi-DOTA formed by β- decay of 212Pb(DOTA)2-

International Nuclear Information System (INIS)

Mirzadeh, S.; Kumar, K.; Gansow, O.A.

1993-01-01

The increasing use of inert metal complexes in radioimmunotherapy prompted us to explore the potential use of 212 Pb chelates. Herein, we report a study of the chemical fate of the 212 Bi-DOTA complex formed by β - decay of 212 Pb(DOTA) 2- (H 4 DOTA = 1,4,7,10-tetraazacyclododecanetetraacetic acid). To assure that both parent and daughter complexes were thermally stable, kinetic studies were performed with 203 Pb(II) and 206 Bi(III) which showed that both lead and bismuth complexes with DOTA undergo chemical exchange only very slowly in aqueous solution at pH 4-10. To investigate whether the complex ion which results from decay of 212 Pb(DOTA) 2- was intact and also stable, solutions initially containing only this ion were analyzed for amounts of DOTA-complexed and uncomplexed 212 Bi after attaining transient equilibrium with 212 Bi. The fraction of 212 Bi radioactivity not complexed to DOTA, vide infra, was found to be 36±2%. This value represents the fraction of breakup of 212 Bi(DOTA) - formed from β - decay of the parent complex. By considering the various extranuclear processes responsible for kinetic and electronic excitation of the 212 Bi daughter, break-up of the 212 Bi-DOTA complex is ascribed to the internal conversion of γ-rays emitted by the excited 212 Bi nuclide. (orig.)

Radioiodinated iodobenzoyl conjugates of a monoclonal antibody Fab fragment. In vivo comparisons with chloramine-T-labeled Fab

International Nuclear Information System (INIS)

Wilbur, D.S.; Hadley, S.W.; Grant, L.M.; Hylarides, M.D.

1991-01-01

A comparative investigation of the biodistributions of radioiodinated p- and m-iodobenzoyl conjugates of a monoclonal antibody Fab fragment, NR-LU-10 Fab, and the same antibody Fab fragment radioiodinated by the chloramine-T (ChT) method has been carried out in mice. Coinjected, dual-isotope studies in athymic mice with tumor xenografts have demonstrated that there are only minor differences in the in vivo distributions of the iodobenzoyl-labeled Fabs, except in the excretory organs, kidneys, and intestines, where major differences were observed. Similarly, coinjection of either the p-iodobenzoyl or m-iodobenzoyl conjugate of NR-LU-10 Fab with the Fab radioiodinated with ChT/radioiodide into BALB/c mice provided additional data that indicated that the two iodobenzoyl conjugates distributed similar in a number of selected tissues. The tissue-distribution differences of the regioisomeric iodobenzoyl conjugates in relation to the ChT-radioiodinated Fab were large for the stomach and neck, consistent with previous studies. The most notable difference between the two iodobenzoyl conjugates was the kidney activity, where the m-iodobenzoyl conjugate was similar to the directly labeled Fab, but the p-iodobenzoyl-conjugated Fab was higher by nearly a factor of 2

Phase II trial of yttrium-90-DOTA-biotin pretargeted by NR-LU-10 antibody/streptavidin in patients with metastatic colon cancer

International Nuclear Information System (INIS)

Knox, S J.; Goris, M L.; Tempero, M.; Weiden, P L.; Gentner, L.; Breitz, H.; Adams, G. P.; Axworthy, D.; Gaffigan, S.; Bryan, K.; Fisher, Darrell R.; Colcher, D; Horak, I D.; Weiner, L M.

1999-01-01

A Phase II study of yttrium-90-tetra-azacyclododecanetetra-acetic acid-biotin (Y-90-DOTA-biotin) pretargeted by NR-LU-10 antibody/streptavidin (SA) was performed. The primary objectives of the study were to evaluate the efficacy and safety of this therapy in patients with metastatic colon cancer. Twenty-five patients were treated with a single dose of 110 mCi/m 2 (mean administered dose, 106.5-10.3 mCi/m 2 ) of Y-90-DOTA-biotin. There were three components of the therapy. Patients first received NR-LU-10/SA on day 1. A clearing agent (biotin-galactose-human serum albumin) was administered 48 h after the NR-LU-10/SA to remove residual circulating unbound NR-LU-10/SA. Lastly, 24 h after administration of clearing agent, patients received biotin-DOTA-labeled with 110 mCi/m 2 Y-90. All three components of the therapy were administered i.v. Both hematological and nonhematological toxicities were observed. Diarrhea was the most frequent grade 4 nonhematological toxicity (16%; with 16% grade 3 diarrhea). Hematological toxicity was less severe with 8% grade 3 and 8% grade 4 neutropenia and 8% grade 3 and 16% grade 4 thrombocytopenia. The overall response rate was 8%. Two partial responders had freedom from progression of 16 weeks. Four patients (16%) had stable disease with freedom from progression of 10-20 weeks. Despite the relatively disappointing results of this study in terms of therapeutic efficacy and toxicity, proof of principle was obtained for the pretargeting approach. In addition, valuable new information was obtained about normal tissue tolerance to low-dose-rate irradiation that will help to provide useful guidelines for future study designs

Legomedicine-A Versatile Chemo-Enzymatic Approach for the Preparation of Targeted Dual-Labeled Llama Antibody-Nanoparticle Conjugates.

Science.gov (United States)

van Lith, Sanne A M; van Duijnhoven, Sander M J; Navis, Anna C; Leenders, William P J; Dolk, Edward; Wennink, Jos W H; van Nostrum, Cornelus F; van Hest, Jan C M

2017-02-15

Conjugation of llama single domain antibody fragments (Variable Heavy chain domains of Heavy chain antibodies, VHHs) to diagnostic or therapeutic nanoparticles, peptides, proteins, or drugs offers many opportunities for optimized targeted cancer treatment. Currently, mostly nonspecific conjugation strategies or genetic fusions are used that may compromise VHH functionality. In this paper we present a versatile modular approach for bioorthogonal VHH modification and conjugation. First, sortase A mediated transPEGylation is used for introduction of a chemical click moiety. The resulting clickable VHHs are then used for conjugation to other groups employing the Cu + -independent strain-promoted alkyne-azide cycloadition (SPAAC) reaction. Using this approach, tail-to-tail bispecific VHHs and VHH-targeted nanoparticles are generated without affecting VHH functionality. Furthermore, this approach allows the bioconjugation of multiple moieties to VHHs for simple and convenient production of VHH-based theranostics.

Stabilised 111In-labelled DTPA- and DOTA-conjugated neurotensin analogues for imaging and therapy of exocrine pancreatic cancer

International Nuclear Information System (INIS)

Visser, M. de; Krenning, E.P.; Jong, M. de; Janssen, P.J.J.M.; Srinivasan, A.; Reubi, J.C.; Waser, B.; Erion, J.L.; Schmidt, M.A.

2003-01-01

Neurotensin (NT) receptors are overexpressed in exocrine pancreatic cancer and Ewing's sarcoma. The potential utility of native NT in cancer diagnosis and therapy is, however, limited by its rapid degradation in vivo. Therefore, NT analogues were synthesised with modified lysine and arginine derivatives to enhance stability and coupled either to DTPA, to enable high specific activity labelling with indium-111 for imaging, or to DOTA, to enable high specific activity labelling with β-emitting radionuclides, such as lutetium-177 and yttrium-90. Based on serum stability (4 h incubation at 37 C in human serum) and receptor binding affinity, the five most promising analogues were selected and further evaluated in in vitro internalisation studies in human colorectal adenocarcinoma HT29 cells, which overexpress NT receptors. All five NT analogues bound with high affinity to NT receptors on human exocrine pancreatic tumour sections. The analogues could be labelled with 111 In to a high specific activity. The 111 In-labelled compounds were found to be very stable in serum. Incubation of HT29 cells with the 111 In-labelled analogues at 37 C showed rapid receptor-mediated uptake and internalisation. The most promising analogue, peptide 2530 [DTPA-(Pip)Gly-Pro-(PipAm)Gly-Arg-Pro-Tyr-tBuGly-Leu-OH] was further tested in vivo in a biodistribution study using HT29 tumour-bearing nude mice. The results of this study showed low percentages of injected dose per gram tissue of this 111 In-labelled 2530 analogue in receptor-negative organs like blood, spleen, pancreas, liver, muscle and femur. Good uptake was found in the receptor-positive HT29 tumour and high uptake was present in the kidneys. Co-injection of excess unlabelled NT significantly reduced tumour uptake, showing that tumour uptake is a receptor-mediated process. With their enhanced stability, maintained high receptor affinity and rapid receptor-mediated internalisation, the 111 In-labelled DTPA- and DOTA-conjugated NT

Somatostatin receptor PET in neuroendocrine tumours: 68Ga-DOTA0,Tyr3-octreotide versus 68Ga-DOTA0-lanreotide

International Nuclear Information System (INIS)

Putzer, Daniel; Kroiss, Alexander; Waitz, Dietmar; Gabriel, Michael; Uprimny, Christian; Guggenberg, Elisabeth von; Decristoforo, Clemens; Warwitz, Boris; Virgolini, Irene Johanna; Traub-Weidinger, Tatjana; Widmann, Gerlig

2013-01-01

The aim of this study was to evaluate the impact of 68 Ga-labelled DOTA 0 -lanreotide ( 68 Ga-DOTA-LAN) on the diagnostic assessment of neuroendocrine tumour (NET) patients with low to moderate uptake on planar somatostatin receptor (SSTR) scintigraphy or 68 Ga-labelled DOTA 0 ,Tyr 3 -octreotide ( 68 Ga-DOTA-TOC) positron emission tomography (PET). Fifty-three patients with histologically confirmed NET and clinical signs of progressive disease, who had not qualified for peptide receptor radionuclide therapy (PRRT) on planar SSTR scintigraphy or 68 Ga-DOTA-TOC PET (n = 38) due to lack of tracer uptake, underwent 68 Ga-DOTA-LAN PET to evaluate a treatment option with 90 Y-labelled lanreotide according to the MAURITIUS trial. The included patients received 150 ± 30 MBq of each radiopharmaceutical intravenously. PET scans were acquired 60-90 min after intravenous bolus injection. Image results from both PET scans were compared head to head, focusing on the intensity of tracer uptake in terms of treatment decision. CT was used for morphologic correlation of tumour lesions. To further evaluate the binding affinities of each tracer, quantitative and qualitative values were calculated for target lesions. 68 Ga-DOTA-LAN and 68 Ga-DOTA-TOC both showed equivalent findings in 24/38 patients when fused PET/CT images were interpreted. The sensitivity, specificity and accuracy of 68 Ga-DOTA-LAN in comparison to CT were 0.63, 0.5 and 0.62 (n = 53; p 68 Ga-DOTA-TOC in comparison to CT 0.78, 0.5 and 0.76 (n = 38; p 68 Ga-DOTA-TOC showed a significantly higher maximum standardized uptake value (SUV max ) regarding the primary tumour in 25 patients (p 68 Ga-DOTA-LAN. Corresponding values of both PET scans for tumour and liver did not show any significant correlation. 68 Ga-DOTA-TOC revealed more tumour sites than 68 Ga-DOTA-LAN (106 vs 53). The tumour to background ratios for tumour and liver calculated from SUV max measurements were significantly higher for 68 Ga-DOTA-TOC than 68 Ga-DOTA

Assessment of blood flow with (68)Ga-DOTA PET in experimental inflammation: a validation study using (15)O-water.

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Autio, Anu; Saraste, Antti; Kudomi, Nobuyuki; Saanijoki, Tiina; Johansson, Jarkko; Liljenbäck, Heidi; Tarkia, Miikka; Oikonen, Vesa; Sipilä, Hannu T; Roivainen, Anne

2014-01-01

Increased blood flow and vascular permeability are key events in inflammation. Based on the fact that Gadolinium-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (Gd-DOTA) is commonly used in magnetic resonance (MR) imaging of blood flow (perfusion), we evaluated the feasibility of its Gallium-68 labeled DOTA analog ((68)Ga-DOTA) for positron emission tomography (PET) imaging of blood flow in experimental inflammation. Adult, male Sprague-Dawley rats with turpentine oil induced sterile skin/muscle inflammation were anesthetized with isoflurane, and imaged under rest and adenosine-induced hyperemia by means of dynamic 2-min Oxygen-15 labeled water (H2 (15)O) and 30-min (68)Ga-DOTA PET. For the quantification of PET data, regions of interest (ROIs) were defined in the focus of inflammation, healthy muscle, myocardium and heart left ventricle. Radioactivity concentration in the ROIs versus time after injection was determined for both tracers and blood flow was calculated using image-derived input. According to the H2 (15)O PET, blood flow was 0.69 ± 0.15 ml/min/g for inflammation and 0.15 ± 0.03 ml/min/g for muscle during rest. The blood flow remained unchanged during adenosine-induced hyperemia 0.67 ± 0.11 and 0.12 ± 0.03 ml/min/g for inflammation and muscle, respectively, indicating that adenosine has little effect on blood flow in peripheral tissues in rats. High focal uptake of (68)Ga-DOTA was seen at the site of inflammation throughout the 30-min PET imaging. According to the (68)Ga-DOTA PET, blood flow measured as the blood-to-tissue transport rate (K1) was 0.60 ± 0.07 ml/min/g for inflammation and 0.14 ± 0.06 ml/min/g for muscle during rest and 0.63 ± 0.08 ml/min/g for inflammation and 0.09 ± 0.04 ml/min/g for muscle during adenosine-induced hyperemia. The H2 (15)O-based blood flow and (68)Ga-DOTA-based K1 values correlated well (r = 0.94, P DOTA PET imaging is useful for the quantification of increased blood flow induced by inflammation.

Assessment of blood flow with 68Ga-DOTA PET in experimental inflammation: a validation study using 15O-water

Science.gov (United States)

Autio, Anu; Saraste, Antti; Kudomi, Nobuyuki; Saanijoki, Tiina; Johansson, Jarkko; Liljenbäck, Heidi; Tarkia, Miikka; Oikonen, Vesa; Sipilä, Hannu T; Roivainen, Anne

2014-01-01

Increased blood flow and vascular permeability are key events in inflammation. Based on the fact that Gadolinium-1,4,7,10-tetraazacyclododecane-N,N‘,N‘‘,N‘‘‘-tetraacetic acid (Gd-DOTA) is commonly used in magnetic resonance (MR) imaging of blood flow (perfusion), we evaluated the feasibility of its Gallium-68 labeled DOTA analog (68Ga-DOTA) for positron emission tomography (PET) imaging of blood flow in experimental inflammation. Adult, male Sprague-Dawley rats with turpentine oil induced sterile skin/muscle inflammation were anesthetized with isoflurane, and imaged under rest and adenosine-induced hyperemia by means of dynamic 2-min Oxygen-15 labeled water (H2 15O) and 30-min 68Ga-DOTA PET. For the quantification of PET data, regions of interest (ROIs) were defined in the focus of inflammation, healthy muscle, myocardium and heart left ventricle. Radioactivity concentration in the ROIs versus time after injection was determined for both tracers and blood flow was calculated using image-derived input. According to the H2 15O PET, blood flow was 0.69 ± 0.15 ml/min/g for inflammation and 0.15 ± 0.03 ml/min/g for muscle during rest. The blood flow remained unchanged during adenosine-induced hyperemia 0.67 ± 0.11 and 0.12 ± 0.03 ml/min/g for inflammation and muscle, respectively, indicating that adenosine has little effect on blood flow in peripheral tissues in rats. High focal uptake of 68Ga-DOTA was seen at the site of inflammation throughout the 30-min PET imaging. According to the 68Ga-DOTA PET, blood flow measured as the blood-to-tissue transport rate (K1) was 0.60 ± 0.07 ml/min/g for inflammation and 0.14 ± 0.06 ml/min/g for muscle during rest and 0.63 ± 0.08 ml/min/g for inflammation and 0.09 ± 0.04 ml/min/g for muscle during adenosine-induced hyperemia. The H2 15O-based blood flow and 68Ga-DOTA-based K1 values correlated well (r = 0.94, P DOTA PET imaging is useful for the quantification of increased blood flow induced by inflammation. PMID

Multivalent peptidic linker enables identification of preferred sites of conjugation for a potent thialanstatin antibody drug conjugate.

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Sujiet Puthenveetil

Full Text Available Antibody drug conjugates (ADCs are no longer an unknown entity in the field of cancer therapy with the success of marketed ADCs like ADCETRIS and KADCYLA and numerous others advancing through clinical trials. The pursuit of novel cytotoxic payloads beyond the mictotubule inhibitors and DNA damaging agents has led us to the recent discovery of an mRNA splicing inhibitor, thailanstatin, as a potent ADC payload. In our previous work, we observed that the potency of this payload was uniquely tied to the method of conjugation, with lysine conjugates showing much superior potency as compared to cysteine conjugates. However, the ADC field is rapidly shifting towards site-specific ADCs due to their advantages in manufacturability, characterization and safety. In this work we report the identification of a highly efficacious site-specific thailanstatin ADC. The site of conjugation played a critical role on both the in vitro and in vivo potency of these ADCs. During the course of this study, we developed a novel methodology of loading a single site with multiple payloads using an in situ generated multi-drug carrying peptidic linker that allowed us to rapidly screen for optimal conjugation sites. Using this methodology, we were able to identify a double-cysteine mutant ADC delivering four-loaded thailanstatin that was very efficacious in a gastric cancer xenograft model at 3mg/kg and was also shown to be efficacious against T-DM1 resistant and MDR1 overexpressing tumor cell lines.

Multivalent peptidic linker enables identification of preferred sites of conjugation for a potent thialanstatin antibody drug conjugate.

Science.gov (United States)

Puthenveetil, Sujiet; He, Haiyin; Loganzo, Frank; Musto, Sylvia; Teske, Jesse; Green, Michael; Tan, Xingzhi; Hosselet, Christine; Lucas, Judy; Tumey, L Nathan; Sapra, Puja; Subramanyam, Chakrapani; O'Donnell, Christopher J; Graziani, Edmund I

2017-01-01

Antibody drug conjugates (ADCs) are no longer an unknown entity in the field of cancer therapy with the success of marketed ADCs like ADCETRIS and KADCYLA and numerous others advancing through clinical trials. The pursuit of novel cytotoxic payloads beyond the mictotubule inhibitors and DNA damaging agents has led us to the recent discovery of an mRNA splicing inhibitor, thailanstatin, as a potent ADC payload. In our previous work, we observed that the potency of this payload was uniquely tied to the method of conjugation, with lysine conjugates showing much superior potency as compared to cysteine conjugates. However, the ADC field is rapidly shifting towards site-specific ADCs due to their advantages in manufacturability, characterization and safety. In this work we report the identification of a highly efficacious site-specific thailanstatin ADC. The site of conjugation played a critical role on both the in vitro and in vivo potency of these ADCs. During the course of this study, we developed a novel methodology of loading a single site with multiple payloads using an in situ generated multi-drug carrying peptidic linker that allowed us to rapidly screen for optimal conjugation sites. Using this methodology, we were able to identify a double-cysteine mutant ADC delivering four-loaded thailanstatin that was very efficacious in a gastric cancer xenograft model at 3mg/kg and was also shown to be efficacious against T-DM1 resistant and MDR1 overexpressing tumor cell lines.

Amperometric Immunosensor for Carbofuran Detection Based on MWCNTs/GS-PEI-Au and AuNPs-Antibody Conjugate

Directory of Open Access Journals (Sweden)

Xiangyou Wang

2013-04-01

Full Text Available In this paper, an amperometric immunosensor for the detection of carbofuran was developed. Firstly, multiwall carbon nanotubes (MWCNTs and graphene sheets-ethyleneimine polymer-Au (GS-PEI-Au nanocomposites were modified onto the surface of a glass carbon electrode (GCE via self-assembly. The nanocomposites can increase the surface area of the GCE to capture a large amount of antibody, as well as produce a synergistic effect in the electrochemical performance. Then the modified electrode was coated with gold nanoparticles-antibody conjugate (AuNPs-Ab and blocked with BSA. The monoclonal antibody against carbofuran was covalently immobilized on the AuNPs with glutathione as a spacer arm. The morphologies of the GS-PEI-Au nanocomposites and the fabrication process of the immunosensor were characterized by X-ray diffraction (XRD, ultraviolet and visible absorption spectroscopy (UV-vis and scanning electron microscopy (SEM, respectively. Under optimal conditions, the immunosensor showed a wide linear range, from 0.5 to 500 ng/mL, with a detection limit of 0.03 ng/mL (S/N = 3. The as-constructed immunosensor exhibited notable performance features such as high specificity, good reproducibility, acceptable stability and regeneration performance. The results are mainly due to the excellent properties of MWCNTs, GS-PEI-Au nanocomposites and the covalent immobilization of Ab with free hapten binding sites for further immunoreaction. It provides a new avenue for amperometric immunosensor fabrication.

Quantification of residual solvents in antibody drug conjugates using gas chromatography

Energy Technology Data Exchange (ETDEWEB)

Medley, Colin D., E-mail: medley.colin@gene.com [Genentech Inc., Small Molecule Pharmaceutical Sciences, 1 DNA Way, South San Francisco, CA 94080 (United States); Kay, Jacob [Research Pharmaceutical Services, 520 Virginia Dr. Fort, Washington, PA (United States); Li, Yi; Gruenhagen, Jason; Yehl, Peter; Chetwyn, Nik P. [Genentech Inc., Small Molecule Pharmaceutical Sciences, 1 DNA Way, South San Francisco, CA 94080 (United States)

2014-11-19

Highlights: • Sensitive residual solvents detection in ADCs. • 125 ppm QL for common conjugation solvents. • Generic and validatable method. - Abstract: The detection and quantification of residual solvents present in clinical and commercial pharmaceutical products is necessary from both patient safety and regulatory perspectives. Head-space gas chromatography is routinely used for quantitation of residual solvents for small molecule APIs produced through synthetic processes; however residual solvent analysis is generally not needed for protein based pharmaceuticals produced through cultured cell lines where solvents are not introduced. In contrast, antibody drug conjugates and other protein conjugates where a drug or other molecule is covalently bound to a protein typically use solvents such as N,N-dimethylacetamide (DMA), N,N‑dimethylformamide (DMF), dimethyl sulfoxide (DMSO), or propylene glycol (PG) to dissolve the hydrophobic small molecule drug for conjugation to the protein. The levels of the solvent remaining following the conjugation step are therefore important to patient safety as these parental drug products are introduced directly into the patients bloodstream. We have developed a rapid sample preparation followed by a gas chromatography separation for the detection and quantification of several solvents typically used in these conjugation reactions. This generic method has been validated and can be easily implemented for use in quality control testing for clinical or commercial bioconjugated products.

Somatostatin-based radiopeptide therapy with [{sup 177}Lu-DOTA]-TOC versus [{sup 90}Y-DOTA]-TOC in neuroendocrine tumours

Energy Technology Data Exchange (ETDEWEB)

Romer, A.; Seiler, D.; Brunner, P.; Ng, Q.K.T.; Mueller-Brand, J. [University Hospital Basel, Institute of Nuclear Medicine, Basel (Switzerland); Marincek, N.; Walter, M.A. [University Hospital Basel, Institute of Nuclear Medicine, Basel (Switzerland); University Hospital Bern, Institute of Nuclear Medicine, Bern (Switzerland); Koller, M.T. [University Hospital Basel, Basel Institute for Clinical Epidemiology and Biostatistics, Basel (Switzerland); Maecke, H.R. [University Hospital Basel, Division of Radiochemistry, Basel (Switzerland); Rochlitz, C. [University Hospital Basel, Department of Oncology, Basel (Switzerland); Briel, M. [University Hospital Bern, Institute of Nuclear Medicine, Bern (Switzerland); University Hospital Basel, Basel Institute for Clinical Epidemiology and Biostatistics, Basel (Switzerland); McMaster University, Department of Clinical Epidemiology and Biostatistics, Hamilton (Canada); Schindler, C. [University of Basel, Swiss Tropical and Public Health Institute, Basel (Switzerland)

2014-02-15

Somatostatin-based radiopeptide treatment is generally performed using the β-emitting radionuclides {sup 90}Y or {sup 177}Lu. The present study aimed at comparing benefits and harms of both therapeutic approaches. In a comparative cohort study, patients with advanced neuroendocrine tumours underwent repeated cycles of [{sup 90}Y-DOTA]-TOC or [{sup 177}Lu-DOTA]-TOC until progression of disease or permanent adverse events. Multivariable Cox regression and competing risks regression were employed to examine predictors of survival and adverse events for both treatment groups. Overall, 910 patients underwent 1,804 cycles of [{sup 90}Y-DOTA]-TOC and 141 patients underwent 259 cycles of [{sup 177}Lu-DOTA]-TOC. The median survival after [{sup 177}Lu-DOTA]-TOC and after [{sup 90}Y-DOTA]-TOC was comparable (45.5 months versus 35.9 months, hazard ratio 0.91, 95 % confidence interval 0.63-1.30, p = 0.49). Subgroup analyses revealed a significantly longer survival for [{sup 177}Lu-DOTA]-TOC over [{sup 90}Y-DOTA]-TOC in patients with low tumour uptake, solitary lesions and extra-hepatic lesions. The rate of severe transient haematotoxicities was lower after [{sup 177}Lu-DOTA]-TOC treatment (1.4 vs 10.1 %, p = 0.001), while the rate of severe permanent renal toxicities was similar in both treatment groups (9.2 vs 7.8 %, p = 0.32). The present results revealed no difference in median overall survival after [{sup 177}Lu-DOTA]-TOC and [{sup 90}Y-DOTA]-TOC. Furthermore, [{sup 177}Lu-DOTA]-TOC was less haematotoxic than [{sup 90}Y-DOTA]-TOC. (orig.)

Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody-Drug Conjugates.

Science.gov (United States)

Vollmar, Breanna S; Wei, Binqing; Ohri, Rachana; Zhou, Jianhui; He, Jintang; Yu, Shang-Fan; Leipold, Douglas; Cosino, Ely; Yee, Sharon; Fourie-O'Donohue, Aimee; Li, Guangmin; Phillips, Gail L; Kozak, Katherine R; Kamath, Amrita; Xu, Keyang; Lee, Genee; Lazar, Greg A; Erickson, Hans K

2017-10-18

The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small molecules to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small molecule and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was observed between disulfide stability and efficacy for the conjugates. We hypothesized that the observed site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol pK a . We measured the cysteine thiol pK a using isothermal titration calorimetry (ITC) and found that the variants with the highest thiol pK a (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homology modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol pK a and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pK a of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol pK a . The influence of cysteine thiol pK a on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering.

Antibody drug conjugates - Trojan horses in the war on cancer.

Science.gov (United States)

Iyer, U; Kadambi, V J

2011-01-01

Antibody drug conjugates (ADCs) consist of an antibody attached to a cytotoxic drug by means of a linker. ADCs provide a way to couple the specificity of a monoclonal antibody (mAb) to the cytotoxicity of a small-molecule drug and, therefore, are promising new therapies for cancer. ADCs are prodrugs that are inactive in circulation but exert their cytotoxicity upon binding to the target cancer cell. Earlier unsuccessful attempts to generate ADCs with therapeutic value have emphasized the important role each component plays in determining the efficacy and safety of the final ADC. Scientific advances in engineering antibodies for maximum efficacy as anticancer agents, identification of highly cytotoxic molecules, and generation of linkers with increased stability in circulation have all contributed to the development of the many ADCs that are currently in clinical trials. This review discusses parameters that guide the selection of the components of an ADC to increase its therapeutic window, provides a brief look at ADCs currently in clinical trials, and discusses future challenges in this field. Copyright © 2011. Published by Elsevier Inc.

Conjugation of E. coli O157:H7 Antibody to CdSe/ZnS Quantum Dots

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N. T. Vo

2015-01-01

Full Text Available The conjugation of antibody to semiconductor quantum dots plays a very important role in many applications such as bioimaging, biomarking, and biosensing. In this research, we present some results of highly luminescent core/shell structure CdSe/ZnS on which the E. coli antibody was conjugated. The CdSe core was synthesized successfully with chemical “green” method. For biological applications, the capping surfactant, trioctylphosphine oxide, was substituted by a new one, mercaptopropionic acid (MPA, before the antibody attachment step. Finally, the E. coli antibody was attached to quantum dots CdSe/ZnS. Morphology, structure, and optical properties were investigated with PL, UV-Vis, TEM, and XRD methods. The successful ligand substitution and antibody attachment were confirmed by zeta potential measurement, FTIR spectroscopy, and TEM. The results showed quantum dots size of 2.3 nm, uniform distribution, and high luminescence. CdSe/ZnS core/shell structure had better stability and enhanced the luminescence efficiency up to threefold compared with the core CdSe. MPA ligand shifted the initial hydrophobic quantum dots to hydrophilic ones, which helped to dissolve them in organic solvents and attach the antibody.

Antibody-Drug Conjugates (ADCs) for Personalized Treatment of Solid Tumors: A Review.

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Lambert, John M; Morris, Charles Q

2017-05-01

Attaching a cytotoxic "payload" to an antibody to form an antibody-drug conjugate (ADC) provides a mechanism for selective delivery of the cytotoxic agent to cancer cells via the specific binding of the antibody to cancer-selective cell surface molecules. The first ADC to receive marketing authorization was gemtuzumab ozogamicin, which comprises an anti-CD33 antibody conjugated to a highly potent DNA-targeting antibiotic, calicheamicin, approved in 2000 for treating acute myeloid leukemia. It was withdrawn from the US market in 2010 following an unsuccessful confirmatory trial. The development of two classes of highly potent microtubule-disrupting agents, maytansinoids and auristatins, as payloads for ADCs resulted in approval of brentuximab vedotin in 2011 for treating Hodgkin lymphoma and anaplastic large cell lymphoma, and approval of ado-trastuzumab emtansine in 2013 for treating HER2-positive breast cancer. Their success stimulated much research into the ADC approach, with >60 ADCs currently in clinical evaluation, mostly targeting solid tumors. Five ADCs have advanced into pivotal clinical trials for treating various solid tumors-platinum-resistant ovarian cancer, mesothelioma, triple-negative breast cancer, glioblastoma, and small cell lung cancer. The level of target expression is a key parameter in predicting the likelihood of patient benefit for all these ADCs, as well as for the approved compound, ado-trastuzumab emtansine. The development of a patient selection strategy linked to target expression on the tumor is thus critically important for identifying the population appropriate for receiving treatment.

PET Imaging of 64Cu-DOTA-scFv-Anti-PSMA Lipid Nanoparticles (LNPs): Enhanced Tumor Targeting over Anti-PSMA scFv or Untargeted LNPs

Science.gov (United States)

Wong, Patty; Li, Lin; Chea, Junie; Delgado, Melissa K.; Crow, Desiree; Poku, Erasmus; Szpikowska, Barbara; Bowles, Nicole; Channappa, Divya; Colcher, David; Wong, Jeffrey Y.C.; Shively, John E.; Yazaki, Paul J.

2017-01-01

Introduction Single chain (scFv) antibodies are ideal targeting ligands due to their modular structure, high antigen specificity and affinity. These monovalent ligands display rapid tumor targeting but have limitations due to their fast urinary clearance. Methods An anti-prostate membrane antigen (PSMA) scFv with a site-specific cysteine was expressed and evaluated in a prostate cancer xenograft model by Cu-64 PET imaging. To enhance tumor accumulation, the scFv-cys was conjugated to the co-polymer DSPE-PEG-maleimide that spontaneously assembled into a homogeneous multivalent lipid nanoparticle (LNP). Results The targeted LNP exhibited a 2-fold increase in tumor uptake compared to the scFv alone using two different thiol ester chemistries. The anti-PSMA scFv-LNP exhibited a 1.6 fold increase in tumor targeting over the untargeted LNP. Conclusions The targeted anti-PSMA scFv-LNP showed enhanced tumor accumulation over the scFv alone or the untargeted DOTA-micelle providing evidence for the development of this system for drug delivery. Advances in Knowledge and implications for patient care Anti-tumor scFv antibody fragments have not achieved their therapeutic potential due to their fast blood clearance. Conjugation to a LNP enables multivalency to the tumor antigen as well as increased molecular size for chemotherapy drug delivery. PMID:28126683

Comparative assay of fluorescent antibody test results among twelve European National Reference Laboratories using various anti-rabies conjugates

DEFF Research Database (Denmark)

Robardet, E.; Andrieu, S.; Rasmussen, Thomas Bruun

2013-01-01

Twelve National Reference Laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies conjugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive...

Production of anti-fullerene C{sub 60} polyclonal antibodies and study of their interaction with a conjugated form of fullerene

Energy Technology Data Exchange (ETDEWEB)

Hendrickson, O. D., E-mail: odhendrick@gmail.com; Fedyunina, N. S. [Russian Academy of Sciences, Institute of Biochemistry (Russian Federation); Martianov, A. A. [Moscow State University (Russian Federation); Zherdev, A. V.; Dzantiev, B. B. [Russian Academy of Sciences, Institute of Biochemistry (Russian Federation)

2011-09-15

The aim of this study was to produce anti-fullerene C{sub 60} antibodies for the development of detection systems for fullerene C{sub 60} derivatives. To produce anti-fullerene C{sub 60} antibodies, conjugates of the fullerene C{sub 60} carboxylic derivative with thyroglobulin, soybean trypsin inhibitor, and bovine serum albumin were synthesized by carbodiimide activation and characterized. Immunization of rabbits by the conjugates led to the production of polyclonal anti-fullerene antibodies. The specificity of the immune response to fullerene was investigated. Indirect competitive immunoenzyme assay was developed for the determination of conjugated fullerene with detection limits of 0.04 ng/mL (calculated for coupled C{sub 60}) and 0.4 ng/mL (accordingly to total fullerene-protein concentration).

A smart T(1)-weighted MRI contrast agent for uranyl cations based on a DNAzyme-gadolinium conjugate.

Science.gov (United States)

Xu, Weichen; Xing, Hang; Lu, Yi

2013-11-07

Rational design of smart MRI contrast agents with high specificity for metal ions remains a challenge. Here, we report a general strategy for the design of smart MRI contrast agents for detecting metal ions based on conjugation of a DNAzyme with a gadolinium complex. The 39E DNAzyme, which has high selectivity for UO2(2+), was conjugated to Gd(III)-DOTA and streptavidin. The binding of UO2(2+) to its 39E DNAzyme resulted in the dissociation of Gd(III)-DOTA from the large streptavidin, leading to a decrease of the T1 correlation time, and a change in the MRI signal.

Peptide receptor radionuclide therapy (PRRT) using Lu-177 DOTA-NOC and Lu-177 DOTA-TATE: Comparative results

International Nuclear Information System (INIS)

Wehrmann, C.; Senftleben, S.; Baum, R.P.

2007-01-01

Full text: Aim: One of the few treatment options for inoperable neuroendocrine tumors (NET) is peptide receptor radiotherapy with somatostatin analogues. DOTA-NOC shows the highest affinity to the somatostatin receptors (sstr) 3 and 5 and a very high affinity to sstr 2. We compared the dosimetric parameters uptake, half-life (kinetics) and mean absorbed organ and tumor doses of 177 Lu DOTANOC and 177 Lu DOTA-TATE. Methods: 139 patients with neuroendocrine tumors with high sstr expression (verified by Ga-68 DOTA-NOC PET/CT) were studied. 130 patients (57m, 73f; aged 60±11a) were treated with 2.5-7.4 GBq Lu-177 DOTA-TATE and 9 patients (3m, 6f, aged 64±10a) with 3.6-7.4 GBq Lu-177 DOTA-NOC. Whole-body scans were performed after 0.5h, 3h, 24h, 48h, 72h and 96h p.i. Blood samples from 23 patients were obtained after therapy. By means of geometric mean and after background correction, ROI results were used to calculate the estimated absorbed organ and tumor doses according to the MIRD-scheme (OLINDA software). Results: Lu-177 DOTA-NOC showed a higher uptake as compared to Lu-177 DOTATATE (=100%): for whole-body about 38% and in normal tissue 36%, in the spleen 17% and in the kidneys 18%. The tumor uptake was about 5% higher for DOTA-TATE. The effective half-life for whole-body was comparable for both peptides (t1/2a NOC 2.9h vs. TATE 2.4h and t1/2b NOC 54h vs. TATE 56h). In normal tissue, t1/2a was similar (NOC 3.3h; TATE 2.6h) but the t1/2b was longer for DOTA-TATE (NOC 43h; TATE 48h). t1/2b was longer for DOTA-NOC in the spleen (NOC 81h; TATE 72h) and in the kidney (NOC 68.1h; TATE 65h). The mean absorbed dose in the kidney (TATE 5Sv; NOC 6Sv) and spleen (TATE 7Sv; NOC 8Sv) was higher for DOTA-NOC. In the tumor, the t1/2b was higher for DOTA-TATE (NOC 65h; TATE 77h). For DOTA-TATE the whole-body dose was lower (0.27Sv) as compared to DOTA-NOC (0.38 Sv) (significant by unpaired sign test). The estimated mean absorbed tumor doses were 47+/-66 Sv for DOTA-TATE and 35

A novel multi-walled carbon nanotube-based antibody conjugate for quantitative and semi-quantitative lateral flow assays.

Science.gov (United States)

Sun, Wenjuan; Hu, Xiaolong; Liu, Jia; Zhang, Yurong; Lu, Jianzhong; Zeng, Libo

2017-10-01

In this study, the multi-walled carbon nanotubes (MWCNTs) were applied in lateral flow strips (LFS) for semi-quantitative and quantitative assays. Firstly, the solubility of MWCNTs was improved using various surfactants to enhance their biocompatibility for practical application. The dispersed MWCNTs were conjugated with the methamphetamine (MET) antibody in a non-covalent manner and then manufactured into the LFS for the quantitative detection of MET. The MWCNTs-based lateral flow assay (MWCNTs-LFA) exhibited an excellent linear relationship between the values of test line and MET when its concentration ranges from 62.5 to 1500 ng/mL. The sensitivity of the LFS was evaluated by conjugating MWCNTs with HCG antibody and the MWCNTs conjugated method is 10 times more sensitive than the one conjugated with classical colloidal gold nanoparticles. Taken together, our data demonstrate that MWCNTs-LFA is a more sensitive and reliable assay for semi-quantitative and quantitative detection which can be used in forensic analysis.

Imaging of Hsp70-positive tumors with cmHsp70.1 antibody-conjugated gold nanoparticles

Directory of Open Access Journals (Sweden)

Gehrmann MK

2015-09-01

Full Text Available Mathias K Gehrmann,1 Melanie A Kimm,2 Stefan Stangl,1 Thomas E Schmid,1 Peter B Noël,2 Ernst J Rummeny,2 Gabriele Multhoff11Department of Radiation Oncology, 2Department of Diagnostic and Interventional Radiology, Klinikum rechts der Isar, Technische Universität München, Munich, GermanyAbstract: Real-time imaging of small tumors is still one of the challenges in cancer diagnosis, prognosis, and monitoring of clinical outcome. Targeting novel biomarkers that are selectively expressed on a large variety of different tumors but not normal cells has the potential to improve the imaging capacity of existing methods such as computed tomography. Herein, we present a novel technique using cmHsp70.1 monoclonal antibody-conjugated spherical gold nanoparticles for quantification of the targeted uptake of gold nanoparticles into membrane Hsp70-positive tumor cells. Upon binding, cmHsp70.1-conjugated gold nanoparticles but not nanoparticles coupled to an isotype-matched IgG1 antibody or empty nanoparticles are rapidly taken up by highly malignant Hsp70 membrane-positive mouse tumor cells. After 24 hours, the cmHsp70.1-conjugated gold nanoparticles are found to be enriched in the perinuclear region. Specificity for membrane Hsp70 was shown by using an Hsp70 knockout tumor cell system. Toxic side effects of the cmHsp70.1-conjugated nanoparticles are not observed at a concentration of 1–10 µg/mL. Experiments are ongoing to evaluate whether cmHsp70.1 antibody-conjugated gold nanoparticles are suitable for the detection of membrane-Hsp70-positive tumors in vivo.Keywords: heat shock protein 70, tumor biomarker, theranostics, multimodal CT, multispectral CT, k-edge

Synthesis of indium-labeled antibody-chelate conjugates for radioassays

Energy Technology Data Exchange (ETDEWEB)

Gokce, A; Nakamura, R M; Tubis, M; Wolf, W

1982-01-01

A method has been developed to achieve rapid and reproducible complexation of indium to transferrin at pH 7.4. The system consists of nitrilotriacetic acid (NTA) as the intermediate carrier ligand, whose function is to allow the /sup 113/m In ion, in a solution in Tris buffer, pH 7.4, to be transferred rapidly to the specific binding sites on transferrin. Just as in the case of iron, this complexation requires the presence of a synergistic ion such as bicarbonate. The present system can be used to allow the binding of /sup 113/mIn to transferrin when coupled to an antibody. This method has been tested by studying the conjugation of an antibody, the IgG fraction of goat anti-rabbit-IgG, with either transferrin or desferoxamine, using glutaraldehyde as the coupling agent. Optimization in terms of total protein concentration and glutaraldehyde levels lead to products where the specific metal binding capacity of the transferrin moiety remains unchanged, and where the antibody retains 70% of its antigenic activity. The present system can be considered an extension of the ELISA techniques and can be used to determine, by a terminal /sup 113/mIn labeling technique, the level of specific binding of an antibody to its antigen.

Somatostatin-based radiotherapy with [90Y-DOTA]-TOC in neuroendocrine tumors: long-term outcome of a phase I dose escalation study

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Marincek Nicolas

2013-01-01

Full Text Available Abstract Background We describe the long-term outcome after clinical introduction and dose escalation of somatostatin receptor targeted therapy with [90Y-DOTA]-TOC in patients with metastasized neuroendocrine tumors. Methods In a clinical phase I dose escalation study we treated patients with increasing [90Y-DOTA]-TOC activities. Multivariable Cox regression and competing risk regression were used to compare efficacy and toxicities of the different dosage protocols. Results Overall, 359 patients were recruited; 60 patients were enrolled for low dose (median: 2.4 GBq/cycle, range 0.9-7.8 GBq/cycle, 77 patients were enrolled for intermediate dose (median: 3.3 GBq/cycle, range: 2.0-7.4 GBq/cycle and 222 patients were enrolled for high dose (median: 6.7 GBq/cycle, range: 3.7-8.1 GBq/cycle [90Y-DOTA]-TOC treatment. The incidences of hematotoxicities grade 1–4 were 65.0%, 64.9% and 74.8%; the incidences of grade 4/5 kidney toxicities were 8.4%, 6.5% and 14.0%, and the median survival was 39 (range: 1–158 months, 34 (range: 1–118 months and 29 (range: 1–113 months. The high dose protocol was associated with an increased risk of kidney toxicity (Hazard Ratio: 3.12 (1.13-8.59 vs. intermediate dose, p = 0.03 and a shorter overall survival (Hazard Ratio: 2.50 (1.08-5.79 vs. low dose, p = 0.03. Conclusions Increasing [90Y-DOTA]-TOC activities may be associated with increasing hematological toxicities. The dose related hematotoxicity profile of [90Y-DOTA]-TOC could facilitate tailoring [90Y-DOTA]-TOC in patients with preexisting hematotoxicities. The results of the long-term outcome suggest that fractionated [90Y-DOTA]-TOC treatment might allow to reduce renal toxicity and to improve overall survival. (ClinicalTrials.gov number NCT00978211.

Somatostatin-based radiotherapy with [90Y-DOTA]-TOC in neuroendocrine tumors: long-term outcome of a phase I dose escalation study

Science.gov (United States)

2013-01-01

Background We describe the long-term outcome after clinical introduction and dose escalation of somatostatin receptor targeted therapy with [90Y-DOTA]-TOC in patients with metastasized neuroendocrine tumors. Methods In a clinical phase I dose escalation study we treated patients with increasing [90Y-DOTA]-TOC activities. Multivariable Cox regression and competing risk regression were used to compare efficacy and toxicities of the different dosage protocols. Results Overall, 359 patients were recruited; 60 patients were enrolled for low dose (median: 2.4 GBq/cycle, range 0.9-7.8 GBq/cycle), 77 patients were enrolled for intermediate dose (median: 3.3 GBq/cycle, range: 2.0-7.4 GBq/cycle) and 222 patients were enrolled for high dose (median: 6.7 GBq/cycle, range: 3.7-8.1 GBq/cycle) [90Y-DOTA]-TOC treatment. The incidences of hematotoxicities grade 1–4 were 65.0%, 64.9% and 74.8%; the incidences of grade 4/5 kidney toxicities were 8.4%, 6.5% and 14.0%, and the median survival was 39 (range: 1–158) months, 34 (range: 1–118) months and 29 (range: 1–113) months. The high dose protocol was associated with an increased risk of kidney toxicity (Hazard Ratio: 3.12 (1.13-8.59) vs. intermediate dose, p = 0.03) and a shorter overall survival (Hazard Ratio: 2.50 (1.08-5.79) vs. low dose, p = 0.03). Conclusions Increasing [90Y-DOTA]-TOC activities may be associated with increasing hematological toxicities. The dose related hematotoxicity profile of [90Y-DOTA]-TOC could facilitate tailoring [90Y-DOTA]-TOC in patients with preexisting hematotoxicities. The results of the long-term outcome suggest that fractionated [90Y-DOTA]-TOC treatment might allow to reduce renal toxicity and to improve overall survival. Trial registration ClinicalTrials.gov number:NCT00978211 PMID:23320604

PURWARUPA PORTAL MEMBER DOTA 2 INDONESIA

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Yogi Anggoro

2017-11-01

Full Text Available Game di Indonesia sangatlah berkembang pesat, dari game offline maupun online. Penggemar game bukan hanya dari kalangan anak kecil, namun dari semua kalangan. Apalagi game online, sampai diperlombakan. Salah satunya yaitu game Dota 2, yang merupakan salah satu game online. Pada gamers bahkan tidak hanya di lingkup Indonesia saja, namun dapat tanding dengan negara manapun. Dari game ini muncul komunitas Dota 2, di Indonesia terdapat website yang memberikan informasi tentang Dota 2. Tetapi dari sekian website yang dikembangkan tidak ditemukan sebuah website yang mengakomodasi kebutuhan dari gamers. Tujuan dari penelitian ini adalah membuat sebuah wadah yang berfungsi sebagai sistem informasi untuk mengatur segala aktivitas gamers dalam bermain Dota 2. Purwarupa dalam penelitian ini mengakomodasi dalam pembuatan grup, mendapatkan pertandingan serta berpartisipasi dalam pertandingan ini. Di dalam purwarupa ini menjadi penting agar mempermudah pemain dalam bermain game Dota 2 ini. Dengan metode pengumpulan data literature, inisiasi, investigasi sampai pengembangan sistem dapat menghasilkan sebuah sistem informasi untuk komunitas Dota 2 di Indonesia.

A comparative study of preliminary dosimetry for human based on distribution data in rats with 111In, 90Y, 153Sm, and 177Lu labeled rituximab

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Radfar Edalat

2012-01-01

Full Text Available Radio immunotherapy is one of the most important and effective therapies for B-cell non Hoddgkin’s lymphoma treatment. Today, anti CD-20 antibodies labeled with beta emitter radionuclides are used in radio immunotherapy. Various radionuclides for labeling anti CD-20 antibodies have been studied and developed for the treatment and diagnosis of malignancies. This paper describes the preparation, bio-distribution and absorbed dose rate of 111In, 90Y, 177Lu, and 153Sm labeled anti CD-20 antibodies (rituximab in human organs, after injection to rats. The macro cyclic bifunctional chelating agent, N-succinimidyl-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA-NHS for conjugation to antibody, was used to prepare DOTA-rituximab. The conjugates were purified by molecular filtration, the average number of DOTA conjugated per mAb was calculated and total concentration was determined by spectrophotometric method. Radio-labeling was performed at 40 °C for 24 hours. After the quality control studies, the final radioactive solution was injected intravenously into rats through their tail vein. The tissue uptakes of each injection were measured. Then we calculated S values for 177Lu and 153Sm by using specific absorbed fractions and data used in the manner of radio-labeled analysis and dosimetry for humans. The absorbed dose rate of each organ was calculated in the specific time by medical internal radiation dose method with linear approximation in the activity measurements.

Preparation of (177Lu-DOTAn-PAMAM-[Nimotuzumab-F(ab’2] as a Therapeutic Radioimmunoconjugate for EGFR Overexpressed Cancer Treatment

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Titis Sekar Humani

2017-12-01

Full Text Available Intact monoclonal antibodies with a high molecular weight tend to have a poor pharmacokinetic profile and tumor penetration, and potential for eliciting host antibody responses. F(ab’2 fragments smaller than intact monoclonal antibodies that still maintain antigen binding could solve this problem. The objective of this study was to optimize the digestion process of nimotuzumab, an anti-EGFR monoclonal antibody, into its F(ab’2 fragment and investigate its potential as a therapeutic radioimmunoconjugate. Optimal conditions for digestion of nimotuzumab to its F(ab’2 fragment were found to be 6 hours of digestion time with a pH of 3.5 and 1:100 mol ratio of pepsin to nimotuzumab. The purity of the F(ab’2-nimotuzumab was confirmed by SDS-PAGE and HPLC analysis. Prior to its labeling with lutetium-177 radionuclide, the nimotuzumab-F(ab’2 was conjugated to DOTA-PAMAM dendrimer [DOTA denotes 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, PAMAM denotes poly(amidoamine] to form conjugate of (DOTAn-PAMAM-[nimotuzumab-F(ab’2]. Radiolabeling of DOTA-PAMAM-[nimotuzumab-F(ab’2] conjugate with 177Lu resulted in (177Lu-DOTAn-PAMAM-[nimotuzumab-F(ab’2] with radiochemical purity > 99% after purification with a PD-10 column. Further studies still need to be performed in order to confirm the potential of this radioimmunoconjugate as a radioimmunotherapeutic agent for EGFR overexpressed cancers.

Targeting of indium 111-labeled bivalent hapten to human melanoma mediated by bispecific monoclonal antibody conjugates: Imaging of tumors hosted in nude mice

International Nuclear Information System (INIS)

Le Doussal, J.M.; Gruaz-Guyon, A.; Martin, M.; Gautherot, E.; Delaage, M.; Barbet, J.

1990-01-01

Antibody conjugates were prepared by coupling F(ab')2 or Fab' fragments of an antibody specific for the human high molecular weight-melanoma associated antigen to Fab' fragments of an antibody specific for indium-diethylenetriaminepentaacetate complexes. Monovalent and bivalent haptens were synthesized by reacting the dipeptide tyrosyl-lysine with diethylenetriaminepentaacetic cyclic anhydride. In vitro, the antibody conjugate mediated binding of the 111In-labeled haptens to melanoma cells. In vivo, it allowed specific localization of the haptens in A375 tumors. The bivalent hapten exhibited much higher efficiency at targeting 111In onto cells, both in vitro and in vivo. Antibody conjugate and hapten doses (2 micrograms and 1 pmol, respectively) and the delay between antibody conjugate and tracer injections (24 h) were adjusted to maximize tumor uptake (4% injected dose/g) and tumor to normal tissue contrast (greater than 3) obtained 3 h after injection of the 111In-labeled bivalent hapten. This two-step technique, when compared to direct targeting of 111In-labeled F(ab')2 fragments, provided lower localization of injected activity into the tumor (x 0.25), but higher tumor/tissue ratios, especially with respect to liver (x 7), spleen (x 8), and kidneys (x 10). In addition, high contrast images were obtained within 3 hours, instead of days. Thus, antibody conjugate-mediated targeting of small bivalent haptens, labeled with short half-life isotopes, is proposed as a general method for improving tumor radioimmunolocalization

Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins

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Rosen, Christian B.; Kodal, Anne L. B.; Nielsen, Jesper S.; Schaffert, David H.; Scavenius, Carsten; Okholm, Anders H.; Voigt, Niels V.; Enghild, Jan J.; Kjems, Jørgen; Tørring, Thomas; Gothelf, Kurt V.

2014-09-01

DNA-protein conjugates are important in bioanalytical chemistry, molecular diagnostics and bionanotechnology, as the DNA provides a unique handle to identify, functionalize or otherwise manipulate proteins. To maintain protein activity, conjugation of a single DNA handle to a specific location on the protein is often needed. However, preparing such high-quality site-specific conjugates often requires genetically engineered proteins, which is a laborious and technically challenging approach. Here we demonstrate a simpler method to create site-selective DNA-protein conjugates. Using a guiding DNA strand modified with a metal-binding functionality, we directed a second DNA strand to the vicinity of a metal-binding site of His6-tagged or wild-type metal-binding proteins, such as serotransferrin, where it subsequently reacted with lysine residues at that site. This method, DNA-templated protein conjugation, facilitates the production of site-selective protein conjugates, and also conjugation to IgG1 antibodies via a histidine cluster in the constant domain.

Development of a lyophilized formulation for preparing the radiopharmaceutical {sup 177}Lu-DOTA-Anti-CD20; Desarrollo de una formulacion liofilizada para la preparacion del radiofarmaco {sup 177}-DOTA-Anti-CD20

Energy Technology Data Exchange (ETDEWEB)

Serrano E, L. A.

2015-07-01

The radiolabeled proteins are molecules of interest in nuclear medicine for their diagnostic and therapeutic application in cancer. Antibodies, such as chimeric monoclonal antibody Anti-CD20 rituximab, have established themselves as suitable vectors of radionuclides (e.g. {sup 177}Lu) , introducing high affinity by the surface antigens over- expressed and widely distributed in cells involved in certain diseases. The aim of this work was to design, optimize and document the production process of radiopharmaceutical {sup 177}Lu-DOTA-Anti-CD20 for sanitary registration request to the Comision Federal para la Proteccion contra Riesgos Sanitarios (COFEPRIS). First, a raw material analysis using the Ft-Mir technique and gamma spectrometry was performed. Then, was carried out the development of the lyophilized formulation for the preparation of {sup 177}Lu-DOTA-Anti-CD20, in which an ANOVA was performed where the dependent variable was the radiochemical purity. The optimal pharmaceutical formulation was: 5 mg DOTA-CD20 and 80 mg Mannitol to be reconstituted with 1 m L of acetate buffer 0.25 M, ph 7, with an incubation time of 15 min at 37 degrees Celsius in a dry bath. Once completed the development of the lyophilized formulation, we proceeded to the optimization of the production process, development and validation of the analytical method. Three batches were prepared under protocols of Good Manufacturing Practice, which met pre-established specifications as sterile and endotoxin-free of bacterial formulations, with greater that 95% of radiochemical purity. Currently, is conducting the study of shelf stability. Upon completion of the stability studies, the legal record of {sup 177}Lu-DOTA-Anti-CD20 will be integrated with documented evidence of the quality and stability of the formulation of this radiopharmaceutical. (Author)

Tumor targeting using {sup 67}Ga-DOTA-Bz-folate - investigations of methods to improve the tissue distribution of radiofolates

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Mueller, Cristina, E-mail: cristina.mueller@psi.ch [Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute, 5232 Villigen-PSI (Switzerland); Vlahov, Iontcho R.; Santhapuram, Hari Krishna R.; Leamon, Christopher P. [Endocyte Inc., West Lafayette, IN 47906 (United States); Schibli, Roger [Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute, 5232 Villigen-PSI (Switzerland); Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich (Switzerland)

2011-07-15

Introduction: Use of folic acid radioconjugates for folate receptor (FR) targeting is a promising strategy for imaging purposes as well as for potential therapy of cancer and inflammatory diseases due to the frequent FR overexpression found on cancer cells and activated macrophages. Herein, we report on preclinical results using a novel DOTA-Bz-EDA-folate conjugate radiolabeled with [{sup 67}Ga]-gallium. Methods: DOTA-Bz-EDA-folate was prepared by conjugation of ethylenediamine-({gamma})-folate with 2-(p-isothiocyanobenzyl)-DOTA. Radiolabeling was carried out with {sup 67}GaCl{sub 3} according to standard procedures. Biodistribution studies of the tracer were performed in mice bearing FR-positive KB tumor xenografts. The effects on radiofolate biodistribution with coadministered renal uptake-blocking amino acids, diuretic agents, antifolates as well as different routes of administration were likewise investigated. Supportive imaging studies were performed using a small-animal single photon emission computed tomography (SPECT)/CT scanner. Results: {sup 67}Ga-DOTA-Bz-EDA-folate showed a high and specific accumulation in tumors (6.30%{+-}0.75% ID/g, 1 h pi and 6.08%{+-}0.89% ID/g, 4 h pi). Nonspecific radioactivity uptake in nontargeted tissues was negligible, but significant accumulation was found in FR-positive kidneys, which resulted in unfavorably low tumor-to-kidney ratios (<0.1). Coadministered amino acids or diuretics did not effectively reduce renal accumulation; in contrast, predosed pemetrexed did significantly reduce kidney uptake (<29% of control values). The SPECT/CT studies confirmed the excellent tumor-to-background contrast of {sup 67}Ga-radiofolate and the favorable reduction in kidney uptake (with improved imaging quality) resulting from pemetrexed administration. Conclusion: Conventional methods to reduce kidney uptake of radiofolates fail. However, the novel {sup 67}Ga-radiolabeled DOTA-Bz-EDA-folate can effectively be used to image FR

Site-specific antibody-liposome conjugation through copper-free click chemistry: a molecular biology approach for targeted photodynamic therapy (Conference Presentation)

Science.gov (United States)

Obaid, Girgis; Wang, Yucheng; Kuriakose, Jerrin; Broekgaarden, Mans; Alkhateeb, Ahmed; Bulin, Anne-Laure; Hui, James; Tsourkas, Andrew; Hasan, Tayyaba

2016-03-01

Nanocarriers, such as liposomes, have the ability to potentiate photodynamic therapy (PDT) treatment regimens by the encapsulation of high payloads of photosensitizers and enhance their passive delivery to tumors through the enhanced permeability and retention effect. By conjugating targeting moieties to the surface of the liposomal nanoconstructs, cellular selectivity is imparted on them and PDT-based therapies can be performed with significantly higher dose tolerances, as off-target toxicity is simultaneously reduced.1 However, the maximal benefits of conventional targeted nanocarriers, including liposomes, are hindered by practical limitations including chemical instability, non-selective conjugation chemistry, poor control over ligand orientation, and loss of ligand functionality following conjugation, amongst others.2 We have developed a robust, physically and chemically stable liposomal nanoplatform containing benzoporphyrin derivative photosensitizer molecules within the phospholipid bilayer and an optimized surface density of strained cyclooctyne moieties for `click' conjugation to azido-functionalized antibodies.3 The clinical chimeric anti-EGFR antibody Cetuximab is site-specifically photocrosslinked to a recombinant bioengineered that recognizes the antibody's Fc region, containing a terminal azide.4 The copper-free click conjugation of the bioengineered Cetuximab derivative to the optimized photosensitizing liposome provides exceptional control over the antibody's optimal orientation for cellular antigen binding. Importantly, the reaction occurs rapidly under physiological conditions, bioorthogonally (selectively in the presence of other biomolecules) and without the need for toxic copper catalysis.3 Such state-of-the-art conjugation strategies push the boundaries of targeted photodynamic therapy beyond the limitations of traditional chemical coupling techniques to produce more robust and effective targeted therapeutics with applications beyond

Vaccination with Shigella flexneri 2a conjugate induces type 2a and cross-reactive type 6 antibodies in humans but not in mice.

Science.gov (United States)

Farzam, Nahid; Ramon-Saraf, Reut; Banet-Levi, Yonit; Lerner-Geva, Liat; Ashkenazi, Shai; Kubler-Kielb, Joanna; Vinogradov, Evgeny; Robbins, John B; Schneerson, Rachel

2017-09-05

Shigella flexneri (S. flexneri) 6 has emerged as an important cause of shigellosis. Our efficacy study of Shigella sonnei and S. flexneri 2a O-specific polysaccharide (O-SP) conjugates in 1-4year-olds had too few S. flexneri 2a cases for efficacy evaluation but surprisingly showed protection of 3-4year-olds, S. flexneri 2a-recipients, from S. flexneri 6 infection. To investigate this cross-protection antibodies to both Shigella types were investigated in all sera remaining from previous studies. Twenty to 30% of 3-44year-old humans injected with S. flexneri 2a conjugate responded with ≥4-fold increases of IgG anti type 6, p<0.00001. The specificity of these antibodies was shown by inhibition studies. S. flexneri 6 infection of 2 children induced besides S. flexneri 6, also S. flexneri 2a antibodies, at levels of S. flexneri 2a vaccinees. S. flexneri 2a antibodies induced by S. flexneri 6 conjugates could not be studied since no such conjugate was assessed in humans and mice responded almost exclusively to the O-SP of the injected conjugate, with no cross-reactive antibodies. Our results indicate induction of cross-reactive protective antibodies. The O-acetylated disaccharide shared by S. flexneri 6 and 2a O-SPs, is the likely basis for their cross-reactivity. S. flexneri 6 O-SP conjugates, alone and in combination with S. flexneri 2a, merit further investigation for broad S. flexneri protection. Published by Elsevier Ltd.

Evaluation and comparison of Ga-68 DOTA-TATE and Ga-68 DOTA-NOC PET/CT imaging in well-differentiated thyroid cancer.

Science.gov (United States)

Ocak, Meltem; Demirci, Emre; Kabasakal, Levent; Aygun, Aslan; Tutar, Rumeysa O; Araman, Ahmet; Kanmaz, Bedii

2013-11-01

Somatostatin receptor (Sstr) scintigraphy with radiolabelled somatostatin analogues has been used extensively for the diagnosis and therapy of Sstr-expressing tumours. It has been shown that well-differentiated thyroid cancer (WDTC) cells have a high expression of Sstr2, Sstr3 and Sstr5. Hence, WDTC cells could be an ideal target for the evaluation of lesion uptake of Ga-68 DOTA-1-NaI3-octreotide (DOTA-NOC), which has a high affinity not only to Sstr2 but also to Sstr3 and Sstr5. The aim of the present study was to evaluate the value of Ga-68 DOTA-NOC as a target for Sstr2-expressing, Sstr3-expressing and Sstr5-expressing tumours in WDTC patients and to compare the results with those of Ga-68 DOTA-TATE in the same patient population. Thirteen patients with WDTC were included in our study: nine with papillary thyroid cancer, three with Hurthle cell carcinoma and one with follicular thyroid carcinoma. All patients had elevated serum thyroglobulin levels and negative post-therapeutic I-131 whole-body scans, which were obtained after the last radioiodine treatment. All patients had undergone two consecutive PET imaging studies with Ga-68 DOTA-D-Phe1-Tyr3-octreotate (DOTA-TATE) and Ga-68 DOTA-NOC, respectively. All images were evaluated visually, and maximum standardized uptake values were calculated. Both Ga-68 DOTA-TATE and Ga-68 DOTA-NOC PET images gave comparable results. Among the 13 patients, imaging with both Ga-68 DOTA-TATE and Ga-68 DOTA-NOC gave negative results in five (38%) patients and positive results in eight (62%) patients. A total of 45 lesions were identified on Ga-68 DOTA-TATE images and 42 on Ga-68 DOTA-NOC images; three lesions were missed. Lesion uptake was significantly higher on Ga-68 DOTA-TATE images. Maximum standardized uptake values of Ga-68 DOTA-TATE and Ga-68 DOTA-NOC were 12.9±9.1 and 6.3±4.1 (n=54, PDOTA-TATE has a higher lesion uptake even in WDTC patients and may have potential advantage over Ga-68 DOTA-NOC.

Preparation and preliminary studies of [64Cu]-antiMUC-1 for breast cancer targeting

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Behrouz Alirezapour

2016-06-01

Full Text Available PR81 is a monoclonal antibody that binds with high affinity to MUC1 that over expressed on breast tumors. PR81 is considered a suitable targeting molecule that was radiolabeled using Cu-64 for positron imaging studies. The monoclonal antibody was conjugated with DOTA moiety and after purification was evaluated for radiochemical purity, immunoreactivity, cell toxicity and structure integrity as well as biodistribution study in normal rats. The radiolabeled antibody prepared with acceptable radiochemical purity (> 93.2 ± 0.6 %, ITLC; specific activity; 4.6 µCi/µg, protein structure integration, significant cytotoxicity and significant immunoreactivity retention was assessed by radioimmunoassay (RIA. Animal biodistribution of the 64Cu-DOTA-PR81 was consistent with other radiolabeled antibodies. The results showed that 64Cu-DOTA-PR81 may be considered for tumor imaging for ultimate diagnosis and follow-up of MUC1 expression in oncology.

The in vivo disposition and in vitro transmembrane transport of two model radiometabolites of DOTA-conjugated receptor-specific peptides labelled with (177) Lu.

Science.gov (United States)

Volková, Marie; Mandíková, Jana; Bárta, Pavel; Navrátilová, Lucie; Lázníčková, Alice; Trejtnar, František

2015-01-01

In vivo metabolism of the radiolabelled receptor-specific peptides has been described; however, information regarding the pharmacokinetic behaviour of the degradation products within the body is very scarce. The present study was designed to obtain new knowledge on the disposition and elimination of low-molecular radiometabolites of receptor-specific peptides in the organism and to reveal the potential involvement of selected membrane transport mechanisms in the cellular uptake of radiometabolites, especially in the kidney. The study compared pharmacokinetics of two radiometabolites: a final metabolite of somatostatin analogues, (177)Lu-DOTA-DPhe, and a tripeptide metabolite of (177)Lu-DOTA-minigastrin 11, (177)Lu-DOTA-DGlu-Ala-Tyr. Their pharmacokinetics was compared with that of respective parent (177)Lu-radiopeptide. Both radiometabolites exhibited relative rapid clearing from most body tissues in rats in vivo along with predominant renal excretion. The long-term renal retention of the smaller radiometabolite (177)Lu-DOTA-DPhe was lower than that of (177)Lu-DOTA-DGlu-Ala-Tyr. An uptake of (177)Lu-DOTA-DPhe by human renal influx transporter organic cation transporter 2 was found in vitro in a cellular model. The study brings the first experimental data on the in vivo pharmacokinetics of radiometabolites of receptor-specific somatostatin and gastrin analogues. The found results may indicate a negative correlation between the degree of decomposition of the parent peptide chain and the renal retention of the metabolite. Copyright © 2015 John Wiley & Sons, Ltd.

Preclinical evaluation of potential infection-imaging probe [68 Ga]Ga-DOTA-K-A9 in sterile and infectious inflammation.

Science.gov (United States)

Nielsen, Karin M; Jørgensen, Nis P; Kyneb, Majbritt H; Borghammer, Per; Meyer, Rikke L; Thomsen, Trine R; Bender, Dirk; Jensen, Svend B; Nielsen, Ole L; Alstrup, Aage K O

2018-05-23

The development of bacteria-specific infection radiotracers is of considerable interest to improve diagnostic accuracy and enabling therapy monitoring. The aim of this study was to determine if the previously reported radiolabelled 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetraacetic acid (DOTA) conjugated peptide, [ 68 Ga]Ga-DOTA-K-A9 could detect a staphylococcal infection in vivo, and distinguish it from aseptic inflammation. An optimised [ 68 Ga]Ga-DOTA-K-A9 synthesis omitting the use of acetone was developed, yielding 93% ± 0.9% radiochemical purity. The in vivo infection binding specificity of [ 68 Ga]Ga-DOTA-K-A9 was evaluated by micro positron emission tomography/magnetic resonance imaging (μPET/MRI) of 15 mice with either subcutaneous S. aureus infection or turpentine induced inflammation and compared with 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG). The scans showed that [ 68 Ga]Ga-DOTA-K-A9 accumulated in all the infected mice at injected doses ≥3.6 MBq. However, the tracer was not found to be selective towards infection, since the [ 68 Ga]Ga-DOTA-K-A9 also accumulated in mice with inflammation. In a concurrent in vitro binding evaluation performed with a 5-Carboxytetramethylrhodamine (TAMRA) fluorescence analogue of the peptide, TAMRA-K-A9, the microscopy results suggested that TAMRA-K-A9 bound to an intracellular epitope and therefore preferentially targeted dead bacteria. Thus, the [ 68 Ga]Ga-DOTA-K-A9 uptake observed in vivo is presumably a combination of local hyperaemia, vascular leakiness and/or binding to an epitope present in dead bacteria. This article is protected by copyright. All rights reserved.

Rapid intracerebroventricular delivery of Cu-DOTA-etanercept after peripheral administration demonstrated by PET imaging

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Chen Xiaoyuan

2009-02-01

Full Text Available Abstract Background The cytokines interleukin-1 and tumor necrosis factor (TNF, and the cytokine blocker interleukin-1 receptor antagonist, all have been demonstrated to enter the cerebrospinal fluid (CSF following peripheral administration. Recent reports of rapid clinical improvement in patients with Alzheimer's disease and related forms of dementia following perispinal administration of etanercept, a TNF antagonist, suggest that etanercept also has the ability to reach the brain CSF. To investigate, etanercept was labeled with a positron emitter to enable visualization of its intracranial distribution following peripheral administration by PET in an animal model. Findings Radiolabeling of etanercept with the PET emitter 64Cu was performed by DOTA (1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid conjugation of etanercept, followed by column purification and 64Cu labeling. MicroPET imaging revealed accumulation of 64Cu-DOTA-etanercept within the lateral and third cerebral ventricles within minutes of peripheral perispinal administration in a normal rat anesthesized with isoflurane anesthesia, with concentration within the choroid plexus and into the CSF. Conclusion Synthesis of 64Cu-DOTA-etanercept enabled visualization of its intracranial distribution by microPET imaging. MicroPET imaging documented rapid accumulation of 64Cu-DOTA-etanercept within the choroid plexus and the cerebrospinal fluid within the cerebral ventricles of a living rat after peripheral administration. Further study of the effects of etanercept and TNF at the level of the choroid plexus may yield valuable insights into the pathogenesis of Alzheimer's disease.

radiolabeling of DOTA-substance P with 177Lu and biodistribution of 177Lu-DOTA-substance P

International Nuclear Information System (INIS)

Liang Jixin; Li Hongyu; Xiang Xueqin; Luo Zhifu; Luo Hongyi; Hu Liansheng; Chen Yang; Zhuang Ling; Deng Xinrong

2012-01-01

The aim of this project is to evaluate the biodistribution of 177 Lu-DOTA-SP in normal mice and in PANC-1 tumor bearing nude mice and to pave the way for its potentially medical application. In this study, 177 Lu-DOTA-SP was successfully prepared with labeling yield of greater than 90% at optimized conditions and more than 98% of radiochemical purity after C18 Sep-Pak purification. 177 Lu-DOTA-SP showed good stability in saline and in 5% serum while it decomposed slowly in 10% serum. Biodistribution studies in normal mice showed high uptake of 177 Lu-DOTA-SP in the kidneys, indicating the excretion mainly by renal pathway. In addition, 177 Lu-DOTA-SP was washed out from the blood quickly. Bio- distribution of 177 Lu-DOTA-SP in PANC-1 tumor bearing mice showed higher uptake in pancreatic tumor than that in normal pancreas, indicating the presence of NK-1 receptors in PANC-1 pancreatic tumor. However, from SPECT image, no radioactivity accumulation was observed in PANC-1 tumor. Further evaluation is needed to confirm its potential application for radiotherapy of pancreatic cancers. (authors)

A Rapid Detection Method of Brucella with Quantum Dots and Magnetic Beads Conjugated with Different Polyclonal Antibodies

Science.gov (United States)

Song, Dandan; Qu, Xiaofeng; Liu, Yushen; Li, Li; Yin, Dehui; Li, Juan; Xu, Kun; Xie, Renguo; Zhai, Yue; Zhang, Huiwen; Bao, Hao; Zhao, Chao; Wang, Juan; Song, Xiuling; Song, Wenzhi

2017-03-01

Brucella spp. are facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide. Traditional methods for detection of Brucella spp. take 48-72 h that does not meet the need of rapid detection. Herein, a new rapid detection method of Brucella was developed based on polyclonal antibody-conjugating quantum dots and antibody-modified magnetic beads. First, polyclonal antibodies IgG and IgY were prepared and then the antibody conjugated with quantum dots (QDs) and immunomagnetic beads (IMB), respectively, which were activated by N-(3-dimethylaminopropyl)- N'-ethylcar-bodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form probes. We used the IMB probe to separate the Brucella and labeled by the QD probe, and then detected the fluorescence intensity with a fluorescence spectrometer. The detection method takes 105 min with a limit of detection of 103 CFU/mL and ranges from 10 to 105 CFU/mL ( R 2 = 0.9983), and it can be well used in real samples.

Representasi Hero Perempuan Dalam Game Dota 2

OpenAIRE

Handoko, Sofyan; Yuwono, Elisabeth Christine; Mardiono, Bambang

2016-01-01

Dota 2 merupakan video game yang sedang berkembang di Indonesia, adalah game yang mempertarungkan pemain-pemain yang terbagi kedalam dua tim yang berbeda. Dota 2 memperlihatkan bahwa meskipun game bertema perang, terdapat karakter-karakter yang biasa disebut hero, terdapat sosok perempuan pada game Dota 2. Studi dilakukan untuk melihat apakah hero perempuan dalam game Dota 2 telah direpresentasikan sesuai dengan atribut mereka masing-masing (Strength, Agility, dan Intellegence) dan melihat ma...

Comparison of 111In-DOTA-DPhe1-Tyr3-octreotide and 111In-DOTA-lanreotide scintigraphy and dosimetry in patients with neuroendocrine tumours

International Nuclear Information System (INIS)

Rodrigues, Margarida; Virgolini, Irene; Traub-Weidinger, Tatjana; Li, Shuren; Ibi, Bettina

2006-01-01

Somatostatin receptor scintigraphy with 111 In-DOTA-DPhe 1 -Tyr 3 -octreotide ( 111 In-DOTA-TOC) and 111 In-DOTA-lanreotide ( 111 In-DOTA-LAN) has been used for staging of neuroendocrine tumours (NETs). However, the comparative diagnostic value of these radioligands on a lesion basis has not yet been established. The aim of this study was to compare the diagnostic capacity of 111 In-DOTA-TOC and 111 In-DOTA-LAN scintigraphy in patients with NETs, evaluating whether significant differences exist in lesion imaging with these radioligands. Furthermore, dosimetric data were compared. Forty-five patients with NETs were investigated with 111 In-DOTA-TOC and 111 In-DOTA-LAN scintigraphy. Scintigraphic results were compared with those of conventional imaging and/or surgery in each patient, and also 18 F-fluorodeoxyglucose positron emission tomography (FDG-PET) in 20 patients. 111 In-DOTA-TOC and 111 In-DOTA-LAN scintigraphy were true positive in 42/45 (93%) and 39/45 (87%) patients, and imaged 74/91 (81%) and 73/91 (80%) tumour lesions, respectively. 111 In-DOTA-TOC and 111 In-DOTA-LAN detected liver metastases in 21 and 14 patients, mediastinal metastases in seven and 11 patients, and bone metastases in two and seven patients, respectively. These radioligands revealed lesions not seen by conventional imaging in seven and eight patients, respectively, or by 18 F-FDG-PET in eight and seven patients, respectively. The estimated tumour absorbed doses for 90 Y-DOTA-TOC were higher than those for 90 Y-DOTA-LAN in 14 patients, whereas the opposite was true in 12 patients. Both 111 In-DOTA-TOC and 111 In-DOTA-LAN are suitable for imaging tumour lesions in patients with NETs and can detect lesions that may not be seen by conventional imaging and 18 F-FDG-PET. Compared with 111 In-DOTA-LAN, 111 In-DOTA-TOC has a superior diagnostic capacity for liver metastases, but a lower diagnostic capacity for metastatic lesions in mediastinum and bone. (orig.)

Preparation and radiolabeling of humanized anti-HER1 monoclonal antibody nimotuzumab Fab' fragment with 68Ga and 90Y

International Nuclear Information System (INIS)

Alonso Martinez, L. M.; Xiques Castillo, A.; Leyva Montanna, R.; Perez-Malo Cruz, M.; Zamora Barrabi, M.; Manresa Sanchez, Y.

2013-01-01

Antibody-based targeted delivery of radioisotopes to malignant tissues is a promising approach in cancer diagnostics and therapy. However, intact antibody molecules are large glycoproteins (∼150 kDa) that have limited application in molecular imaging and therapy due to their relatively slow clearance from the circulation leading to a high background signal rather both cases the sensitivity can be increased with the use of enzymatically produced Fab' fragments. In this work, the ability to get labeled with 62 Ga and 90 Y of a monoclonal antibody (mAb) Fab' fragment against the transmembrane receptor tyrosine kinase HER-1 was studied for future applications in PET imaging and radioimmunotherapy of tumors. In order to obtain the Fab' fragment the mAb was cleaved with pepsin in molar excess. After separating the reaction mixture in two steps using affinity and ion-exchange chromatography, the Fab' fragment was finally obtained by reduction of the F(ab') 2 with a molar excess of 2-mercaptoethanol followed by a size exclusion purification step. The Fab' fragment was derivatized with 1,4,7,10-tetraaza cyclododecane-1,4,7,10-tetraacetic acid mono N-hydroxysuccinimide commercial ester (DOTA-NHS-ester) applying a simple procedure and the number of DOTA groups linked to Fab' were determinate. The labeling of the conjugate with 68 Ga and 90 Y from 'in-house generators yielded radiochemically pure probes that can become a suitable radioimmunoconjugated in a near future. (Author)

Sensitivity-Enhancement of FRET Immunoassays by Multiple-Antibody Conjugation on Quantum Dots.

Science.gov (United States)

Annio, Giacomo; Jennings, Travis; Tagit, Oya; Hildebrandt, Niko

2018-05-23

Quantum dots (QDs) are not only advantageous for color-tuning, improved brightness, and high stability, but their nanoparticle surfaces also allow for the attachment of many biomolecules. Because IgG antibodies (ABs) are in the same size range of biocompatible QDs and the AB orientation after conjugation to the QD is often random, it is difficult to predict if few or many ABs per QD will lead to an efficient AB-QD conjugate. This is particularly true for homogeneous Förster resonance energy transfer (FRET) sandwich immunoassays, for which the ABs on the QD must bind a biomarker that needs to bind a second AB-FRET-conjugate. Here, we investigate the performance of Tb-to-QD FRET immunoassays against total prostate specific antigen (TPSA) by changing the number of ABs per QD while leaving all the other assay components unchanged. We first characterize the AB-QD conjugation by various spectroscopic, microscopic, and chromatographic techniques and then quantify the TPSA immunoassay performance regarding sensitivity, limit of detection, and dynamic range. Our results show that an increasing conjugation ratio leads to significantly enhanced FRET immunoassays. These findings will be highly important for developing QD-based immunoassays in which the concentrations of both ABs and QDs can significantly influence the assay performance.

Preparation and preclinical evaluation of 68Ga-DOTA-amlodipine for L-type calcium channel imaging

Science.gov (United States)

Firuzyar, Tahereh; Jalilian, Amir Reza; Aboudzadeh, Mohammad Reza; Sadeghpour, Hossein; Shafiee-Ardestani, Mahdi; Khalaj, Ali

2016-01-01

Aim: In order to develop a possible tracer for L-type calcium channel imaging, we here report the development of a Ga-68 amlodipine derivative for possible PET imaging. Materials and Methods: Amlodipine DOTA conjugate was synthesized, characterized and went through calcium channel blockade, toxicity, apoptosis/necrosis tests. [68Ga] DOTA AMLO was prepared at optimized conditions followed by stability tests, partition coefficient determination and biodistribution studies using tissue counting and co incidence imaging up to 2 h. Results: [68Ga] DOTA AMLO was prepared at pH 4–5 in 7–10 min at 95°C in high radiochemical purity (>99%, radio thin layer chromatography; specific activity: 1.9–2.1 GBq/mmol) and was stable up to 4 h with a log P of −0.94. Calcium channel rich tissues including myocardium, and tissues with smooth muscle cells such as colon, intestine, and lungs demonstrated significant uptake. Co incidence images supported the biodistribution data up to 2 h. Conclusions: The complex can be a candidate for further positron emission tomography imaging for L type calcium channels. PMID:27833311

Preparation and radiolabeling of a lyophilized (kit) formulation of DOTA-rituximab with ⁹⁰Y and ¹¹¹In for domestic radioimmunotherapy and radioscintigraphy of non-Hodgkin's lymphoma.

Science.gov (United States)

Gholipour, Nazila; Jalilian, Amir Reza; Khalaj, Ali; Johari-Daha, Fariba; Yavari, Kamal; Sabzevari, Omid; Khanchi, Ali Reza; Akhlaghi, Mehdi

2014-07-29

On the basis of results of our previous investigations on 90Y-DTPA-rituximab and in order to fulfil national demands to radioimmunoconjugates for radioscintigraphy and radioimmunotherapy of Non-Hodgkin's Lymphoma (NHL), preparation and radiolabeling of a lyophilized formulation (kit) of DOTA-rituximab with 111In and 90Y was investigated. 111In and 90Y with high radiochemical and radionuclide purity were prepared by 112Cd (p,2n)111In nuclear reaction and a locally developed 90Sr/90Y generator, respectively. DOTA-rituximab immunoconjugates were prepared by the reaction of solutions of p-SCN-Bz-DOTA and rituximab in carbonate buffer (pH = 9.5) and the number of DOTA per molecule of conjugates were determined by transchelation reaction between DOTA and arsenaso yttrium(III) complex. DOTA-rituximab immunoconjugates were labeled with 111In and 90Y and radioimmunoconjugates were checked for radiochemical purity by chromatography methods and for immunoreactivity by cell-binding assay using Raji cell line. The stability of radiolabeled conjugate with the approximate number of 7 DOTA molecules per one rituximab molecule which was prepared in moderate yield and showed moderate immunoreactivity, compared to two other prepared radioimmunoconjugates, was determined at different time intervals and against EDTA and human serum by chromatography methods and reducing SDS-polyacrylamide gel electrophoresis, respectively. The biodistribution of the selected radioimmunoconjugate in rats was determined by measurement of the radioactivity of different organs after sacrificing the animals by ether asphyxiation. The radioimmunoconjugate with approximate DOTA/rituximab molar ratio of 7 showed stability after 24 h at room temperature, after 96 h at 4°C, as the lyophilized formulation after six months storage and against EDTA and human serum. This radioimmunoconjugate had a biodistribution profile similar to that of 90Y-ibritumomab, which is approved by FDA for radioimmunotherapy of NHL

¹¹¹In-DOTA-Annexin V for imaging of apoptosis during HSV1-tk/GCV prodrug activation gene therapy in mice with NG4TL4 sarcoma.

Science.gov (United States)

Lin, Ming-Hsien; Wu, Shih-Yen; Wang, Hsin-Ell; Liu, Ren-Shyan; Chen, Jyh-Cheng

2016-02-01

Apoptosis has been suggested as a cytocidal mechanism of the HSV1-tk-expressing cells when exposed to ganciclovir (GCV). This study evaluated the efficacy of (111)In-labeled Annexin V for monitoring tumor responses during prodrug activation gene therapy with HSV1-tk and GCV. Annexin V was conjugated to DOTA using N-hydroxysulfosuccinimide (sulfo-NHS) and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), labeled with (111)In-InCl3 and purified using size exclusion chromatography to give (111)In-DOTA-Annexin V conjugate. The radiochemical yield and the radiochemical purity of (111)In-DOTA-Annexin V were 74±12% and 98±3%, respectively (n=10). (111)In-DOTA-BSA was prepared similarly. An in vitro study to demonstrate the apoptosis of NG4TL4-STK cells after GCV treatment has been performed. Mice bearing NG4TL4-STK and NG4TL4-WT tumors were treated with GCV (10 mg/kg daily) by i.p. injection for 7 consecutive days. Before and during the GCV treatment, biodistribution studies and scintigraphic imaging were performed at 2h post injection of the radiotracers. The uptake of (111)In-DOTA-Annexin V in treated cells (13.41±1.30%) was 4.1 times higher than that in untreated cells (3.21±0.37%). The GCV-induced cell apoptosis in NG4TL4-STK tumor resulted in a significantly increasing accumulation of (111)In-DOTA-Annexin V (1.92±0.32%ID/g at day 0, 4.79±0.86%ID/g at day 2, 4.56±0.58%ID/g at day 4) was observed, but not for that of (111)In-DOTA-BSA. During consecutive GCV treatment, scintigraphic imaging with (111)In-DOTA-Annexin V revealed high uptake in NG4TL4-STK tumor compared with that in NG4TL4-WT tumor. However, no specific (111)In-DOTA-BSA accumulation in NG4TL4-STK and NG4TL4-WT tumors was observed throughout the course of GCV treatment. This study demonstrated that (111)In-DOTA-Annexin V can be used for monitoring tumor cell apoptosis during prodrug activation gene therapy with HSV1-tk and GCV for cancer treatment. Copyright © 2015 Elsevier Ltd. All rights

68Ga-DOTA-NGR as a novel molecular probe for APN-positive tumor imaging using MicroPET.

Science.gov (United States)

Zhang, Jun; Lu, Xiaoli; Wan, Nan; Hua, Zichun; Wang, Zizheng; Huang, Hongbo; Yang, Min; Wang, Feng

2014-03-01

Aminopeptidase N (APN) is selectively expressed on many tumors and the endothelium of tumor neovasculature, and may serve as a promising target for cancer diagnosis and therapy. Asparagine-glycine-arginine (NGR) peptides have been shown to bind specifically to the APN receptor and have served as vehicles for the delivery of various therapeutic drugs in previous studies. The purpose of this study was to synthesize and evaluate the efficacy of a (68)Ga-labeled NGR peptide as a new molecular probe that binds to APN. NGR peptide was conjugated with 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) and labeled with (68)Ga at 95°C for 10 min. In vitro uptake and binding analysis was performed with A549 and MDA-MB231 cells. Biodistribution of (68)Ga-DOTA-NGR was determined in normal mice by dissection method. (68)Ga-DOTA-NGR PET was performed in A549 and MDA-MB231 xenografts, and included dynamic and static imaging. APN expression in tumors and new vasculatures was analyzed by immunohistochemistry. The radiochemical purity of (68)Ga-DOTA-NGR was 98.0% ± 1.4% with a specific activity of about 17.49 MBq/nmol. The uptake of (68)Ga-DOTA-NGR in A549 cells increased with longer incubation times, and could be blocked by cold DOTA-NGR, while no specific uptake was found in MDA-MB231 cells. In vivo biodistribution studies showed that (68)Ga-DOTA-NGR was mainly excreted from the kidney, and rapidly cleared from blood and nonspecific organs. MicroPET imaging showed that high focal accumulation had occurred in the tumor site at 1 h post-injection (pi) in A549 tumor xenografts. A significant reduction of tumor uptake was observed following coinjection with a blocking dose of DOTA-NGR, whereas only mild uptake was found in MDA-MB231 tumor xenografts. Tumor uptake, measured as the tumor/lung ratio, increased with time peaking at 12.58 ± 1.26 at 1.5 h pi. Immunohistochemical staining confirmed that APN was overexpressed on A549 cells and neovasculature. (68)Ga-DOTA

Treatment with belimumab in systemic lupus erythematosus does not impair antibody response to 13-valent pneumococcal conjugate vaccine.

Science.gov (United States)

Nagel, J; Saxne, T; Geborek, P; Bengtsson, A A; Jacobsen, S; Svaerke Joergensen, C; Nilsson, J-Å; Skattum, L; Jönsen, A; Kapetanovic, M C

2017-09-01

Background/purpose The objective of this study was to explore the impact of systemic lupus erythematosus and belimumab given in addition to standard of care therapy on 13-valent conjugated pneumococcal vaccine (PCV13) response. Methods Forty-seven systemic lupus erythematosus patients and 21 healthy controls were immunized with a single dose of 13-valent conjugated pneumococcal vaccine. Forty systemic lupus erythematosus patients were treated with traditional disease-modifying anti rheumatic drugs, 11 of those received belimumab in addition, and 32 patients were treated with concomitant prednisolone. Quantification of serotype specific IgG levels to 12 pneumococcal capsular polysaccharides was performed in serum taken before and four to six weeks after vaccination using multiplex fluorescent microsphere immunoassay. IgG levels against serotypes 23F and 6B were also analyzed using standard enzyme-linked immunosorbent assays. Opsonophagocytic assay was performed on serotype 23F to evaluate the functionality of the antibodies. Pre- and post-vaccination log transformed antibody levels were compared to determine the impact of systemic lupus erythematosus diagnosis and different treatments on antibody response. Results Systemic lupus erythematosus patients as a group showed lower post-vaccination antibody levels and lower fold increase of antibody levels after vaccination compared to controls ( p = 0.02 and p = 0.009, respectively). Systemic lupus erythematosus patients treated with belimumab in addition to standard of care therapy or with only hydroxychloroquine did not differ compared to controls, whereas the other treatment groups had significantly lower fold increase of post-vaccination antibody levels. Higher age was associated with lower post-vaccination antibody levels among systemic lupus erythematosus patients. Conclusion Belimumab given in addition to traditional disease-modifying anti rheumatic drugs or prednisolone did not further impair antibody

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

Science.gov (United States)

Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

2008-08-22

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

Visualization of Tumor Angiogenesis Using MR Imaging Contrast Agent Gd-DTPA-anti-VEGF Receptor 2 Antibody Conjugate in a Mouse Tumor Model

International Nuclear Information System (INIS)

Jun, Hong Young; Yin, Hong Hua; Kim, Sun Hee; Park, Seong Hoon; Kim, Hun Soo; Yoon Kwon Ha Yoon

2010-01-01

To visualize tumor angiogenesis using the MRI contrast agent, Gd- DTPA-anti-VEGF receptor 2 antibody conjugate, with a 4.7-Tesla MRI instrument in a mouse model. We designed a tumor angiogenesis-targeting T1 contrast agent that was prepared by the bioconjugation of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) and an anti-vascular endothelial growth factor receptor-2 (VEGFR2) antibody. The specific binding of the agent complex to cells that express VEGFR2 was examined in cultured murine endothelial cells (MS-1 cells) with a 4.7-Tesla magnetic resonance imaging scanner. Angiogenesis-specific T1 enhancement was imaged with the Gd-DTPA-anti-VEGFR2 antibody conjugate using a CT-26 adenocarcinoma tumor model in eight mice. As a control, the use of the Gd-DTPA-anti-rat immunoglobulin G (Gd-DTPA-anti-rat IgG) was imaged with a tumor model in eight mice. Statistical significance was assessed using the Mann-Whitney test. Tumor tissue was examined by immunohistochemical analysis. The Gd-DTPA-anti-VEGFR2 antibody conjugate showed predominant binding to cultured endothelial cells that expressed a high level of VEGFR2. Signal enhancement was approximately three-fold for in vivo T1-weighted MR imaging with the use of the Gd-DTPA-anti-VEGFR2 antibody conjugate as compared with the Gd-DTPA-rat IgG in the mouse tumor model (p < 0.05). VEGFR2 expression in CT-26 tumor vessels was demonstrated using immunohistochemical staining. MR imaging using the Gd-DTPA-anti-VEGFR2 antibody conjugate as a contrast agent is useful in visualizing noninvasively tumor angiogenesis in a murine tumor model

Visualization of Tumor Angiogenesis Using MR Imaging Contrast Agent Gd-DTPA-anti-VEGF Receptor 2 Antibody Conjugate in a Mouse Tumor Model

Energy Technology Data Exchange (ETDEWEB)

Jun, Hong Young; Yin, Hong Hua; Kim, Sun Hee; Park, Seong Hoon; Kim, Hun Soo; Yoon Kwon Ha Yoon [Wonkwang University School of Medicine, Iksan (Korea, Republic of)

2010-08-15

To visualize tumor angiogenesis using the MRI contrast agent, Gd- DTPA-anti-VEGF receptor 2 antibody conjugate, with a 4.7-Tesla MRI instrument in a mouse model. We designed a tumor angiogenesis-targeting T1 contrast agent that was prepared by the bioconjugation of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) and an anti-vascular endothelial growth factor receptor-2 (VEGFR2) antibody. The specific binding of the agent complex to cells that express VEGFR2 was examined in cultured murine endothelial cells (MS-1 cells) with a 4.7-Tesla magnetic resonance imaging scanner. Angiogenesis-specific T1 enhancement was imaged with the Gd-DTPA-anti-VEGFR2 antibody conjugate using a CT-26 adenocarcinoma tumor model in eight mice. As a control, the use of the Gd-DTPA-anti-rat immunoglobulin G (Gd-DTPA-anti-rat IgG) was imaged with a tumor model in eight mice. Statistical significance was assessed using the Mann-Whitney test. Tumor tissue was examined by immunohistochemical analysis. The Gd-DTPA-anti-VEGFR2 antibody conjugate showed predominant binding to cultured endothelial cells that expressed a high level of VEGFR2. Signal enhancement was approximately three-fold for in vivo T1-weighted MR imaging with the use of the Gd-DTPA-anti-VEGFR2 antibody conjugate as compared with the Gd-DTPA-rat IgG in the mouse tumor model (p < 0.05). VEGFR2 expression in CT-26 tumor vessels was demonstrated using immunohistochemical staining. MR imaging using the Gd-DTPA-anti-VEGFR2 antibody conjugate as a contrast agent is useful in visualizing noninvasively tumor angiogenesis in a murine tumor model

Development of a lyophilized formulation for preparing the radiopharmaceutical 177Lu-DOTA-Anti-CD20

International Nuclear Information System (INIS)

Serrano E, L. A.

2015-01-01

The radiolabeled proteins are molecules of interest in nuclear medicine for their diagnostic and therapeutic application in cancer. Antibodies, such as chimeric monoclonal antibody Anti-CD20 rituximab, have established themselves as suitable vectors of radionuclides (e.g. 177 Lu) , introducing high affinity by the surface antigens over- expressed and widely distributed in cells involved in certain diseases. The aim of this work was to design, optimize and document the production process of radiopharmaceutical 177 Lu-DOTA-Anti-CD20 for sanitary registration request to the Comision Federal para la Proteccion contra Riesgos Sanitarios (COFEPRIS). First, a raw material analysis using the Ft-Mir technique and gamma spectrometry was performed. Then, was carried out the development of the lyophilized formulation for the preparation of 177 Lu-DOTA-Anti-CD20, in which an ANOVA was performed where the dependent variable was the radiochemical purity. The optimal pharmaceutical formulation was: 5 mg DOTA-CD20 and 80 mg Mannitol to be reconstituted with 1 m L of acetate buffer 0.25 M, ph 7, with an incubation time of 15 min at 37 degrees Celsius in a dry bath. Once completed the development of the lyophilized formulation, we proceeded to the optimization of the production process, development and validation of the analytical method. Three batches were prepared under protocols of Good Manufacturing Practice, which met pre-established specifications as sterile and endotoxin-free of bacterial formulations, with greater that 95% of radiochemical purity. Currently, is conducting the study of shelf stability. Upon completion of the stability studies, the legal record of 177 Lu-DOTA-Anti-CD20 will be integrated with documented evidence of the quality and stability of the formulation of this radiopharmaceutical. (Author)

Experimental MR imaging with Gd-DOTA: Preliminary results

International Nuclear Information System (INIS)

Schouman-Claeys, E.; Kien, P.; Caille, J.M.; Bonnemain, B.; Frija, G.

1986-01-01

To evaluate the paramagnetic properties of a new gadolinium chelate, Gd-DOTA, in vitro and in vivo MR imaging was performed with a 0.5-T supraconductive magnet. The in vitro study consisted in measuring the MR signal obtained with various concentrations of Gd-DOTA and Gd-DTPA in different solutions. Potentialization of the paramagnetic properties of both DOTA and DTPA can be achieved by deuterium, glycerol, and protein solutions. The in vivo study was performed in rabbits with various experimental lesions. Enhancement of anatomic details was obtained with both Gd-DOTA and Gd-DTPA. There was no significant difference between Gd-DOTA and Gd-DTPA, both for in vitro and in vivo experiments. Gd-DOTA appears to be a potential paramagnetic agent for MR imaging

The ubiquitous DOTA and its derivatives: the impact of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid on biomedical imaging.

Science.gov (United States)

Stasiuk, Graeme J; Long, Nicholas J

2013-04-07

Over the last twenty-five years 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) has made a significant impact on the field of diagnostic imaging. DOTA is not the only metal chelate in use in medical diagnostics, but it is the only one to significantly impact on all of the major imaging modalities Magnetic Resonance (MR), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), and Fluorescence imaging. This crossover of modalities has been possible due to the versatility of DOTA firstly, to complex a variety of metal ions and secondly, the ease with which it can be modified for different disease states. This has driven research over the last two decades into the chemistry of DOTA and the modification of the substituent pendant arms of this macrocycle to create functional, targeted and dual-modal imaging agents. The primary use of DOTA has been with the lanthanide series of metals, gadolinium for MRI, europium and terbium for fluorescence and neodymium for near infra-red imaging. There are now many research groups dedicated to the use of lanthanides with DOTA although other chelates such as DTPA and NOTA are being increasingly employed. The ease with which DOTA can be conjugated to peptides has given rise to targeted imaging agents seen in the PET, SPECT and radiotherapy fields. These modalities use a variety of radiometals that complex with DOTA, e.g.(64)Cu and (68)Ga which are used in clinical PET scans, (111)In, and (90)Y for SPECT and radiotherapy. In this article, we will demonstrate the remarkable versatility of DOTA, how it has crossed the imaging modality boundaries and how it has been successfully transferred into the clinic.

Multiscale Modeling of Antibody Drug Conjugates: Connecting tissue and cellular distribution to whole animal pharmacokinetics and potential implications for efficacy

Science.gov (United States)

Cilliers, Cornelius; Guo, Hans; Liao, Jianshan; Christodolu, Nikolas; Thurber, Greg M.

2016-01-01

Antibody drug conjugates exhibit complex pharmacokinetics due to their combination of macromolecular and small molecule properties. These issues range from systemic concerns, such as deconjugation of the small molecule drug during the long antibody circulation time or rapid clearance from non-specific interactions, to local tumor tissue heterogeneity, cell bystander effects, and endosomal escape. Mathematical models can be used to study the impact of these processes on overall distribution in an efficient manner, and several types of models have been used to analyze varying aspects of antibody distribution including physiologically based pharmacokinetic (PBPK) models and tissue-level simulations. However, these processes are quantitative in nature and cannot be handled qualitatively in isolation. For example, free antibody from deconjugation of the small molecule will impact the distribution of conjugated antibodies within the tumor. To incorporate these effects into a unified framework, we have coupled the systemic and organ-level distribution of a PBPK model with the tissue-level detail of a distributed parameter tumor model. We used this mathematical model to analyze new experimental results on the distribution of the clinical antibody drug conjugate Kadcyla in HER2 positive mouse xenografts. This model is able to capture the impact of the drug antibody ratio (DAR) on tumor penetration, the net result of drug deconjugation, and the effect of using unconjugated antibody to drive ADC penetration deeper into the tumor tissue. This modeling approach will provide quantitative and mechanistic support to experimental studies trying to parse the impact of multiple mechanisms of action for these complex drugs. PMID:27287046

A novel antibody-drug conjugate targeting SAIL for the treatment of hematologic malignancies.

Science.gov (United States)

Kim, S Y; Theunissen, J-W; Balibalos, J; Liao-Chan, S; Babcock, M C; Wong, T; Cairns, B; Gonzalez, D; van der Horst, E H; Perez, M; Levashova, Z; Chinn, L; D'Alessio, J A; Flory, M; Bermudez, A; Jackson, D Y; Ha, E; Monteon, J; Bruhns, M F; Chen, G; Migone, T-S

2015-05-29

Although several new therapeutic approaches have improved outcomes in the treatment of hematologic malignancies, unmet need persists in acute myeloid leukemia (AML), multiple myeloma (MM) and non-Hodgkin's lymphoma. Here we describe the proteomic identification of a novel cancer target, SAIL (Surface Antigen In Leukemia), whose expression is observed in AML, MM, chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). While SAIL is widely expressed in CLL, AML, MM, DLBCL and FL patient samples, expression in cancer cell lines is mostly limited to cells of AML origin. We evaluated the antitumor activity of anti-SAIL monoclonal antibodies, 7-1C and 67-7A, conjugated to monomethyl auristatin F. Following internalization, anti-SAIL antibody-drug conjugates (ADCs) exhibited subnanomolar IC50 values against AML cell lines in vitro. In pharmacology studies employing AML cell line xenografts, anti-SAIL ADCs resulted in significant tumor growth inhibition. The restricted expression profile of this target in normal tissues, the high prevalence in different types of hematologic cancers and the observed preclinical activity support the clinical development of SAIL-targeted ADCs.

Indication of viruses and virus-specific antibodies by ELISA using conjugates based on β-lactamase obtained by genetic engineering

International Nuclear Information System (INIS)

Kharitonenkov, I.G.; Kordym, V.A.; Khristova, M.L.; Leonov, S.V.; Kirillova, V.S.; Chernykh, S.I.

1987-01-01

The method of enzyme-linked immunosorbent assay (ELISA), by means of which antigens and antibodies of different origin can be detected with high sensitivity and specificity, is an immunoenzymatic technique based on the use of conjugates, or macromolecular complexes formed by covalent attachment of enzyme molecules to antigen or antibody molecules. Conjugates based on peroxidase, alkaline phosphatase, and beta-galactosidase are most frequently used to construct immunoenzymatic test systems. The use of these enzymes in ELISA, however, is complicated by the fact that they are often present in free or bound form in the biological material under study, and that their substrates either possess low stability, are difficult to synthesize, or are toxic. In this paper, in order to avoid these shortcomings, the authors develop a method for the biosynthesis of lactamase conjugates which is based on genetic engineering, and demonstrate the viability and stability of these conjugates in radioimmunoenzymatic assay of viruses

Strong Antibody Responses Induced by Protein Antigens Conjugated onto the Surface of Lecithin-Based Nanoparticles

Science.gov (United States)

Sloat, Brian R.; Sandoval, Michael A.; Hau, Andrew M.; He, Yongqun; Cui, Zhengrong

2009-01-01

An accumulation of research over the years has demonstrated the utility of nanoparticles as antigen carriers with adjuvant activity. Herein we defined the adjuvanticity of a novel lecithin-based nanoparticle engineered from emulsions. The nanoparticles were spheres of around 200 nm. Model protein antigens, bovine serum albumin (BSA) or Bacillus anthracis protective antigen (PA) protein, were covalently conjugated onto the nanoparticles. Mice immunized with the BSA-conjugated nanoparticles developed strong anti-BSA antibody responses comparable to that induced by BSA adjuvanted with incomplete Freund's adjuvant and 6.5-fold stronger than that induced by BSA adsorbed onto aluminum hydroxide. Immunization of mice with the PA-conjugated nanoparticles elicited a quick, strong, and durable anti-PA antibody response that afforded protection of the mice against a lethal dose of anthrax lethal toxin challenge. The potent adjuvanticity of the nanoparticles was likely due to their ability to move the antigens into local draining lymph nodes, to enhance the uptake of the antigens by antigen-presenting cells (APCs), and to activate APCs. This novel nanoparticle system has the potential to serve as a universal protein-based vaccine carrier capable of inducing strong immune responses. PMID:19729045

In Vitro Cytotoxicity of Low-Dose-Rate Radioimmunotherapy by the Alpha-Emitting Radioimmunoconjugate Thorium-227-DOTA-Rituximab

International Nuclear Information System (INIS)

Dahle, Jostein; Krogh, Cecilie; Melhus, Katrine B.; Borrebaek, Jorgen; Larsen, Roy H.; Kvinnsland, Yngve

2009-01-01

Purpose: To determine whether the low-dose-rate α-particle-emitting radioimmunoconjugate 227 Th-1,4,7,10-p-isothiocyanato-benzyl-tetraazacyclododecane-1,4,7, 10-tetraacetic acid (DOTA)-rituximab can be used to inactivate lymphoma cells growing as single cells and small colonies. Methods and Materials: CD20-positive lymphoma cell lines were treated with 227 Th-DOTA-rituximab for 1-5 weeks. To simulate the in vivo situation with continuous but decreasing supply of radioimmunoconjugates from the blood pool, the cells were not washed after incubation with 227 Th-DOTA-rituximab, but half of the medium was replaced with fresh medium, and cell concentration and cell-bound activity were determined every other day after start of incubation. A microdosimetric model was established to estimate the average number of hits in the nucleus for different localizations of activity. Results: There was a specific targeted effect on cell growth of the 227 Th-DOTA-rituximab treatment. Although the cells were not washed after incubation with 227 Th-DOTA-rituximab, the average contribution of activity in the medium to the mean dose was only 6%, whereas the average contribution from activity on the cells' own surface was 78%. The mean dose rates after incubation with 800 Bq/mL 227 Th-DOTA-rituximab varied from 0.01 to 0.03 cGy/min. The average delay in growing from 10 5 to 10 7 cells/mL was 15 days when the cells were treated with a mean absorbed radiation dose of 2 Gy α-particle radiation from 227 Th-DOTA-rituximab, whereas it was 11 days when the cells were irradiated with 6 Gy of X-radiation. The relative biologic effect of the treatment was estimated to be 2.9-3.4. Conclusions: The low-dose-rate radioimmunoconjugate 227 Th-DOTA-rituximab is suitable for inactivation of single lymphoma cells and small colonies of lymphoma cells.

Synthesis of novel bifunctional chelators and their use in preparing monoclonal antibody conjugates for tumor targeting

International Nuclear Information System (INIS)

Westerberg, D.A.; Carney, P.L.; Rogers, P.E.; Kline, S.J.; Johnson, D.K.

1989-01-01

Bifunctional derivatives of the chelating agents ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid, in which a p-isothiocyanatobenzyl moiety is attached at the methylene carbon atom of one carboxymethyl arm, was synthesized by reductive alkylation of the relevant polyamine with (p-nitrophenyl)pyruvic acid followed by carboxymethylation, reduction of the nitro group, and reaction with thiophosgene. The resulting isothiocyanate derivatives reacted with monoclonal antibody B72.3 to give antibody-chelator conjugates containing 3 mol of chelator per mole of immunoglobulin, without significant loss of immunological activity. Such conjugates, labeled with the radioisotopic metal indium-111, selectively bound a human colorectal carcinoma implanted in nude mice when given intravenously. Uptake into normal tissues was comparable to or lower than that reported for analogous conjugates with known bifunctional chelators. It is concluded that substitution with a protein reactive group at this position in polyaminopolycarboxylate chelators does not alter the chelating properties of these molecules to a sufficient extent to adversely affect biodistribution and thus provides a general method for the synthesis of such chelators

Comparison of Ga-68 DOTA-TATE and Ga-68 DOTA-LAN PET/CT imaging in the same patient group with neuroendocrine tumours: preliminary results.

Science.gov (United States)

Demirci, Emre; Ocak, Meltem; Kabasakal, Levent; Araman, Ahmet; Ozsoy, Yildiz; Kanmaz, Bedii

2013-08-01

Recent studies have suggested that PET imaging with Ga-68-labelled DOTA-somatostatin analogues such as octreotide and octreotate is useful in diagnosing neuroendocrine tumours (NETs) and has superior value over both computed tomography and planar and SPECT somatostatin receptor scintigraphy. The aim of the present study was to evaluate the role of Ga-68 DOTA-lanreotide (Ga-68-DOTA-LAN) in patients with somatostatin receptor (sst)-expressing tumours and to compare the results of Ga-68 DOTA-D-Phe1-Tyr3-octreotate (Ga-68-DOTA-TATE) in the same patient population. Twelve patients with NETs who were referred to our department for somatostatin receptor scintigraphy were included in the study. There were four patients with well-differentiated neuroendocrine tumour (WDNET) grade 1, two patients with WDNET grade 2, and three patients with poorly differentiated neuroendocrine carcinoma (PDNEC) grade 3. There was also one patient with medullary thyroid cancer, one patient with meningioma and one patient with MEN-1. All patients underwent two consecutive PET imaging studies with Ga-68-DOTA-TATE and Ga-68 DOTA-LAN. All images were evaluated visually, and maximum standardized uptake value was calculated for quantitative evaluation. On visual examination of maximum intensity projection images, GA-68 DOTA-LAN was seen to have high background activity and high bone marrow uptake. Both tracers defined 67 lesions. Ga-68 DOTA-TATE images revealed 63 (94%) clearly defined lesions, missing four lesions. In contrast, Ga-68 DOTA-LAN images defined only 23 (44%) lesions, missing 44 (56%) lesions. Thirty-two bone lesions were detected on Ga-68-DOTA-TATE images. Among them, only 11 (34%) were positive on Ga-68 DOTA-LAN images, whereas 21 (66%) were negative. When we evaluated liver, mediastinum and gastrointestinal tract lesions, Ga-68 DOTA-LAN was seen to be positive for 12 (34%) lesions and negative for 23 (66%) lesions. Although the results are preliminary, the image quality obtained by

Treatment with tandem [90Y]DOTA-TATE and [177Lu]DOTA-TATE of neuroendocrine tumours refractory to conventional therapy.

Science.gov (United States)

Seregni, E; Maccauro, M; Chiesa, C; Mariani, L; Pascali, C; Mazzaferro, V; De Braud, F; Buzzoni, R; Milione, M; Lorenzoni, A; Bogni, A; Coliva, A; Lo Vullo, S; Bombardieri, E

2014-02-01

Peptide receptor radionuclide therapy (PRRT) with radiolabelled somatostatin analogues has been demonstrated to be an effective therapeutic option in patients with disseminated neuroendocrine tumours (NET). Treatment with tandem [(90)Y]DOTA-TATE and [(177)Lu]DOTA-TATE may improve the efficacy of PRRT without increasing the toxicity. In a phase II study we evaluated the feasibility of combined PPRT with a high-energy beta emitter ((90)Y) and a medium-energy beta/gamma emitter ([(177)Lu) in patients with metastatic NET refractory to conventional therapy. A group of 26 patients with metastatic NET were treated with four therapeutic cycles of alternating [[(177)Lu]DOTA-TATE (5.55 GBq) and [(90)Y]DOTA-TATE (2.6 GBq). A dosimetric evaluation was carried out after administration of [[(177)Lu]DOTA-TATE to calculate the absorbed doses in healthy organs. The acute and long-term toxicities of repeated treatment were analysed. PRRT efficacy was evaluated according to RECIST. Administration of tandem [(90)Y]DOTA-TATE and [[(177)Lu]DOTA-TATE induced objective responses in 42.3 % of patients with metastatic NET with a median progression-free survival longer than 24 months. Of patients with pretreatment carcinoid syndrome, 90 % showed a symptomatic response or a reduction in tumour-associated pain. The cumulative biologically effective doses (BED) were below the toxicity limit in the majority of patients, in the absence of renal function impairment. The results of our study indicates that combined [(90)Y]DOTA-TATE and [(177)Lu]DOTA-TATE therapy is a feasible and effective therapeutic option in NET refractory to conventional therapy. Furthermore, the absence of kidney damage and the evaluated cumulative BEDs suggest that increasing the number of tandem administrations is an interesting approach.

Design, Synthesis, and Biological Evaluation of 68Ga-DOTA-PA1 for Lung Cancer: A Novel PET Tracer for Multiple Somatostatin Receptor Imaging.

Science.gov (United States)

Liu, Fei; Liu, Teli; Xu, Xiaoxia; Guo, Xiaoyi; Li, Nan; Xiong, Chiyi; Li, Chun; Zhu, Hua; Yang, Zhi

2018-02-05

Most of the radiolabeled somatostatin analogues (SSAs) are specific for subtype somatostatin receptor 2 (SSTR 2 ). Lack of ligands targeting other subtypes of SSTRs, especially SSTR 1, SSTR 3 , and SSTR 5 , limited their applications in tumors of low SSTR 2 expression, including lung tumor. In this study, we aimed to design and synthesize a positron emission tomography (PET) radiotracer targeting multi-subtypes of SSTRs for PET imaging. PA1 peptide and its conjugate with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator or fluorescein isothiocyanate (FITC) at the N-terminal of the lysine position were synthesized. 68 Ga was chelated to DOTA-PA1 to obtain 68 Ga-DOTA-PA1 radiotracer. The stability, lipophilicity, binding affinity, and binding specificity of 68 Ga-DOTA-PA1 and FITC-PA1 were evaluated by various in vitro experiments. Micro-PET imaging of 68 Ga-DOTA-PA1 was performed in nude mice bearing A549 lung adenocarcinoma, as compared with 68 Ga-DOTA-(Tyr3)-octreotate ( 68 Ga-DOTA-TATE). Histological analysis of SSTR expression in A549 tumor tissues and human tumor tissues was conducted using immunofluorescence staining and immunohistochemical assay. 68 Ga-DOTA-PA1 had high radiochemical yield and radiochemical purity of over 95% and 99%, respectively. The radiotracer was stable in vitro in different buffers over a 2 h incubation period. Cell uptake of 68 Ga-DOTA-PA1 was 1.31-, 1.33-, and 1.90-fold that of 68 Ga-DOTA-TATE, which has high binding affinity only for SSTR 2 , after 2 h incubation in H520, PG, and A549 lung cancer cell lines, respectively. Micro-PET images of 68 Ga-DOTA-PA1 showed that the PET imaging signal correlated with the total expression of SSTRs, instead of SSTR 2 only, which was measured by Western blotting and immunofluorescence analysis in mice bearing A549 tumors. In summary, a novel PET radiotracer, 68 Ga-DOTA-PA1, targeting multi-subtypes of SSTRs, was successfully synthesized and was confirmed to be useful for PET

Dosimetric Studies in Normal Mice of 177Lu-DOTA-SP and 177Lu-DOTA-His2-MG

International Nuclear Information System (INIS)

Puerta Yepes, N.; Rojo, A.M.; Lopez Bularte, A.C.; Nevares, N.; Zapata, M.; Perez, J.H.; Crudo, J.

2010-01-01

DOTA-Substance-P (SP) and DOTA-minigastrin (His2-MG) labeled with 177 Lu could be used in peptide receptor radionuclide therapy (PRRT) for treatment of various tumour species. Biodistribution studies of both radiopharmaceuticals in normal mice were performed at different times. Absorbed doses in mouse organs were estimated and extrapolated to humans. Dosimetric calculations showed that kidneys received the highest dose, for both radiopharmaceuticals. The Maximum Tolerated Activity (MTA) of 177 Lu-DOTA-SP that can be administered without kidney toxicity are 414 and 422 MBq/kg for the standard adult man and woman, respectively. In the same way, the MTA of 177 Lu-DOTA-His2-MG are 488 and 518 MBq/kg for the standard adult man and woman, respectively. (authors)

68Ga-DOTA-TOC Uptake in Pleomorphic Adenoma.

Science.gov (United States)

Laurens, S Tom; Netea-Maier, Romana T; Aarntzen, Erik J H G

2018-07-01

A 56-year-old man who was recently diagnosed with a carcinoid tumor of the os petrosum was referred for a Ga-DOTA-TOC PET/CT scan. Besides the moderately increased Ga-DOTA-TOC accumulation in the carcinoid tumor, the scan showed strongly increased and focal Ga-DOTA-TOC uptake in an additional lesion in the right parotid gland. The markedly different Ga-DOTA-TOC avidity suggested a different etiology, and histological examination demonstrated a pleomorphic adenoma.

Optimization of a methamphetamine conjugate vaccine for antibody production in mice.

Science.gov (United States)

Stevens, Misty W; Gunnell, Melinda G; Tawney, Rachel; Owens, S Michael

2016-06-01

There are still no approved medications for treating patients who abuse methamphetamine. Active vaccines for treating abuse of nicotine and cocaine are in clinical studies, but have not proven effective seemingly due to inadequate anti-drug antibody production. The current studies aimed to optimize the composition, adjuvant and route of administration of a methamphetamine conjugate vaccine, ICKLH-SMO9, in mice with the goal of generating significantly higher antibody levels. A range of hapten epitope densities were compared, as were the adjuvants Alhydrogel and a new Toll-like receptor 4 (TLR4) agonist called GLA-SE. While methamphetamine hapten density did not strongly affect the antibody response, the adjuvant did. Glucopyranosyl lipid A in a stable oil-in-water emulsion (GLA-SE) produced much higher levels of antibody in response to immunization compared with Alhydrogel; immunization with GLA-SE also produced antibodies with higher affinities for methamphetamine. GLA-SE has been used in human studies of vaccines for influenza among others and like some other clinical TLR4 agonists, it is safe and elicits a strong immune response. GLA-SE adjuvanted vaccines are typically administered by intramuscular injection and this also proved effective in these mouse studies. Clinical studies of the ICKLH-SMO9 methamphetamine vaccine adjuvanted with GLA-SE have the potential for demonstrating efficacy by generating much higher levels of antibody than substance abuse vaccines that have unsuccessfully used aluminum-based adjuvants. Copyright © 2016 Elsevier B.V. All rights reserved.

Assembly of High-Potency Photosensitizer-Antibody Conjugates through Application of Dendron Multiplier Technology.

Science.gov (United States)

Bryden, Francesca; Maruani, Antoine; Rodrigues, João M M; Cheng, Miffy H Y; Savoie, Huguette; Beeby, Andrew; Chudasama, Vijay; Boyle, Ross W

2018-01-17

Exploitation of photosensitizers as payloads for antibody-based anticancer therapeutics offers a novel alternative to the small pool of commonly utilized cytotoxins. However, existing bioconjugation methodologies are incompatible with the requirement of increased antibody loading without compromising antibody function, stability, or homogeneity. Herein, we describe the first application of dendritic multiplier groups to allow the loading of more than 4 porphyrins to a full IgG antibody in a site-specific and highly homogeneous manner. Photophysical evaluation of UV-visible absorbance and singlet oxygen quantum yields highlighted porphyrin-dendron 14 as the best candidate for bioconjugation; with subsequent bioconjugation producing a HER2-targeted therapeutic with average loading ratios of 15.4:1. In vitro evaluation of conjugate 18 demonstrated a nanomolar photocytotoxic effect in a target cell line, which overexpresses HER2, with no observed photocytotoxicity at the same concentration in a control cell line which expresses native HER2 levels, or in the absence of irradiation with visible light.

Development of Fluorophore-Labeled Thailanstatin Antibody-Drug Conjugates for Cellular Trafficking Studies.

Science.gov (United States)

Kulkarni, Chethana; Finley, James E; Bessire, Andrew J; Zhong, Xiaotian; Musto, Sylvia; Graziani, Edmund I

2017-04-19

As the antibody-drug conjugate (ADC) field grows increasingly important for cancer treatment, it is vital for researchers to establish a firm understanding of how ADCs function at the molecular level. To gain insight into ADC uptake, trafficking, and catabolism-processes that are critical to ADC efficacy and toxicity-imaging studies have been performed with fluorophore-labeled conjugates. However, such labels may alter the properties and behavior of the ADC under investigation. As an alternative approach, we present here the development of a "clickable" ADC bearing an azide-functionalized linker-payload (LP) poised for "click" reaction with alkyne fluorophores; the azide group represents a significantly smaller structural perturbation to the LP than most fluorophores. Notably, the clickable ADC shows excellent potency in target-expressing cells, whereas the fluorophore-labeled product ADC suffers from a significant loss of activity, underscoring the impact of the label itself on the payload. Live-cell confocal microscopy reveals robust uptake of the clickable ADC, which reacts selectively in situ with a derivatized fluorescent label. Time-course trafficking studies show greater and more rapid net internalization of the ADCs than the parent antibody. More generally, the application of chemical biology tools to the study of ADCs should improve our understanding of how ADCs are processed in biological systems.

Targeting of [[sup 111]In]biocytin to cultured ovarian adenocarcinoma cells using covalent monoclonal antibody -streptavidin conjugates

Energy Technology Data Exchange (ETDEWEB)

Sheldon, K.; Marks, A. (Toronto Univ., ON (Canada). Banting and Best Dept. of Medical Research); Baumal, R. (Hospital for Sick Children, Toronto, ON (Canada). Dept. of Pathology)

1992-11-01

Three monoclonal antibodies (mAb) directed against the human ovarian adenocarcinoma cell line HEY, were substituted with maleimide and covalently bonded to thiolated streptavidin. The conjugates were separated from unreacted reagents by successive affinity chromatography on protein A-Sepharose and iminobiotin columns. Purified conjugates consisted of an immunoglobulin (Ig) monomer bound to a streptavidin tetramer through a covalent bond between the Ig molecule and one of the streptavidin subunits. The conjugates were able to specifically target [[sup 111]In]biocytin to HEY cells in vitro in the presence of human serum and ascitic fluid from ovarian cancer patients. (Author).

Monoclonal antibody-dendrimer conjugates enable radiolabeling of antibody with markedly high specific activity with minimal loss of immunoreactivity

Energy Technology Data Exchange (ETDEWEB)

Kobayashi, H.; Togashi, K. [Kyoto Univ. (Japan). School of Medicine; Sato, N.; Saga, T.; Nakamoto, Y.; Ishimori, T.; Konishi, J. [Dept. of Nuclear Medicine and Medical Imaging, Kyoto Univ. Graduate School of Medicine, Kyoto (Japan); Toyama, S. [Inst. for Virus Research, Kyoto Univ., Kyoto (Japan); Brechbiel, M.W. [Chemistry Section, Radiation Oncology Branch, National Cancer Inst., National Inst. of Health, Bethesda, Md. (United States)

2000-09-01

For the purpose of radioimmunotherapy, labelling of monoclonal antibody with high specific activity is often necessary, especially when using a radionuclide with a shorter half-life. Polyamine dendrimers (PAMAM) are novel synthetic polymeric molecules with large numbers of amine residues on their spherical surface. In order to bind large numbers of radiometals to single antibody molecules, the generation-4 PAMAM (G4), which has 64 amines, was conjugated with 43 molecules of 2-(p-isothiocyanatobenzyl)-6-methyl-diethylene triamine penta-acetic acid (1B4M), a derivative of DTPA. This product [G4-(1B4M){sub 43}] was then conjugated with OST7, a murine monoclonal IgG{sub 1}. We evaluated the achievable specific activity for {sup 111}In labeling, immunore-activity, biodistribution, and tumor targeting in mice of the {sup 111}In- or {sup 153}Gd-OST7-G4-(1B4M){sub 43} as compared with radiolabeled OST7-1B4M or 56C-1B4M. The maximum specific activity of {sup 111}In-OST7-G4-(1B4M){sub 43} and {sup 111}In-OST7-1B4M was 470 and 8.7 GBq/mg (12,700 and 263 mCi/mg), respectively. Immunoreactivity of radiolabeled OST7-G4-(1B4M){sub 43} and OST7-1B4M, as determined by the binding to KT005 cells expressing the antigen, was respectively 91% and 84% of that of {sup 125}I-labelled OST7. Biodistribution studies for preparations with maximum specific activity in normal mice 3 h after injection showed that {sup 111}In- or {sup 153}Gd-OST7-G4-(1B4M){sub 43} cleared faster from the blood and accumulated more in the liver than did {sup 111}In- or {sup 153}Gd-OST7-1B4M. The dendrimer 1B4M [G4-(1B4M){sub 64}] itself showed similar saturation effects with metals. The radioactivity in all the other organs reflected the rapid clearance of radioactivity from the blood. {sup 153}Gd-OST7-G4-(1B4M){sub 43} showed specific accumulation in the KT005 tumor. In conclusion, we could successfully bind 49 times as many metal atoms to an antibody molecule as is possible with conventional metal labeling for

Improved Tumor Penetration and Single-Cell Targeting of Antibody-Drug Conjugates Increases Anticancer Efficacy and Host Survival.

Science.gov (United States)

Cilliers, Cornelius; Menezes, Bruna; Nessler, Ian; Linderman, Jennifer; Thurber, Greg M

2018-02-01

Current antibody-drug conjugates (ADC) have made advances in engineering the antibody, linker, conjugation site, small-molecule payload, and drug-to-antibody ratio (DAR). However, the relationship between heterogeneous intratumoral distribution and efficacy of ADCs is poorly understood. Here, we compared trastuzumab and ado-trastuzumab emtansine (T-DM1) to study the impact of ADC tumor distribution on efficacy. In a mouse xenograft model insensitive to trastuzumab, coadministration of trastuzumab with a fixed dose of T-DM1 at 3:1 and 8:1 ratios dramatically improved ADC tumor penetration and resulted in twice the improvement in median survival compared with T-DM1 alone. In this setting, the effective DAR was lowered, decreasing the amount of payload delivered to each targeted cell but increasing the number of cells that received payload. This result is counterintuitive because trastuzumab acts as an antagonist in vitro and has no single-agent efficacy in vivo , yet improves the effectiveness of T-DM1 in vivo Novel dual-channel fluorescence ratios quantified single-cell ADC uptake and metabolism and confirmed that the in vivo cellular dose of T-DM1 alone exceeded the minimum required for efficacy in this model. In addition, this technique characterized cellular pharmacokinetics with heterogeneous delivery after 1 day, degradation and payload release by 2 days, and in vitro cell killing and in vivo tumor shrinkage 2 to 3 days later. This work demonstrates that the intratumoral distribution of ADC, independent of payload dose or plasma clearance, plays a major role in ADC efficacy. Significance: This study shows how lowering the drug-to-antibody ratio during treatment can improve the intratumoral distribution of a antibody-drug conjugate, with implications for improving the efficacy of this class of cancer drugs. Cancer Res; 78(3); 758-68. ©2017 AACR . ©2017 American Association for Cancer Research.

Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies

OpenAIRE

Davidson, Edgar; Bryan, Christopher; Fong, Rachel H.; Barnes, Trevor; Pfaff, Jennifer M.; Mabila, Manu; Rucker, Joseph B.; Doranz, Benjamin J.

2015-01-01

Cocktails of monoclonal antibodies (MAbs) that target the surface glycoprotein (GP) of Ebola virus (EBOV) are effective in nonhuman primate models and have been used under emergency compassionate-treatment protocols in human patients. However, the amino acids that form the detailed binding epitopes for the MAbs in the ZMapp, ZMAb, and the related MB-003 cocktails have yet to be identified. Other binding properties that define how each MAb functionally interacts with GP—such as affinity, epito...

Localized conformational interrogation of antibody and antibody-drug conjugates by site-specific carboxyl group footprinting.

Science.gov (United States)

Pan, Lucy Yan; Salas-Solano, Oscar; Valliere-Douglass, John F

Establishing and maintaining conformational integrity of monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) during development and manufacturing is critical for ensuring their clinical efficacy. As presented here, we applied site-specific carboxyl group footprinting (CGF) for localized conformational interrogation of mAbs. The approach relies on covalent labeling that introduces glycine ethyl ester tags onto solvent-accessible side chains of protein carboxylates. Peptide mapping is used to monitor the labeling kinetics of carboxyl residues and the labeling kinetics reflects the conformation or solvent-accessibility of side chains. Our results for two case studies are shown here. The first study was aimed at defining the conformational changes of mAbs induced by deglycosylation. We found that two residues in C H 2 domain (D268 and E297) show significantly enhanced side chain accessibility upon deglycosylation. This site-specific result highlighted the advantage of monitoring the labeling kinetics at the amino acid level as opposed to the peptide level, which would result in averaging out of highly localized conformational differences. The second study was designed to assess conformational effects brought on by conjugation of mAbs with drug-linkers. All 59 monitored carboxyl residues displayed similar solvent-accessibility between the ADC and mAb under native conditions, which suggests the ADC and mAb share similar side chain conformation. The findings are well correlated and complementary with results from other assays. This work illustrated that site-specific CGF is capable of pinpointing local conformational changes in mAbs or ADCs that might arise during development and manufacturing. The methodology can be readily implemented within the industry to provide comprehensive conformational assessment of these molecules.

New peptide receptor radionuclide therapy of invasive cancer cells: in vivo studies using 177Lu-DOTA-AE105 targeting uPAR in human colorectal cancer xenografts

DEFF Research Database (Denmark)

Persson, Morten; Rasmussen, Palle; Madsen, Jacob

2012-01-01

-of-concept for a theranostic approach as treatment modality in a human xenograft colorectal cancer model. MethodsA DOTA-conjugated 9-mer high affinity uPAR binding peptide (DOTA-AE105) was radiolabeled with 64Cu and 177Lu, for PET imaging and targeted radionuclide therapy study, respectively. Human uPAR-positive CRC HT-29...... for the first time the in vivo efficacy of an uPAR-targeted radionuclide therapeutic intervention on both tumor size and its content of uPAR expressing cells thus setting the stage for future translation into clinical use. © 2012 Elsevier Inc. All rights reserved....

Emergence of Ebola Virus Escape Variants in Infected Nonhuman Primates Treated with the MB-003 Antibody Cocktail

Directory of Open Access Journals (Sweden)

Jeffrey R. Kugelman

2015-09-01

Full Text Available MB-003, a plant-derived monoclonal antibody cocktail used effectively in treatment of Ebola virus infection in non-human primates, was unable to protect two of six animals when initiated 1 or 2 days post-infection. We characterized a mechanism of viral escape in one of the animals, after observation of two clusters of genomic mutations that resulted in five nonsynonymous mutations in the monoclonal antibody target sites. These mutations were linked to a reduction in antibody binding and later confirmed to be present in a viral isolate that was not neutralized in vitro. Retrospective evaluation of a second independent study allowed the identification of a similar case. Four SNPs in previously identified positions were found in this second fatality, suggesting that genetic drift could be a potential cause for treatment failure. These findings highlight the importance selecting different target domains for each component of the cocktail to minimize the potential for viral escape.

Evaluation of (64)Cu-labeled DOTA-D-Phe(1)-Tyr (3)-octreotide ((64)Cu-DOTA-TOC) for imaging somatostatin receptor-expressing tumors.

Science.gov (United States)

Hanaoka, Hirofumi; Tominaga, Hideyuki; Yamada, Keiich; Paudyal, Pramila; Iida, Yasuhiko; Watanabe, Shigeki; Paudyal, Bishnuhari; Higuchi, Tetsuya; Oriuchi, Noboru; Endo, Keigo

2009-08-01

In-111 ((111)In)-labeled octreotide has been clinically used for imaging somatostatin receptor-positive tumors, and radiolabeled octreotide analogs for positron emission tomography (PET) have been developed. Cu-64 ((64)Cu; half-life, 12.7 h) is an attractive radionuclide for PET imaging and is produced with high specific activity using a small biomedical cyclotron. The aim of this study is to produce and fundamentally examine a (64)Cu-labeled octreotide analog, (64)Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-D: -Phe(1)-Tyr(3)-octreotide ((64)Cu-DOTA-TOC). (64)Cu produced using a biomedical cyclotron was reacted with DOTA-TOC for 30 min at 45 degrees C. The stability of (64)Cu-DOTA-TOC was evaluated in vitro (incubated with serum) and in vivo (blood collected after administration) by HPLC analysis. Biodistribution studies were performed in normal mice by administration of mixed solution of (64)Cu-DOTA-TOC and (111)In-DOTA-TOC and somatostatin receptor-positive U87MG tumor-bearing mice by administration of (64)Cu-DOTA-TOC or (64)Cu-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid-octreotide ((64)Cu-TETA-OC). The tumor was imaged using (64)Cu-DOTA-TOC, (64)Cu-TETA-OC, and FDG with an animal PET scanner. (64)Cu-DOTA-TOC can be produced in amounts sufficient for clinical study with high radiochemical yield. (64)Cu-DOTA-TOC was stable in vitro, but time-dependent transchelation to protein was observed after injection into mice. In biodistribution studies, the radioactivity of (64)Cu was higher than that of (111)In in all organs except kidney. In tumor-bearing mice, (64)Cu-DOTA-TOC showed a high accumulation in the tumor, and the tumor-to-blood ratio reached as high as 8.81 +/- 1.17 at 6 h after administration. (64)Cu-DOTA-TOC showed significantly higher accumulation in the tumor than (64)Cu-TETA-OC. (64)Cu-DOTA-TOC PET showed a very clear image of the tumor, which was comparable to that of (18)F-FDG PET and very similar to that of (64)Cu

Evaluation of 64Cu-labeled DOTA-D-Phe1-Tyr3-octreotide (64Cu-DOTA-TOC) for imaging somatostatin receptor-expressing tumors

International Nuclear Information System (INIS)

Hanaoka, Hirofumi; Tominaga, Hideyuki; Yamada, Keiich

2009-01-01

In-111 ( 111 In)-labeled octreotide has been clinically used for imaging somatostatin receptor-positive tumors, and radiolabeled octreotide analogs for positron emission tomography (PET) have been developed. Cu-64 ( 64 Cu; half-life, 12.7 h) is an attractive radionuclide for PET imaging and is produced with high specific activity using a small biomedical cyclotron. The aim of this study is to produce and fundamentally examine a 64 Cu-labeled octreotide analog, 64 Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-D-Phe 1 -Tyr 3 -octreotide ( 64 Cu-DOTA-TOC). 64 Cu produced using a biomedical cyclotron was reacted with DOTA-TOC for 30 min at 45 deg C. The stability of 64 Cu-DOTA-TOC was evaluated in vitro (incubated with serum) and in vivo (blood collected after administration) by high performance liquid chromatography (HPLC) analysis. Biodistribution studies were performed in normal mice by administration of mixed solution of 64 Cu-DOTA-TOC and 111 In-DOTA-TOC and somatostatin receptor-positive U87MG tumor-bearing mice by administration of 64 Cu-DOTA-TOC or 64 Cu-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid-octreotide ( 64 Cu-TETA-OC). The tumor was imaged using 64 Cu-DOTA-TOC, 64 Cu-TETA-OC, and fluorodeoxyglucose (FDG) with an animal PET scanner. 64 Cu-DOTA-TOC can be produced in amounts sufficient for clinical study with high radiochemical yield. 64 Cu-DOTA-TOC was stable in vitro, but time-dependent transchelation to protein was observed after injection into mice. In biodistribution studies, the radioactivity of 64 Cu was higher than that of 111 In in all organs except kidney. In tumor-bearing mice, 64 Cu-DOTA-TOC showed a high accumulation in the tumor, and the tumor-to-blood ratio reached as high as 8.81±1.17 at 6 h after administration. 64 Cu-DOTA-TOC showed significantly higher accumulation in the tumor than 64 Cu-TETA-OC. 64 Cu-DOTA-TOC PET showed a very clear image of the tumor, which was comparable to that of 18 F-FDG PET and

Kinetics of antibody responses after primary immunization with meningococcal serogroup C conjugate vaccine or secondary immunization with either conjugate or polysaccharide vaccine in adults

NARCIS (Netherlands)

de Voer, Richarda M.; van der Klis, Fiona R. M.; Engels, Carla W. A. M.; Schepp, Rutger M.; van de Kassteele, Jan; Sanders, Elisabeth A. M.; Rijkers, Ger T.; Berbers, Guy A. M.

2009-01-01

In the Netherlands the meningococcal serogroup C conjugate (MenCC) vaccine is administered as a single dose at 14 months. We evaluated the kinetics of isotype-specific antibodies in adults (n = 21) after primary immunization with MenCC or secondary immunization with MenCC or plain MenC

In vitro and in vivo imaging of prostate cancer angiogenesis using anti-vascular endothelial growth factor receptor 2 antibody-conjugated quantum dot

International Nuclear Information System (INIS)

Kwon, Haejin; Lee, Jiyeon; Song, Rita; Lee, Jung Han; Hwang, Sung Il; Lee, Hak Jong; Kim, Young Hwa

2013-01-01

Authors aimed to determine the targeting ability of vascular endothelial growth factor receptor 2 (VEGFR2)-conjugated quantum dots (QDs) in vitro, and apply it for a xenograft prostate cancer mouse model. Conjugation reaction of QDs was performed by using the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and sulfo-(N-hydroxysulfosuccinimide) (Sulfo-NHS). The human umbilical vein cord endothelial cells (HUVECs) were incubated with QDs, conjugated with antiVGFR2, to see a specific binding in vitro. Fluorescent cell images were taken by a confocal microscope. The human prostate cancer cells (PC3) were injected to five nude mice on hind limbs to make the xenograft tumor model. QD-antiVEGFR2 antibody complex was injected into the tumor model and fluorescence measurements were performed at 1, 4, 9, 12, 15, and 24 hours after the injection. The specific interaction between HUVECs and QD-antiVEGFR2 antibody was clearly shown in vitro. The in vivo fluorescence image disclosed that there was an increased signal of tumor, 12 hours after the injection of QDs. By showing endothelial cells binding with QDs-antiVEGFR2 antibodyand an experimental application of the antibody for VEGFR2 imaging in the prostate cancer xenograft mouse model, we suggests that the antibody-conjugated QDs can be a potential imaging tool for angiogenesis of the cancer.

Comparison of {sup 111}In-DOTA-DPhe{sup 1}-Tyr{sup 3}-octreotide and {sup 111}In-DOTA-lanreotide scintigraphy and dosimetry in patients with neuroendocrine tumours

Energy Technology Data Exchange (ETDEWEB)

Rodrigues, Margarida; Virgolini, Irene [Lainz Hospital, Institute of Nuclear Medicine, Vienna (Austria); University Clinic for Nuclear Medicine, Innsbruck (Austria); Traub-Weidinger, Tatjana [University Clinic for Nuclear Medicine, Innsbruck (Austria); University Hospital, Department of Nuclear Medicine, Vienna (Austria); Li, Shuren [University Hospital, Department of Nuclear Medicine, Vienna (Austria); Ibi, Bettina [Lainz Hospital, Institute of Physics, Vienna (Austria)

2006-05-15

Somatostatin receptor scintigraphy with {sup 111}In-DOTA-DPhe{sup 1}-Tyr{sup 3}-octreotide ({sup 111}In-DOTA-TOC) and {sup 111}In-DOTA-lanreotide ({sup 111}In-DOTA-LAN) has been used for staging of neuroendocrine tumours (NETs). However, the comparative diagnostic value of these radioligands on a lesion basis has not yet been established. The aim of this study was to compare the diagnostic capacity of {sup 111}In-DOTA-TOC and {sup 111}In-DOTA-LAN scintigraphy in patients with NETs, evaluating whether significant differences exist in lesion imaging with these radioligands. Furthermore, dosimetric data were compared. Forty-five patients with NETs were investigated with {sup 111}In-DOTA-TOC and {sup 111}In-DOTA-LAN scintigraphy. Scintigraphic results were compared with those of conventional imaging and/or surgery in each patient, and also {sup 18}F-fluorodeoxyglucose positron emission tomography (FDG-PET) in 20 patients. {sup 111}In-DOTA-TOC and {sup 111}In-DOTA-LAN scintigraphy were true positive in 42/45 (93%) and 39/45 (87%) patients, and imaged 74/91 (81%) and 73/91 (80%) tumour lesions, respectively. {sup 111}In-DOTA-TOC and {sup 111}In-DOTA-LAN detected liver metastases in 21 and 14 patients, mediastinal metastases in seven and 11 patients, and bone metastases in two and seven patients, respectively. These radioligands revealed lesions not seen by conventional imaging in seven and eight patients, respectively, or by {sup 18}F-FDG-PET in eight and seven patients, respectively. The estimated tumour absorbed doses for {sup 90}Y-DOTA-TOC were higher than those for {sup 90}Y-DOTA-LAN in 14 patients, whereas the opposite was true in 12 patients. Both {sup 111}In-DOTA-TOC and {sup 111}In-DOTA-LAN are suitable for imaging tumour lesions in patients with NETs and can detect lesions that may not be seen by conventional imaging and {sup 18}F-FDG-PET. Compared with {sup 111}In-DOTA-LAN, {sup 111}In-DOTA-TOC has a superior diagnostic capacity for liver metastases, but a

Treatment with tandem [{sup 90}Y]DOTA-TATE and [{sup 177}Lu]DOTA-TATE of neuroendocrine tumours refractory to conventional therapy

Energy Technology Data Exchange (ETDEWEB)

Seregni, E.; Maccauro, M.; Chiesa, C.; Pascali, C.; Lorenzoni, A.; Bogni, A.; Coliva, A.; Bombardieri, E. [Fondazione IRCCS Istituto Nazionale Tumori, Nuclear Medicine, Milan (Italy); Mariani, L.; Vullo, S.Lo [Fondazione IRCCS Istituto Nazionale Tumori, Statistics and Biometry Unit, Milan (Italy); Mazzaferro, V. [Fondazione IRCCS Istituto Nazionale Tumori, Surgery and Liver Transplantation, Milan (Italy); De Braud, F.; Buzzoni, R. [Fondazione IRCCS Istituto Nazionale Tumori, Medical Oncology, Milan (Italy); Milione, M. [Fondazione IRCCS Istituto Nazionale Tumori, Pathology Department, Milan (Italy)

2014-02-15

Peptide receptor radionuclide therapy (PRRT) with radiolabelled somatostatin analogues has been demonstrated to be an effective therapeutic option in patients with disseminated neuroendocrine tumours (NET). Treatment with tandem [{sup 90}Y]DOTA-TATE and [{sup 177}Lu]DOTA-TATE may improve the efficacy of PRRT without increasing the toxicity. In a phase II study we evaluated the feasibility of combined PPRT with a high-energy beta emitter ({sup 90}Y) and a medium-energy beta/gamma emitter ({sup 177}Lu) in patients with metastatic NET refractory to conventional therapy. A group of 26 patients with metastatic NET were treated with four therapeutic cycles of alternating [{sup 177}Lu]DOTA-TATE (5.55 GBq) and [{sup 90}Y]DOTA-TATE (2.6 GBq). A dosimetric evaluation was carried out after administration of [{sup 177}Lu]DOTA-TATE to calculate the absorbed doses in healthy organs. The acute and long-term toxicities of repeated treatment were analysed. PRRT efficacy was evaluated according to RECIST. Administration of tandem [{sup 90}Y]DOTA-TATE and [{sup 177}Lu]DOTA-TATE induced objective responses in 42.3 % of patients with metastatic NET with a median progression-free survival longer than 24 months. Of patients with pretreatment carcinoid syndrome, 90 % showed a symptomatic response or a reduction in tumour-associated pain. The cumulative biologically effective doses (BED) were below the toxicity limit in the majority of patients, in the absence of renal function impairment The results of our study indicates that combined [{sup 90}Y]DOTA-TATE and [{sup 177}Lu]DOTA-TATE therapy is a feasible and effective therapeutic option in NET refractory to conventional therapy. Furthermore, the absence of kidney damage and the evaluated cumulative BEDs suggest that increasing the number of tandem administrations is an interesting approach. (orig.)

Hyperpolarized 89Y NMR spectroscopic detection of yttrium ion and DOTA macrocyclic ligand complexation: pH dependence and Y-DOTA intermediates

Science.gov (United States)

Ferguson, Sarah; Kiswandhi, Andhika; Niedbalski, Peter; Parish, Christopher; Kovacs, Zoltan; Lumata, Lloyd

Dissolution dynamic nuclear polarization (DNP) is a rapidly emerging physics technique used to enhance the signal strength in nuclear magnetic resonance (NMR) and imaging (MRI) experiments for nuclear spins such as yttrium-89 by >10,000-fold. One of the most common and stable MRI contrast agents used in the clinic is Gd-DOTA. In this work, we have investigated the binding of the yttrium and DOTA ligand as a model for complexation of Gd ion and DOTA ligand. The macrocyclic ligand DOTA is special because its complexation with lanthanide ions such as Gd3+ or Y3+ is highly pH dependent. Using this physics technology, we have tracked the complexation kinetics of hyperpolarized Y-triflate and DOTA ligand in real-time and detected the Y-DOTA intermediates. Different kinds of buffers were used (lactate, acetate, citrate, oxalate) and the pseudo-first order complexation kinetic calculations will be discussed. The authors would like to acknowledge the support by US Dept of Defense Award No. W81XWH-14-1-0048 and Robert A. Welch Foundation Grant No. AT-1877.

The added value of 68Ga-DOTA-TATE-PET to contrast-enhanced CT for primary site detection in CUP of neuroendocrine origin.

Science.gov (United States)

Kazmierczak, Philipp M; Rominger, Axel; Wenter, Vera; Spitzweg, Christine; Auernhammer, Christoph; Angele, Martin K; Rist, Carsten; Cyran, Clemens C

2017-04-01

To quantify the additional value of 68 Ga-DOTA-TATE PET/CT in comparison with contrast-enhanced CT alone for primary tumour detection in neuroendocrine cancer of unknown primary (CUP-NET). In total, 38 consecutive patients (27 men, 11 women; mean age 62 years) with histologically proven CUP-NET who underwent a contrast-enhanced 68 Ga-DOTA-TATE PET/CT scan for primary tumour detection and staging between 2010 and 2014 were included in this IRB-approved retrospective study. Two blinded readers independently analysed the contrast-enhanced CT and 68 Ga-DOTA-TATE PET datasets separately and noted from which modality they suspected a primary tumour. Consensus was reached if the results were divergent. Postoperative histopathology (24 patients) and follow-up 68 Ga-DOTA-TATE PET/CT imaging (14 patients) served as the reference standards and statistical measures of diagnostic accuracy were calculated accordingly. The majority of confirmed primary tumours were located in the abdomen (ileum in 19 patients, pancreas in 12, lung in 2, small pelvis in 1). High interobserver agreement was noted regarding the suspected primary tumour site (Cohen's k 0.90, p DOTA-TATE PET demonstrated a significantly higher sensitivity (94 % vs. 63 %, p = 0.005) and a significantly higher accuracy (87 % vs. 68 %, p = 0.003) than contrast-enhanced CT. Ga-DOTA-TATE PET/CT compared with contrast-enhanced CT alone provides an improvement in sensitivity of 50 % and an improvement in accuracy of 30 % in primary tumour detection in CUP-NET. • 68 Ga-DOTA-TATE PET augments the sensitivity of contrast-enhanced CT by 50 % • 68 Ga-DOTA-TATE PET augments the accuracy of contrast-enhanced CT by 30 % • Somatostatin receptor-targeted hybrid imaging optimizes primary tumour detection in CUP-NET.

Comparative evaluation of synthetic anti-HER2 Affibody molecules site-specifically labelled with {sup 111}In using N-terminal DOTA, NOTA and NODAGA chelators in mice bearing prostate cancer xenografts

Energy Technology Data Exchange (ETDEWEB)

Malmberg, Jennie; Varasteh, Zohreh; Orlova, Anna [Uppsala University, Preclinical PET Platform, Department of Medicinal Chemistry, Uppsala (Sweden); Perols, Anna; Braun, Alexis; Eriksson Karlstroem, Amelie [AlbaNova University Centre, Division of Molecular Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm (Sweden); Altai, Mohamed; Tolmachev, Vladimir [Uppsala University, Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala (Sweden); Sandstroem, Mattias [Uppsala University Hospital, Section of Medical Physics, Department of Oncology, Uppsala (Sweden); Garske, Ulrike [Uppsala University Hospital, Department of Medical Sciences, Section of Nuclear Medicine, Uppsala (Sweden)

2012-03-15

In disseminated prostate cancer, expression of human epidermal growth factor receptor type 2 (HER2) is one of the pathways to androgen independence. Radionuclide molecular imaging of HER2 expression in disseminated prostate cancer might identify patients for HER2-targeted therapy. Affibody molecules are small (7 kDa) targeting proteins with high potential as tracers for radionuclide imaging. The goal of this study was to develop an optimal Affibody-based tracer for visualization of HER2 expression in prostate cancer. A synthetic variant of the anti-HER2 Z{sub HER2:342} Affibody molecule, Z{sub HER2:S1}, was N-terminally conjugated with the chelators DOTA, NOTA and NODAGA. The conjugated proteins were biophysically characterized by electrospray ionization mass spectroscopy (ESI-MS), circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR)-based biosensor analysis. After labelling with {sup 111}In, the biodistribution was assessed in normal mice and the two most promising conjugates were further evaluated for tumour targeting in mice bearing DU-145 prostate cancer xenografts. The HER2-binding equilibrium dissociation constants were 130, 140 and 90 pM for DOTA-Z{sub HER2:S1}, NOTA-Z{sub HER2:S1} and NODAGA-Z{sub HER2:S1}, respectively. A comparative study of {sup 111}In-labelled DOTA-Z{sub HER2:S1}, NOTA-Z{sub HER2:S1} and NODAGA-Z{sub HER2:S1} in normal mice demonstrated a substantial influence of the chelators on the biodistribution properties of the conjugates. {sup 111}In-NODAGA-Z{sub HER2:S1} had the most rapid clearance from blood and healthy tissues. {sup 111}In-NOTA-Z{sub HER2:S1} showed high hepatic uptake and was excluded from further evaluation. {sup 111}In-DOTA-Z{sub HER2:S1} and {sup 111}In-NODAGA-Z{sub HER2:S1} demonstrated specific uptake in DU-145 prostate cancer xenografts in nude mice. The tumour uptake of {sup 111}In-NODAGA-Z{sub HER2:S1}, 5.6 {+-} 0.4%ID/g, was significantly lower than the uptake of {sup 111}In-DOTA-Z{sub HER2:S1

Biokinetics and dosimetry with 177Lu-DOTA-TATE in athymic mice with induced pancreatic malignant tumours

Science.gov (United States)

Rodríguez-Cortés, J.; de Murphy, C. Arteaga; Ferro-Flores, Ge; Pedraza-López, M.; Murphy-Stack, E.

Malignant pancreatic tumours induced in athymic mice are a good model for peptide receptor targeted radiotherapy. The objective of this research was to determine biokinetic parameters in mice, in order to estimate the induced pancreatic tumour absorbed doses and to evaluate an `in house' 177Lu-DOTA-TATE radiopharmaceutical as part of preclinical studies for targeted therapy in humans. AR42J murine pancreas cancer cells expressing somatostatin receptors, were implanted in athymic mice (nD22) to obtain biokinetic and dosimetric data of 177Lu-DOTA-TATE. The mean tumour uptake 2 h post injection was 14.76±1.9% I.A./g; kidney and pancreas uptake, at the same time, were 7.27±1.1% I.A./g (1.71±0.90%/organ) and 4.20±0.98% I.A./g (0.42±0.03%/organ), respectively. The mean absorbed dose to tumour, kidney and pancreas was 0.58±0.02 Gy/MBq; 0.23±0.01 Gy/MBq and 0.14±0.01 Gy/MBq, respectively. These studies justify further dosimetric estimations to ensure that 177Lu-DOTA-TATE will act as expected in humans.

Quantitative PET of EGFR expression in xenograft-bearing mice using 64Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody

International Nuclear Information System (INIS)

Cai, Weibo; Chen, Kai; He, Lina; Cao, Qizhen; Chen, Xiaoyuan; Koong, Albert

2007-01-01

Cetuximab, a chimeric monoclonal antibody targeting epidermal growth factor receptor (EGFR) on the surface of cancer cells, was approved by the FDA to treat patients with metastatic colorectal cancer. It is currently also in advanced-stage development for the treatment of several other solid tumors. Here we report for the first time the quantitative positron emission tomography (PET) imaging of EGFR expression in xenograft-bearing mice using 64 Cu-labeled cetuximab. We conjugated cetuximab with macrocyclic chelating agent 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA), labeled with 64 Cu, and tested the resulting 64 Cu-DOTA-cetuximab in seven xenograft tumor models. The tracer uptake measured by PET was correlated with the EGFR expression quantified by western blotting. The estimated human dosimetry based on the PET data in Sprague-Dawley rats was also calculated. MicroPET imaging showed that 64 Cu-DOTA-cetuximab had increasing tumor activity accumulation over time in EGFR-positive tumors but relatively low uptake in EGFR-negative tumors at all times examined ( 2 0.80) between the tracer uptake (measured by PET) and the EGFR expression level (measured by western blotting). Human dosimetry estimation indicated that the tracer may be safely administered to human patients for tumor diagnosis, with the dose-limiting organ being the liver. The success of EGFR-positive tumor imaging using 64 Cu-DOTA-cetuximab can be translated into the clinic to characterize the pharmacokinetics, to select the right population of patients for EGFR-targeted therapy, to monitor the therapeutic efficacy of anti-EGFR treatment, and to optimize the dosage of either cetuximab alone or cetuximab in combination with other therapeutic agents. (orig.)

Gold Nanotheranostics: Photothermal Therapy and Imaging of Mucin 7 Conjugated Antibody Nanoparticles for Urothelial Cancer

Directory of Open Access Journals (Sweden)

Chieh Hsiao Chen

2015-01-01

Full Text Available Objective. To kill urothelial cancer cells while preserving healthy cells, this study used photothermal therapy (PTT. PTT techniques target urothelial cancer cells using gold nanoparticles (GNPs and a green light laser. Materials and Methods. The GNPs were conjugated with anti-Mucin 7 antibodies, which acted as a probe for targeting tumor cells. Conjugated GNPs were exposed to a green light laser (532 nm with sufficient thermal energy to kill the transitional cell carcinomas (TCCs. Results. According to our results, nanoparticles conjugated with Mucin 7 antibodies damaged all types of cancer cells (MBT2, T24, 9202, and 8301 at relatively low energy levels (i.e., 500 laser shots at 10 W/cm2 in power, 1.6 Hz in frequency, and 300 ms in duration. Nonconjugated nanoparticles required 30 W/cm2 or more to achieve the same effect. Cell damage was directly related to irradiation time and applied laser energy. Conclusions. The minimally invasive PTT procedure combined with Mucin 7 targeted GNPs is able to kill cancer cells and preserve healthy cells. The success of this treatment technique can likely be attributed to the lower amount of energy required to kill targeted cancer cells compared with that required to kill nontargeted cancer cells. Our in vitro pilot study yielded promising results; however, additional animal studies are required to confirm these findings.

68Ga-autoclabeling of DOTA-TATE and DOTA-NOC

DEFF Research Database (Denmark)

Blom, Elisabeth; Koziorowski, Jacek

2012-01-01

A new method combining (68)Ga-labeling and steam sterilization, here called autoclabeling, has been evaluated for two somatostatin receptor binding tracers used for positron emission tomography (PET) imaging of neuroendocrine tumors; DOTA-TATE and -NOC....

Development of a potent DOTA-conjugated bombesin antagonist for targeting GRPr-positive tumours

Energy Technology Data Exchange (ETDEWEB)

Mansi, Rosalba; Maecke, Helmut R. [University Hospital Basel, Division of Radiological Chemistry, Basel (Switzerland); University of Freiburg, Department of Nuclear Medicine, Freiburg (Germany); Wang, Xuejuan [University Hospital Basel, Division of Radiological Chemistry, Basel (Switzerland); Forrer, Flavio [University Hospital Basel, Institute of Nuclear Medicine, Basel (Switzerland); Erasmus Medical Centre, Nuclear Medicine, Rotterdam (Netherlands); Waser, Beatrice; Cescato, Renzo; Reubi, Jean Claude [University of Berne, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, Berne (Switzerland); Graham, Keith; Borkowski, Sandra [Bayer Schering Pharma AG, Global Drug Discovery, Berlin (Germany)

2011-01-15

Radiolabelled somatostatin-based antagonists show a higher uptake in tumour-bearing mouse models than agonists of similar or even distinctly higher receptor affinity. Very similar results were obtained with another family of G protein-coupled receptor ligands, the bombesin family. We describe a new conjugate, RM2, with the chelator DOTA coupled to D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH{sub 2} via the cationic spacer 4-amino-1-carboxymethyl-piperidine for labelling with radiometals such as {sup 111}In and {sup 68}Ga. RM2 was synthesized on a solid support and evaluated in vitro in PC-3 cells. IC{sub 50} and K{sub d} values were determined. The antagonist potency was evaluated by immunofluorescence-based internalization and Ca{sup 2+} mobilization assays. Biodistribution studies were performed in PC-3 and LNCaP tumour-bearing mice with {sup 111}In-RM2 and {sup 68}Ga-RM2, respectively. PET/CT studies were performed on PC-3 and LNCaP tumour-bearing nude mice with {sup 68}Ga-RM2. RM2 and {sup 111}In-RM2 are high-affinity and selective ligands for the GRP receptor (7.7{+-}3.3 nmol/l for RM2; 9.3{+-}3.3 nmol/l for {sup nat}In-RM2). The potent antagonistic properties were confirmed by an immunofluorescence-based internalization and Ca{sup 2+} mobilization assays. {sup 68}Ga- and {sup 111}In-RM2 showed high and specific uptake in both the tumour and the pancreas. Uptake in the tumour remained high (15.2{+-}4.8%IA/g at 1 h; 11.7{+-}2.4%IA/g at 4 h), whereas a relatively fast washout from the pancreas and the other abdominal organs was observed. Uptake in the pancreas decreased rapidly from 22.6{+-}4.7%IA/g at 1 h to 1.5{+-}0.5%IA/g at 4 h. RM2 was shown to be a potent GRPr antagonist. Pharmacokinetics and imaging studies indicate that {sup 111}In-RM2 and {sup 68}Ga-RM2 are ideal candidates for clinical SPECT and PET studies. (orig.)

Development of a potent DOTA-conjugated bombesin antagonist for targeting GRPr-positive tumours

International Nuclear Information System (INIS)

Mansi, Rosalba; Maecke, Helmut R.; Wang, Xuejuan; Forrer, Flavio; Waser, Beatrice; Cescato, Renzo; Reubi, Jean Claude; Graham, Keith; Borkowski, Sandra

2011-01-01

Radiolabelled somatostatin-based antagonists show a higher uptake in tumour-bearing mouse models than agonists of similar or even distinctly higher receptor affinity. Very similar results were obtained with another family of G protein-coupled receptor ligands, the bombesin family. We describe a new conjugate, RM2, with the chelator DOTA coupled to D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH 2 via the cationic spacer 4-amino-1-carboxymethyl-piperidine for labelling with radiometals such as 111 In and 68 Ga. RM2 was synthesized on a solid support and evaluated in vitro in PC-3 cells. IC 50 and K d values were determined. The antagonist potency was evaluated by immunofluorescence-based internalization and Ca 2+ mobilization assays. Biodistribution studies were performed in PC-3 and LNCaP tumour-bearing mice with 111 In-RM2 and 68 Ga-RM2, respectively. PET/CT studies were performed on PC-3 and LNCaP tumour-bearing nude mice with 68 Ga-RM2. RM2 and 111 In-RM2 are high-affinity and selective ligands for the GRP receptor (7.7±3.3 nmol/l for RM2; 9.3±3.3 nmol/l for nat In-RM2). The potent antagonistic properties were confirmed by an immunofluorescence-based internalization and Ca 2+ mobilization assays. 68 Ga- and 111 In-RM2 showed high and specific uptake in both the tumour and the pancreas. Uptake in the tumour remained high (15.2±4.8%IA/g at 1 h; 11.7±2.4%IA/g at 4 h), whereas a relatively fast washout from the pancreas and the other abdominal organs was observed. Uptake in the pancreas decreased rapidly from 22.6±4.7%IA/g at 1 h to 1.5±0.5%IA/g at 4 h. RM2 was shown to be a potent GRPr antagonist. Pharmacokinetics and imaging studies indicate that 111 In-RM2 and 68 Ga-RM2 are ideal candidates for clinical SPECT and PET studies. (orig.)

Preparation and immunological properties of procaine-protein conjugates

International Nuclear Information System (INIS)

Liakopoulou, A.

1981-01-01

Procaine was conjugated to BSA and rat and rabbit Gf using the carbodiimide method and 14 C-procaine as tracer. The composition of the conjugates could be varied depending on the time of incubation and the concentration of procaine in the reaction mixtures. Procaine-BSA conjugates were soluble in water or saline. However, procaine conjugates to rat or rabbit Gf were not readily soluble in saline. These conjugates were good for immunization purposes, but it was cumbersome to work with them when clear solutions were needed, as in the immunochemical procedures used in this study. The immunological properties of the conjugates were studied in rats and rabbits. Rats responded with production of IgGa and precipitating antibodies to the procaine group, but IgE antibodies to the immunogen could not be detected. Furthermore, precipitating antibodies towards the procaine group were raised in rabbits. When BSA was the protein carrier, antibodies to the carrier molecule were also detected in both rats and rabbits. The conjugates of procaine to rat or rabbit Gf did not elicit antibody response to the carrier molecule when used in the homologous species. Hapten inhibition studies suggested that, in the rabbit, antibodies were also produced with specificity directed towards the molecular configuration of the hapten-carrier bond. (author)

Peptide receptor radionuclide therapy (PRRT) of neuroendocrine tumors: First comparative results using the somatostatin analogues Lu-177 DOTA-NOC and Lu-177 DOTA-TATE

International Nuclear Information System (INIS)

Wehrmann, C.; Senftleben, S.; Baum, R.P.

2005-01-01

Peptide receptor radionuclide therapy (PRRT) is used in our department since 5 years (approx. 400 applications) for the treatment of patients with metastatic neuroendocrine tumors. Of all known peptides, the somatostatin analogue DOTA-NOC shows in vitro the highest affinity to somatostatin receptors (sstr) 3 and 5 and a very high affinity to sstr 2. We studied the in vivo behaviour of the two peptides DOTA-NOC and DOTA-TATE (highest affinity to sstr 2) by the use of different parameters like tumor and organ uptake, effective half-lifes (kinetics) and mean absorbed organ and tumor doses. We studied 27 patients with metastatic neuroendocrine tumors with high somatostatin expression, as verified prior to treatment by Ga-68 DOTA-NOC receptor PET/CT or somatostatin receptor scintigraphy (Tc-99m EDDA-Hynic TOC or In-111 OctreoScan, planar and SPECT). 22 patients (8M and 14F; aged 619 years) were treated with 2500 6790 MBq Lu-177 DOTA-TATE. Another 5 patients (1M and 4F, aged 6310 years) were treated with 4000 7400 MBq Lu-177 DOTA-NOC. Labelling efficiency and radiochemical purity using Lutetium-177 chloride (obtained from PerkinElmer Life Sciences, USA) were constantly over 99.5%. Whole-body scans (anterior/posterior) were performed at 0.5h, 3h, 24h, 48h, 72h and 96h p.i. ROIs were drawn over the whole-body, organs, and different metastases (mainly in the liver). Blood samples were obtained in 12 patients after therapy with Lu-177 DOTA-TATE over 5 days for calculating the kinetics in blood. The ROI results were used to determine the uptake and effective half-life in different organs (kidney, spleen, liver, bone etc.) and the tumor residence times. By means of geometric mean, and after background correction, the ROI results were also used to calculate the estimated absorbed organ and tumor doses using the OLINDA software. Compared to Lu-177 DOTA-TATE (=100%), the uptake of Lu-177 DOTA-NOC was higher for the whole-body (45%) and for normal tissues (28%), and also in the

Gold nanorod-mediated hyperthermia enhances the efficacy of HPMA copolymer-90Y conjugates in treatment of prostate tumors

International Nuclear Information System (INIS)

Buckway, Brandon; Frazier, Nick; Gormley, Adam J.; Ray, Abhijit; Ghandehari, Hamidreza

2014-01-01

Introduction: The treatment of prostate cancer using a radiotherapeutic 90 Y labeled N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer can be enhanced with localized tumor hyperthermia. An 111 In labeled HPMA copolymer system for single photon emission computerized tomography (SPECT) was developed to observe the biodistribution changes associated with hyperthermia. Efficacy studies were conducted in prostate tumor bearing mice using the 90 Y HPMA copolymer with hyperthermia. Methods: HPMA copolymers containing 1, 4, 7, 10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were synthesized by reversible addition-fragmentation transfer (RAFT) copolymerization and subsequently labeled with either 111 In for imaging or 90 Y for efficacy studies. Radiolabel stability was characterized in vitro with mouse serum. Imaging and efficacy studies were conducted in DU145 prostate tumor bearing mice. Imaging was performed using single photon emission computerized tomography (SPECT). Localized mild tumor hyperthermia was achieved by plasmonic photothermal therapy using gold nanorods. Results: HPMA copolymer-DOTA conjugates demonstrated efficient labeling and stability for both radionuclides. Imaging analysis showed a marked increase of radiolabeled copolymer within the hyperthermia treated prostate tumors, with no significant accumulation in non-targeted tissues. The greatest reduction in tumor growth was observed in the hyperthermia treated tumors with 90 Y HPMA copolymer conjugates. Histological analysis confirmed treatment efficacy and safety. Conclusion: HPMA copolymer-DOTA conjugates radiolabeled with both the imaging and treatment radioisotopes, when combined with hyperthermia can serve as an image guided approach for efficacious treatment of prostate tumors

Antibody response to Haemophilus influenzae type b capsular polysaccharide conjugated to tetanus toxoid in preterm infants

DEFF Research Database (Denmark)

Kristensen, Kim; Gyhrs, A; Lausen, B

1996-01-01

OBJECTIVE: To evaluate the antibody response to a Haemophilus influenzae type b capsular polysaccharide (HibCP) tetanus toxoid (TT) conjugate vaccine (HibCP-TT) in preterm infants. SUBJECTS: Thirty-five healthy preterm infants with gestational ages (GA) from 27 to 36 weeks and birth weights from...

Positron emission tomography study on pancreatic somatostatin receptors in normal and diabetic rats with {sup 68}Ga-DOTA-octreotide: A potential PET tracer for beta cell mass measurement

Energy Technology Data Exchange (ETDEWEB)

Sako, Takeo [Division of Bio-function Dynamics Imaging, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 (Japan); Division of Molecular Imaging, Institute of Biomedical Research and Innovation, 2-2 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 (Japan); Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 (Japan); Hasegawa, Koki; Nishimura, Mie; Kanayama, Yousuke; Wada, Yasuhiro; Hayashinaka, Emi; Cui, Yilong; Kataoka, Yosky [Division of Bio-function Dynamics Imaging, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 (Japan); Senda, Michio [Division of Bio-function Dynamics Imaging, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 (Japan); Division of Molecular Imaging, Institute of Biomedical Research and Innovation, 2-2 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 (Japan); Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017 (Japan); Watanabe, Yasuyoshi, E-mail: yywata@riken.jp [Division of Bio-function Dynamics Imaging, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047 (Japan)

2013-12-06

Highlights: •PET images showed high uptake of {sup 68}Ga-DOTA-octreotide in the normal pancreas. •{sup 68}Ga-DOTA-octreotide specifically binds to somatostatin receptors in the pancreas. •The pancreatic uptake of {sup 68}Ga-DOTA-octreotide was decreased in the diabetic rats. •{sup 68}Ga-DOTA-octreotide could be a candidate PET probe to measure the beta cell mass. -- Abstract: Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia, and the loss or dysfunction of pancreatic beta cells has been reported before the appearance of clinical symptoms and hyperglycemia. To evaluate beta cell mass (BCM) for improving the detection and treatment of DM at earlier stages, we focused on somatostatin receptors that are highly expressed in the pancreatic beta cells, and developed a positron emission tomography (PET) probe derived from octreotide, a metabolically stable somatostatin analog. Octreotide was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a chelating agent, and labeled with {sup 68}Gallium ({sup 68}Ga). After intravenous injection of {sup 68}Ga-DOTA-octreotide, a 90-min emission scan of the abdomen was performed in normal and DM model rats. The PET studies showed that {sup 68}Ga-DOTA-octreotide radioactivity was highly accumulated in the pancreas of normal rats and that the pancreatic accumulation was significantly reduced in the rats administered with an excess amount of unlabeled octreotide or after treatment with streptozotocin, which was used for the chemical induction of DM in rats. These results were in good agreement with the ex vivo biodistribution data. These results indicated that the pancreatic accumulation of {sup 68}Ga-DOTA-octreotide represented specific binding to the somatostatin receptors and reflected BCM. Therefore, PET imaging with {sup 68}Ga-DOTA-octreotide could be a potential tool for evaluating BCM.

99mTC-dextran-antibody conjugates. Labelling procedures

International Nuclear Information System (INIS)

Marquez, M.; Westlin, J.E.; Nilsson, S.; Holmberg, A.R.

1996-01-01

Dextran forms stable chelates with 99m Tc, a radionuclide with ideal properties for planar scintigraphic and tomographic imaging. This study investigates some of the factors of importance to the formation of 99m Tc-dextran. The complex was used for the technetium labelling of a monoclonal antibody. Two radiolabelling methods were studied: Direct dextran labelling with the reductant dissolved in HCl and labelling via a weak 'transfer' chelator (tartaric acid) with the reductant dissolved in ethanol. Different conditions during the labelling reaction were studied. Finally, dextran was coupled to a monoclonal anticytokeratin antibody and the conjugate was subsequently radiolabelled with 99m Tc. Gel filtration (GFR) and thin layer chromatography (TLC) were compared as methods for estimation of the labelling efficiency. When using 10-500 μM of ligand, 5-100 μM SnC1 2 with 10-500 MBq of technetium at pH7 incubated for 10-15 min, the radiolabelling seemed optimal (70-75% labelling efficiency). It was found that 100 μM tartaric acid used as a weak intermediate chelator with SnCl 2 dissolved in ethanol improved the reproducibility of the labelling. The labelling efficiency was not affected by either the presence of oxygen or the addition of an oxygen scavenger during the labelling incubation. In general, TLC showed higher labelling efficiencies than GFR, indicating inadequate separation of the different moieties. (orig.)

Tumor Targeting via Sialic Acid: [68Ga]DOTA-en-pba as a New Tool for Molecular Imaging of Cancer with PET.

Science.gov (United States)

Tsoukalas, Charalambos; Geninatti-Crich, Simonetta; Gaitanis, Anastasios; Tsotakos, Theodoros; Paravatou-Petsotas, Maria; Aime, Silvio; Jiménez-Juárez, Rogelio; Anagnostopoulos, Constantinos D; Djanashvili, Kristina; Bouziotis, Penelope

2018-02-20

The aim of this study was to demonstrate the potential of Ga-68-labeled macrocycle (DOTA-en-pba) conjugated with phenylboronic vector for tumor recognition by positron emission tomography (PET), based on targeting of the overexpressed sialic acid (Sia). The imaging reporter DOTA-en-pba was synthesized and labeled with Ga-68 at high efficiency. Cell binding assay on Mel-C and B16-F10 melanoma cells was used to evaluate melanin production and Sia overexpression to determine the best model for demonstrating the capability of [ 68 Ga]DOTA-en-pba to recognize tumors. The in vivo PET imaging was done with B16-F10 tumor-bearing SCID mice injected with [ 68 Ga]DOTA-en-pba intravenously. Tumor, blood, and urine metabolites were assessed to evaluate the presence of a targeting agent. The affinity of [ 68 Ga]DOTA-en-pba to Sia was demonstrated on B16-F10 melanoma cells, after the production of melanin as well as Sia overexpression was proved to be up to four times higher in this cell line compared to that in Mel-C cells. Biodistribution studies in B16-F10 tumor-bearing SCID mice showed blood clearance at the time points studied, while uptake in the tumor peaked at 60 min post-injection (6.36 ± 2.41 % ID/g). The acquired PET images were in accordance with the ex vivo biodistribution results. Metabolite assessment on tumor, blood, and urine samples showed that [ 68 Ga]DOTA-en-pba remains unmetabolized up to at least 60 min post-injection. Our work is the first attempt for in vivo imaging of cancer by targeting overexpression of sialic acid on cancer cells with a radiotracer in PET.

Evaluation of 68Ga-DOTA-TOC PET/CT for the detection of duodenopancreatic neuroendocrine tumors in patients with MEN1

International Nuclear Information System (INIS)

Morgat, Clement; Mazere, Joachim; Hindie, Elif; Fernandez, Philippe; Velayoudom-Cephise, Fritz-Line; Nunes, Marie-Laure; Tabarin, Antoine; Schwartz, Paul; Guyot, Martine; Gaye, Delphine; Vimont, Delphine; Schulz, Juergen; Smith, Denis

2016-01-01

Somatostatin receptor scintigraphy with 111 In-pentetreotide (SRS) is used to detect duodenopancreatic neuroendocrine tumors (dpNETs) in multiple endocrine neoplasia type 1 (MEN1). However, SRS has limited sensitivity for this purpose. Positron emission tomography/computed tomography (PET/CT) with 68 Ga-DOTA-TOC has a higher rate of sporadic dpNETs detection than SRS but there is little data for dpNETs detection in MEN1. To compare the performances of 68 Ga-DOTA-TOC PET/CT, SRS and contrast-enhanced computed tomography (CE-CT) to diagnose dpNETs in MEN1. Single-institution prospective comparative study Nineteen consecutive MEN1 patients (aged 47 ± 13 years) underwent 68 Ga-DOTA-TOC PET/CT, SRS, and CE-CT within 2 months in random order. Blinded readings of images were performed separately by experienced physicians. Unblinded analysis of CE-CT, combined with additional magnetic resonance imaging, endoscopic-ultrasound, 18 F-2-fluoro-deoxy-d-glucose ( 18 F-FDG) PET/CT or histopathology results served as reference standard for dpNETs diagnosis. The sensitivity of 68 Ga-DOTA-TOC PET/CT, SRS, and CE-CT was 76, 20, and 60 %, respectively (p < 0.0001). All the true-positive lesions detected by SRS were also depicted on 68 Ga-DOTA-TOC PET/CT. 68 Ga-DOTA-TOC PET/CT detected lesions of smaller size than SRS (10.7 ± 7.6 and 15.2 ± 5.9 mm, respectively, p < 0.03). False negatives of 68 Ga-DOTA-TOC PET/CT included small dpNETs (<10 mm) and 18 F-FDG PET/CT positive aggressive dpNETs. No false positives were recorded. In addition, whole-body mapping with 68 Ga-DOTA-TOC PET/CT identified extra-abdominal MEN1-related tumors including one neuroendocrine thymic carcinoma identified by the three imaging procedures, one bronchial carcinoid undetected by CE-CT and three meningiomas undetected by SRS. Owing to higher diagnostic performance, 68 Ga-DOTA-TOC PET/CT (or alternative 68 Ga-labeled somatostatin analogues) should replace 111 In-pentetreotide in the investigation of MEN1

Control Strategy for Small Molecule Impurities in Antibody-Drug Conjugates.

Science.gov (United States)

Gong, Hai H; Ihle, Nathan; Jones, Michael T; Kelly, Kathleen; Kott, Laila; Raglione, Thomas; Whitlock, Scott; Zhang, Qunying; Zheng, Jie

2018-04-01

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceuticals. As such, there are no specific guidelines addressing impurity limits and qualification requirements. The current ICH guidelines on impurities, Q3A (Impurities in New Drug Substances), Q3B (Impurities in New Drug Products), and Q6B (Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) do not adequately address how to assess small molecule impurities in ADCs. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) formed an impurities working group (IWG) to discuss this issue. This white paper presents a strategy for evaluating the impact of small molecule impurities in ADCs. This strategy suggests a science-based approach that can be applied to the design of control systems for ADC therapeutics. The key principles that form the basis for this strategy include the significant difference in molecular weights between small molecule impurities and the ADC, the conjugation potential of the small molecule impurities, and the typical dosing concentrations and dosing schedule. The result is that exposure to small impurities in ADCs is so low as to often pose little or no significant safety risk.

Synthesis and evaluation of 99mTc/99Tc-MAG3-biotin conjugates for antibody pretargeting strategies

International Nuclear Information System (INIS)

Gog, Frank B. van; Visser, Gerard W.M.; Gowrising, Radjish W.A.; Snow, Gordon B.; Dongen, Guus A.M.S. van

1998-01-01

Four 99m Tc-MAG3-biotin conjugates were synthesized to determine their potential use in antibody pretargeting strategies for radioimmunoscintigraphy (RIS). To use these 99m Tc-MAG3-biotin conjugates as model compounds for 186 Re-MAG3-biotin conjugates for radioimmunotherapy (RIT), nanomolar amounts of 99 Tc were added as carrier to 99m Tc. The biotin derivatives used for the preparation of the conjugates - biocytin, biotin hydrazide, biotinyl-piperazine, and biotinyl-diaminosuccinic acid - differed at the site that is regarded to be susceptible to hydrolysis by biotinidase present in human plasma. All four conjugates were produced with high radiochemical purity, were stable in PBS, and demonstrated full binding capacity to streptavidin. The 99m Tc/ 99 Tc-MAG3-labeled biotinyl-piperazine and biotinyl-diaminosuccinic acid conjugates were stable in mouse as well as human plasma, whereas the corresponding biocytin and biotin hydrazide conjugates were rapidly degraded. The biodistribution in nude mice at 30 min after injection was similar for all conjugates, and a rapid blood clearance and high intestinal excretion were both observed. It is concluded that the metabolic routing of a conjugate containing biotin and MAG3 is dominated by these two moieties. For this reason, MAG3-biotin conjugates do not seem suited for pretargeted RIT, for which quantitative and fast renal excretion is a prerequisite to minimize radiation toxicity. However, in a pretargeted RIS approach the 99m Tc-MAG3-biotin conjugates might have potential

Photoluminescence of double core/shell infrared (CdSeTe)/ZnS quantum dots conjugated to Pseudo rabies virus antibodies

Science.gov (United States)

Torchynska, T. V.; Casas Espinola, J. L.; Jaramillo Gómez, J. A.; Douda, J.; Gazarian, K.

2013-06-01

Double core CdSeTe/ZnS quantum dots (QDs) with emission at 800 nm (1.60 eV) have been studied by photoluminescence (PL) and Raman scattering methods in the non-conjugated state and after the conjugation to the Pseudo rabies virus (PRV) antibodies. The transformation of PL spectra, stimulated by the electric charge of antibodies, has been detected for the bioconjugated QDs. Raman scattering spectra are investigated with the aim to reveal the CdSeTe core compositions. The double core QD energy diagrams were designed that help to analyze the PL spectra and their transformation at the bioconjugation. It is revealed that the interface in double core QDs has the type II quantum well character that permits to explain the near IR optical transition (1.60 eV) in the double core QDs. It is shown that the essential transformation of PL spectra is useful for the study of QD bioconjugation with specific antibodies and can be a powerful technique in early medical diagnostics.

Comparison of tumour and whole body absorbed doses of 177-Lu-DOTA-TATE and Lu-177-DOTA-NOC treatment in the same patient group

International Nuclear Information System (INIS)

Yeyin, N.; Kabasakal, L.; Akyel, R.; Demir, M.; Kanmaz, B.; Ocak, M.; Toklu, T.; Selcuk, N.

2015-01-01

Full text of publication follows. Peptide Receptor Radionuclide Therapy (PRRT) with Lu-177 labelled peptides in patients with neuroendocrine tumours (NETs) aroused great interest. An estimation of actual radiation doses to tumours is very important for therapy planning. There are several radiolabelled peptides, which can be used for PRRT with different biological behaviour. Aim: the aim of the study was to compare the tumour and normal organ absorbed doses in patients who have received Lu-177-DOTA-TATE and Lu-177 DOTA-NOC. Materials and methods: study was composed of 20 patients (M/F: 10/10, mean age: 51.5 ± 14.9) with histologically proven inoperable NETs. All patients received Lu-177-DOTA-NOC treatment 6 to 12 weeks after last Lu-177-DOTA-TATE treatment. Dosimetric calculations were performed using MIRD scheme and lesion doses were calculated using post therapy whole body images obtained at 4, 20, 44, and 68 hours after injection. Tumour volumes were determined from CT images. Thirteen blood samples beginning from time zero to 4 days after injection were obtained for bone marrow and whole body dosimetry. Results: There were 53 lesions in Lu-177-DOTA-TATE post-therapy whole body images and 49 lesions in Lu-177 DOTA-NOC post therapy images. Lesions were selected according to lesion delineation and superimposed lesions were excluded. Mean lesion absorbed dose is calculated to be 47.4 ± 53.4 and 42.9 ± 52.8 Gy per 370 MBq for Lu-177-DOTA-TATE and DOTA-NOC respectively (p>0.5). There were significantly higher absorbed doses for kidney and bone marrow after Lu-177-DOTA-NOC treatment as compared to Lu-177-DOTA-TATE treatment, which were 6.9 ± 2.7 vs 3.9 ± 1.7 (p<0.05) and 0.12 ± 0.0 vs 0.10 ± 0.0 (p<0.05) Gy, respectively. There was not any difference in plasma elimination times between two tracers. On the other hand the whole body absorbed dose was significantly higher after Lu-177-DOTA-NOC treatment, which was 0.24 ± 0.07 vs 0.20 ± 0.06 Gy (p<0

An enzymatic deconjugation method for the analysis of small molecule active drugs on antibody-drug conjugates.

Science.gov (United States)

Li, Yi; Gu, Christine; Gruenhagen, Jason; Yehl, Peter; Chetwyn, Nik P; Medley, Colin D

2016-01-01

Antibody-drug conjugates (ADCs) are complex therapeutic agents that use the specific targeting properties of antibodies and the highly potent cytotoxicity of small molecule drugs to selectively eliminate tumor cells while limiting the toxicity to normal healthy tissues. Two critical quality attributes of ADCs are the purity and stability of the active small molecule drug linked to the ADC, but these are difficult to assess once the drug is conjugated to the antibody. In this study, we report a enzyme deconjugation approach to cleave small molecule drugs from ADCs, which allows the drugs to be subsequently characterized by reversed-phase high performance liquid chromatography. The model ADC we used in this study utilizes a valine-citrulline linker that is designed to be sensitive to endoproteases after internalization by tumor cells. We screened several proteases to determine the most effective enzyme. Among the 3 cysteine proteases evaluated, papain had the best efficiency in cleaving the small molecule drug from the model ADC. The deconjugation conditions were further optimized to achieve complete cleavage of the small molecule drug. This papain deconjugation approach demonstrated excellent specificity and precision. The purity and stability of the active drug on an ADC drug product was evaluated and the major degradation products of the active drug were identified. The papain deconjugation method was also applied to several other ADCs, with the results suggesting it could be applied generally to ADCs containing a valine-citrulline linker. Our results indicate that the papain deconjugation method is a powerful tool for characterizing the active small molecule drug conjugated to an ADC, and may be useful in ensuring the product quality, efficacy and the safety of ADCs.

Synthesis and Characterization of a Gd-DOTA-D-Permeation Peptide for Magnetic Resonance Relaxation Enhancement of Intracellular Targets

Directory of Open Access Journals (Sweden)

Andrew M. Prantner

2003-10-01

Full Text Available Many MR contrast agents have been developed and proven effective for extracellular nontargeted applications, but exploitation of intracellular MR contrast agents has been elusive due to the permeability barrier of the plasma membrane. Peptide transduction domains can circumvent this permeability barrier and deliver cargo molecules to the cell interior. Based upon enhanced cellular uptake of permeation peptides with D-amino acid residues, an all-D Tat basic domain peptide was conjugated to DOTA and chelated to gadolinium. Gd-DOTA-D-Tat peptide in serum at room temperature showed a relaxivity of 7.94 ± 0.11 mM−1 sec−1 at 4.7 T. The peptide complex displayed no significant binding to serum proteins, was efficiently internalized by human Jurkat leukemia cells resulting in intracellular T1 relaxation enhancement, and in preliminary T1-weighted MRI experiments, significantly enhanced liver, kidney, and mesenteric signals.

Development of an inflammation imaging tracer, 111In-DOTA-DAPTA, targeting chemokine receptor CCR5 and preliminary evaluation in an ApoE-/- atherosclerosis mouse model.

Science.gov (United States)

Wei, Lihui; Petryk, Julia; Gaudet, Chantal; Kamkar, Maryam; Gan, Wei; Duan, Yin; Ruddy, Terrence D

2018-02-07

Chemokine receptor 5 (CCR5) plays an important role in atherosclerosis. Our objective was to develop a SPECT tracer targeting CCR5 for imaging plaque inflammation by radiolabeling D-Ala-peptide T-amide (DAPTA), a CCR5 antagonist, with 111 In. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated DAPTA (DOTA-DAPTA) was labeled with 111 In. Cell uptake studies were conducted in U87-CD4-CCR5 and U87-MG cells. Biodistribution was determined in C57BL/6 mice. Autoradiography, en face and Oil Red O (ORO) imaging studies were performed in ApoE -/- mice. DOTA-DAPTA was radiolabeled with 111 In with high radiochemical purity (> 98%) and specific activity (70 MBq·nmol). 111 In-DOTA-DAPTA exhibited fast blood and renal clearance and high spleen uptake. The U87-CD4-CCR5 cells had significantly higher uptake in comparison to the U87-MG cells. The cell uptake was reduced by three times with DAPTA, indicating the receptor specificity of the uptake. Autoradiographic images showed significantly higher lesion uptake of 111 In-DOTA-DAPTA in ApoE -/- mice than that in C57BL/6 mice. The tracer uptake in 4 month old ApoE -/- high fat diet (HFD) mice with blocking agent was twofold lower than the same mice without the blocking agent, demonstrating the specificity of the tracer for the CCR5 receptor. 111 In-DOTA-DAPTA, specifically targeting chemokine receptor CCR5, is a potential SPECT agent for imaging inflammation in atherosclerosis.

Two Rab5 Homologs Are Essential for the Development and Pathogenicity of the Rice Blast Fungus Magnaporthe oryzae

Directory of Open Access Journals (Sweden)

Cheng D. Yang

2017-05-01

Full Text Available The rice blast fungus, Magnaporthe oryzae, infects many economically important cereal crops, particularly rice. It has emerged as an important model organism for studying the growth, development, and pathogenesis of filamentous fungi. RabGTPases are important molecular switches in regulation of intracellular membrane trafficking in all eukaryotes. MoRab5A and MoRab5B are Rab5 homologs in M. oryzae, but their functions in the fungal development and pathogenicity are unknown. In this study, we have employed a genetic approach and demonstrated that both MoRab5A and MoRab5B are crucial for vegetative growth and development, conidiogenesis, melanin synthesis, vacuole fusion, endocytosis, sexual reproduction, and plant pathogenesis in M. oryzae. Moreover, both MoRab5A and MoRab5B show similar localization in hyphae and conidia. To further investigate possible functional redundancy between MoRab5A and MoRab5B, we overexpressed MoRAB5A and MoRAB5B, respectively, in MoRab5B:RNAi and MoRab5A:RNAi strains, but neither could rescue each other’s defects caused by the RNAi. Taken together, we conclude that both MoRab5A and MoRab5B are necessary for the development and pathogenesis of the rice blast fungus, while they may function independently.

Comparative Evaluation of Using NOTA and DOTA Derivatives as Bifunctional Chelating Agents in the Preparation of 68Ga-Labeled Porphyrin: Impact on Pharmacokinetics and Tumor Uptake in a Mouse Model.

Science.gov (United States)

Guleria, Mohini; Das, Tapas; Amirdhanayagam, Jeyachitra; Sarma, Haladhar D; Dash, Ashutosh

2018-02-01

Both NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) derivatives have been used as bifunctional chelating agents (BFCAs) for the preparation of 68 Ga-labeled target-specific agents having potential for positron emission tomography (PET) imaging of cancerous lesions. In the present work, the authors have attempted a comparative pharmacokinetic evaluation between 68 Ga-labeled porphyrins prepared using NOTA and DOTA derivatives as the BFCAs. A symmetrical porphyrin derivative, 5,10,15,20-tetrakis(p-carboxymethyleneoxyphenyl)porphyrin, was synthesized and coupled with two different BFCAs viz. p-NH 2 -benzyl-NOTA and p-NH 2 -benzyl-DOTA. Both the porphyrin-BFCA conjugates were radiolabeled with 68 Ga. A comparative bioevaluation involving pharmacokinetics and tumor affinity was performed in a tumor-bearing small animal model. Gallium-68-labeled porphyrin-amido-benzyl-NOTA and porphyrin-amido-benzyl-DOTA complexes were prepared with high radiochemical purity. Both radiolabeled complexes exhibited almost similar stability in human serum and near-identical tumor affinity and pharmacokinetic behavior in animal studies. The present study demonstrates that the pharmacokinetic behavior of 68 Ga-labeled porphyrin derivatives, prepared using either NOTA or DOTA derivatives as BFCAs, remains almost identical and hence both NOTA and DOTA derivatives could be considered equivalent for developing 68 Ga-based PET agents for imaging of tumorous lesions.

Synthesis and evaluation of Lys{sup 1}(α, γ-Folate)Lys{sup 3}({sup 177}Lu-DOTA)-Bombesin(1-14) as a potential theranostic radiopharmaceutical for breast cancer

Energy Technology Data Exchange (ETDEWEB)

Aranda L, L.; Ferro F, G.; Azorin V, E.; Ramirez, F. M.; Ocampo G, B.; Santos C, C.; Jimenez M, N. [ININ, Carretera Mexico-Toluca s/n, 52750 Ocoyoacac, Estado de Mexico (Mexico); Issac O, K. [Universidad Autonoma del Estado de Mexico, Facultad de Medicina, 50180 Toluca, Estado de Mexico (Mexico)

2015-10-15

Full text: Lutetium-177 labeled hetero bivalent molecules that interact with different targets on tumor cells have been proposed as a new class of theranostic radiopharmaceuticals. The aim of this work was to synthesize Lys{sup 1} (α,γ-Folate)-Lys{sup 3}({sup 177}Lu-DOTA)-Bombesin (1-14) ({sup 177}LuFolate-Bn), as well as to assess its in vitro and in vivo potential for molecular imaging and targeted radiotherapy of breast tumors expressing folate receptors (Fr) and gastrin releasing peptide receptors (GRPR). Lys{sup 1} Lys{sup 3} (DOTA)-Bombesin (1-14) was conjugated to the terminal carboxylic group of the folic acid and the product purified by size-exclusion HPLC. Chemical characterization was carried out by UV-vis, Ft-IR spectroscopies and MALDI-TOF mass spectrometry. {sup 177}Lu labeling was performed by reaction of {sup 177}LuCl{sub 3} with the Lys{sup 1} (α,γ-Folate)-Lys{sup 3} (DOTA)-Bombesin (Folate-Bn) conjugate. In vitro binding studies were carried out in T47D breast cancer cells (positive to Fr and GRPR). Biokinetic studies and micro-SPECT/CT images were obtained using athymic mice with T47D induced tumors. Spectroscopic studies and HPLC analyses indicated that the conjugate was obtained with high chemical and radiochemical purity (98 ± 1.3%). T47D-tumors were clearly visible with high contrast at 2 h after radiopharmaceutical administration. The {sup 177}Lu-absorbed dose delivered to tumors was 23.9 ± 2.1 Gy (74 MBq, intravenously administered) {sup 177}Lu-Folate-Bn demonstrated properties suitable as a theranostic radiopharmaceutical for breast tumors expressing Fr s and GRPR s. (Author)

Evaluation of {sup 68}Ga-DOTA-TOC PET/CT for the detection of duodenopancreatic neuroendocrine tumors in patients with MEN1

Energy Technology Data Exchange (ETDEWEB)

Morgat, Clement; Mazere, Joachim; Hindie, Elif; Fernandez, Philippe [CNRS, INCIA, Bordeaux (France); University of Bordeaux, INCIA, Bordeaux (France); University Hospital of Bordeaux, Department of Nuclear Medicine, Bordeaux (France); Velayoudom-Cephise, Fritz-Line; Nunes, Marie-Laure; Tabarin, Antoine [USN Haut-Leveque, Department of Endocrinology, Pessac (France); Schwartz, Paul; Guyot, Martine [University Hospital of Bordeaux, Department of Nuclear Medicine, Bordeaux (France); Gaye, Delphine [University Hospital of Bordeaux, Department of Radiology, Pessac (France); Vimont, Delphine; Schulz, Juergen [CNRS, INCIA, Bordeaux (France); University of Bordeaux, INCIA, Bordeaux (France); Smith, Denis [University Hospital of Bordeaux, Department of Oncology, Bordeaux (France)

2016-07-15

Somatostatin receptor scintigraphy with {sup 111}In-pentetreotide (SRS) is used to detect duodenopancreatic neuroendocrine tumors (dpNETs) in multiple endocrine neoplasia type 1 (MEN1). However, SRS has limited sensitivity for this purpose. Positron emission tomography/computed tomography (PET/CT) with {sup 68}Ga-DOTA-TOC has a higher rate of sporadic dpNETs detection than SRS but there is little data for dpNETs detection in MEN1. To compare the performances of {sup 68}Ga-DOTA-TOC PET/CT, SRS and contrast-enhanced computed tomography (CE-CT) to diagnose dpNETs in MEN1. Single-institution prospective comparative study Nineteen consecutive MEN1 patients (aged 47 ± 13 years) underwent {sup 68}Ga-DOTA-TOC PET/CT, SRS, and CE-CT within 2 months in random order. Blinded readings of images were performed separately by experienced physicians. Unblinded analysis of CE-CT, combined with additional magnetic resonance imaging, endoscopic-ultrasound, {sup 18}F-2-fluoro-deoxy-d-glucose ({sup 18}F-FDG) PET/CT or histopathology results served as reference standard for dpNETs diagnosis. The sensitivity of {sup 68}Ga-DOTA-TOC PET/CT, SRS, and CE-CT was 76, 20, and 60 %, respectively (p < 0.0001). All the true-positive lesions detected by SRS were also depicted on {sup 68}Ga-DOTA-TOC PET/CT. {sup 68}Ga-DOTA-TOC PET/CT detected lesions of smaller size than SRS (10.7 ± 7.6 and 15.2 ± 5.9 mm, respectively, p < 0.03). False negatives of {sup 68}Ga-DOTA-TOC PET/CT included small dpNETs (<10 mm) and {sup 18}F-FDG PET/CT positive aggressive dpNETs. No false positives were recorded. In addition, whole-body mapping with {sup 68}Ga-DOTA-TOC PET/CT identified extra-abdominal MEN1-related tumors including one neuroendocrine thymic carcinoma identified by the three imaging procedures, one bronchial carcinoid undetected by CE-CT and three meningiomas undetected by SRS. Owing to higher diagnostic performance, {sup 68}Ga-DOTA-TOC PET/CT (or alternative {sup 68}Ga-labeled somatostatin analogues

The impact of administration of conjugate vaccines containing cross reacting material on Haemophilus influenzae type b antibody responses in infants: A systematic review and meta-analysis of randomised controlled trials.

Science.gov (United States)

Voysey, Merryn; Sadarangani, Manish; Clutterbuck, Elizabeth; Bolgiano, Barbara; Pollard, Andrew J

2016-07-25

Protein-polysaccharide conjugate vaccines such as Haemophilus influenzae type b (Hib), meningococcal, and pneumococcal vaccine, induce immunological memory and longer lasting protection than plain polysaccharide vaccines. The most common proteins used as carriers are tetanus toxoid (TT) and cross reacting material-197 (CRM), a mutant form of diphtheria toxoid. CRM conjugate vaccines have been reported to suppress antibody responses to co-administered Hib-TT vaccine. We conducted a systematic review and meta-analysis of randomised controlled trials in which infants were randomised to receive meningococcal or pneumococcal conjugate vaccines along with Hib-TT. Trials of licensed vaccines with different carrier proteins were included for group C meningococcal (MenC), quadrivalent ACWY meningococcal (MenACWY), and pneumococcal vaccines. Twenty-three trials were included in the meta-analyses. Overall, administration of MenC-CRM in a 2 or 3 dose schedule resulted in a 45% reduction in Hib antibody concentrations (GMR 0.55, 95% CI 0.49-0.62). MenACWY-CRM boosted Hib antibody responses by 22% (GMR 1.22, 95% CI 1.06-1.41) whilst pneumococcal CRM conjugate vaccines had no impact on Hib antibody responses (GMR 0.91, 95% CI 0.68-1.22). The effect of CRM protein-polysaccharide conjugate vaccines on Hib antibody responses varies greatly between vaccines. Co-administration of a CRM conjugate vaccine can produce either positive or negative effects on Hib antibody responses. These inconsistencies suggest that CRM itself may not be the main driver of variability in Hib responses, and challenge current perspectives on this issue. Copyright © 2016 Elsevier Ltd. All rights reserved.

Antibody-drug conjugates: Promising and efficient tools for targeted cancer therapy.

Science.gov (United States)

Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Majidi, Jafar

2018-09-01

Over the recent decades, the use of antibody-drug conjugates (ADCs) has led to a paradigm shift in cancer chemotherapy. Antibody-based treatment of various human tumors has presented dramatic efficacy and is now one of the most promising strategies used for targeted therapy of patients with a variety of malignancies, including hematological cancers and solid tumors. Monoclonal antibodies (mAbs) are able to selectively deliver cytotoxic drugs to tumor cells, which express specific antigens on their surface, and has been suggested as a novel category of agents for use in the development of anticancer targeted therapies. In contrast to conventional treatments that cause damage to healthy tissues, ADCs use mAbs to specifically attach to antigens on the surface of target cells and deliver their cytotoxic payloads. The therapeutic success of future ADCs depends on closely choosing the target antigen, increasing the potency of the cytotoxic cargo, improving the properties of the linker, and reducing drug resistance. If appropriate solutions are presented to address these issues, ADCs will play a more important role in the development of targeted therapeutics against cancer in the next years. We review the design of ADCs, and focus on how ADCs can be exploited to overcome multiple drug resistance (MDR). © 2018 Wiley Periodicals, Inc.

Radioimmunodetection of tumor with Ga-67 labeled antibodies

International Nuclear Information System (INIS)

Furukawa, Takako; Endo, Keigo; Ohmomo, Yoshiro

1986-01-01

Antibodies against tumor associated antigen; anti-AFP polyclonal antibody, anti-thyroglobulin monoclonal antibody and anti-hCG monoclonal antibody, were labeled with Ga-67, using deferoxamine (DF) as a bifunctional chelating agent. The immunoreactivity and in vivo stability of the Ga-67 labeled antibodies were examined. The effect of DF conjugation to antibodies on the antigen-binding activity was evaluated by RIA and Scatchard analysis or tanned sheep red blood cell hemagglutination technique. When DF was conjugated to antibody at the molar ratio of 1 : 1, the antibody activity of the DF-conjugated antibodies was fully retained. Whereas, in heavily conjugated antibodies, the maximum antigen binding capacity was reduced. Biodistribution study in normal mice demonstrated the high in vivo stability of Ga-67 labeled antibodies. The labeling of DF-antibody conjugated with Ga-67 was performed easily and quickly, with a high labeling efficiency, requiring no further purification. Thus, this labeling method, providing in vivo stability of Ga-67 labeled antibody and full retention of immunoreactivity, would be useful for the radioimmunodetection of various cancers. (author)

Antitenascin antibody 81C6 armed with {sup 177}Lu: in vivo comparison of macrocyclic and acyclic ligands

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Yordanov, Alexander T. [Department of Radiology, Duke University Medical Center Durham, NC 27710 (United States); Hens, Marc [Department of Radiology, Duke University Medical Center Durham, NC 27710 (United States); Pegram, Charles [Department of Pathology, Duke University Medical Center Durham, NC 27710 (United States); Bigner, Darell D. [Department of Pathology, Duke University Medical Center Durham, NC 27710 (United States); Zalutsky, Michael R. [Department of Radiology, Duke University Medical Center Durham, NC 27710 (United States)]. E-mail: zalut001@mc.duke.edu

2007-02-15

Introduction: When labeled with iodine-131, the antitenascin monoclonal antibody (mAb) 81C6 has shown promise as a targeted radiotherapeutic in patients with brain tumors. Because of its more favorable {gamma}-ray properties, lutetium-177 might be a better low-energy {beta}-emitter for this type of therapy. Materials and Methods: Chimeric 81C6 (ch81C6) was labeled with {sup 177}Lu using the acyclic 1B4M ligand and the macrocyclic ligands NHS-DOTA and MeO-DOTA and evaluated for binding to tenascin. Three paired-label tissue distribution experiments were performed in normal mice receiving one of the {sup 177}Lu-labeled immunoconjugates plus {sup 125}I-labeled ch81C6 labeled using Iodogen. Paired-label experiments in athymic mice bearing subcutaneous D54 MG human glioma xenografts were done to directly compare the biodistribution of ch81C6-1B4M-{sup 177}Lu and {sup 125}I-labeled ch81C6, and ch81C6-MeO-DOTA-{sup 177}Lu and {sup 125}I-labeled ch81C6. Similar comparisons were done using murine (mu) instead of ch81C6. The primary parameter utilized for evaluation was the {sup 177}Lu/{sup 125}I uptake ratio in each tissue. Results: In the studies performed in normal mice, the NHS-DOTA ligand yielded the highest {sup 177}Lu/{sup 125}I uptake ratios in tissues indicative of loss of label from the chelate; for this reason, only 1B4M and MeO-DOTA were evaluated further. The {sup 177}Lu/{sup 125}I ratio in bone increased gradually with time for the chimeric conjugates; however, there were no significant differences between ch81C6-1B4M-DTPA-{sup 177}Lu and ch81C6-MeO-DOTA-{sup 177}Lu. In contrast, mu81C6-1B4M-DTPA-{sup 177}Lu and mu81C6-MeO-DOTA-{sup 177}Lu showed a more dramatic increase in the {sup 177}Lu/{sup 125}I ratio in bone - from 2.4{+-}0.3 and 1.7{+-}0.2 at Day 1 to 8.5{+-}1.1 and 4.2{+-}0.5 at Day 7, respectively. Conclusion: With these antitenascin constructs, the nature of the mAb had a profound influence on the relative degree of loss of {sup 177}Lu from these

Quantification and imaging of HER2 protein using nanocrystals conjugated with single-domain antibodies

International Nuclear Information System (INIS)

Glukhov, S; Berestovoy, M; Nabiev, I; Sukhanova, A; Chames, P; Baty, D

2017-01-01

This study dealt with quantification and imaging of human epidermal growth factor receptor 2 (HER2), an important prognostic marker for cancer diagnosis and treatment, using specific quantum-dot-based conjugates. Fluorescent inorganic nanocrystals or quantum dots (QDs) are extremely highly resistant to photobleaching and have a high emission quantum yield and a continuous range of emission spectra, from the ultraviolet to the infrared regions. Ultrasmall nanoprobes consisting of highly affine anti-HER2 single-domain antibodies (sdAbs or 'nanobodies') conjugated with QDs in a strictly oriented manner have been designed. QDs with a fluorescence peak maxima at wavelengths of 562 nm, 569 nm, 570 nm or in the near-infrared region were used. Here, we present our results of ISA quantification of HER2 protein, in situ imaging of HER2 protein on the surface of HER2-positive SK-BR-3 cells in immunohistochemical experiments, and counting of stained with anti-HER2 conjugates HER2-positive SK-BR-3 cells in their mixture with unstained cells of the same culture in flow cytometry experiments. The data demonstrate that the anti-HER2 QD–sdAb conjugates obtained are highly specific and sensitive and could be used in numerous applications for advanced integrated diagnosis. (paper)

Quantification and imaging of HER2 protein using nanocrystals conjugated with single-domain antibodies

Science.gov (United States)

Glukhov, S.; Berestovoy, M.; Chames, P.; Baty, D.; Nabiev, I.; Sukhanova, A.

2017-01-01

This study dealt with quantification and imaging of human epidermal growth factor receptor 2 (HER2), an important prognostic marker for cancer diagnosis and treatment, using specific quantum-dot-based conjugates. Fluorescent inorganic nanocrystals or quantum dots (QDs) are extremely highly resistant to photobleaching and have a high emission quantum yield and a continuous range of emission spectra, from the ultraviolet to the infrared regions. Ultrasmall nanoprobes consisting of highly affine anti-HER2 single-domain antibodies (sdAbs or "nanobodies") conjugated with QDs in a strictly oriented manner have been designed. QDs with a fluorescence peak maxima at wavelengths of 562 nm, 569 nm, 570 nm or in the near-infrared region were used. Here, we present our results of ISA quantification of HER2 protein, in situ imaging of HER2 protein on the surface of HER2-positive SK-BR-3 cells in immunohistochemical experiments, and counting of stained with anti-HER2 conjugates HER2-positive SK-BR-3 cells in their mixture with unstained cells of the same culture in flow cytometry experiments. The data demonstrate that the anti-HER2 QD-sdAb conjugates obtained are highly specific and sensitive and could be used in numerous applications for advanced integrated diagnosis.

Novel Phospholipid-Protein Conjugates Allow Improved Detection of Antibodies in Patients with Autoimmune Diseases.

Directory of Open Access Journals (Sweden)

Simone V Samuelsen

Full Text Available Reliable measurement of clinically relevant autoimmune antibodies toward phospholipid-protein conjugates is highly desirable in research and clinical assays. To date, the development in this field has been limited to the use of natural heterogeneous antigens. However, this approach does not take structural features of biologically active antigens into account and leads to low reliability and poor scientific test value. Here we describe novel phospholipid-protein conjugates for specific detection of human autoimmune antibodies. Our synthetic approach includes mild oxidation of synthetic phospholipid cardiolipin, and as the last step, coupling of the product with azide-containing linker and copper-catalyzed click chemistry with β2-glycoprotein I and prothrombin. To prove utility of the product antigens, we used enzyme-linked immunosorbent assay and three cohorts of samples obtained from patients in Denmark (n = 34 and the USA (n = 27 and n = 14. Afterwards we analyzed correlation of the obtained autoantibody titers with clinical parameters for each patient. Our results prove that using novel antigens clinically relevant autoantibodies can be detected with high repeatability, sensitivity and specificity. Unlike previously used antigens the obtained autoantibody titers strongly correlate with high disease activity and in particular, with arthritis, renal involvement, anti-Smith antibodies and high lymphocyte count. Importantly, chemical composition of antigens has a strong influence on the correlation of detected autoantibodies with disease activity and manifestations. This confirms the crucial importance of antigens' composition on research and diagnostic assays, and opens up exciting perspectives for synthetic antigens in future studies of autoimmunity.

The added value of {sup 68}Ga-DOTA-TATE-PET to contrast-enhanced CT for primary site detection in CUP of neuroendocrine origin

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Kazmierczak, Philipp M. [Klinikum der Universitaet Muenchen, Institut fuer Klinische Radiologie, Muenchen (Germany); Ludwig-Maximilians-University Hospital Munich, Institute for Clinical Radiology, Muenchen (Germany); Rominger, Axel; Wenter, Vera [Ludwig-Maximilians-University Hospital Munich, Department of Nuclear Medicine, Muenchen (Germany); Spitzweg, Christine; Auernhammer, Christoph [Ludwig-Maximilians-University Hospital Munich, Department of Internal Medicine II, Muenchen (Germany); Angele, Martin K. [Ludwig-Maximilians-University Hospital Munich, Department of General, Visceral, Transplantation, Vascular and Thoracic Surgery, Muenchen (Germany); Rist, Carsten; Cyran, Clemens C. [Ludwig-Maximilians-University Hospital Munich, Institute for Clinical Radiology, Muenchen (Germany)

2017-04-15

To quantify the additional value of {sup 68}Ga-DOTA-TATE PET/CT in comparison with contrast-enhanced CT alone for primary tumour detection in neuroendocrine cancer of unknown primary (CUP-NET). In total, 38 consecutive patients (27 men, 11 women; mean age 62 years) with histologically proven CUP-NET who underwent a contrast-enhanced {sup 68}Ga-DOTA-TATE PET/CT scan for primary tumour detection and staging between 2010 and 2014 were included in this IRB-approved retrospective study. Two blinded readers independently analysed the contrast-enhanced CT and {sup 68}Ga-DOTA-TATE PET datasets separately and noted from which modality they suspected a primary tumour. Consensus was reached if the results were divergent. Postoperative histopathology (24 patients) and follow-up {sup 68}Ga-DOTA-TATE PET/CT imaging (14 patients) served as the reference standards and statistical measures of diagnostic accuracy were calculated accordingly. The majority of confirmed primary tumours were located in the abdomen (ileum in 19 patients, pancreas in 12, lung in 2, small pelvis in 1). High interobserver agreement was noted regarding the suspected primary tumour site (Cohen's k 0.90, p < 0.001). {sup 68}Ga-DOTA-TATE PET demonstrated a significantly higher sensitivity (94 % vs. 63 %, p = 0.005) and a significantly higher accuracy (87 % vs. 68 %, p = 0.003) than contrast-enhanced CT. Ga-DOTA-TATE PET/CT compared with contrast-enhanced CT alone provides an improvement in sensitivity of 50 % and an improvement in accuracy of 30 % in primary tumour detection in CUP-NET. (orig.)

Target-mediated drug disposition model and its approximations for antibody-drug conjugates.

Science.gov (United States)

Gibiansky, Leonid; Gibiansky, Ekaterina

2014-02-01

Antibody-drug conjugate (ADC) is a complex structure composed of an antibody linked to several molecules of a biologically active cytotoxic drug. The number of ADC compounds in clinical development now exceeds 30, with two of them already on the market. However, there is no rigorous mechanistic model that describes pharmacokinetic (PK) properties of these compounds. PK modeling of ADCs is even more complicated than that of other biologics as the model should describe distribution, binding, and elimination of antibodies with different toxin load, and also the deconjugation process and PK of the released toxin. This work extends the target-mediated drug disposition (TMDD) model to describe ADCs, derives the rapid binding (quasi-equilibrium), quasi-steady-state, and Michaelis-Menten approximations of the TMDD model as applied to ADCs, derives the TMDD model and its approximations for ADCs with load-independent properties, and discusses further simplifications of the system under various assumptions. The developed models are shown to describe data simulated from the available clinical population PK models of trastuzumab emtansine (T-DM1), one of the two currently approved ADCs. Identifiability of model parameters is also discussed and illustrated on the simulated T-DM1 examples.

Comparison of sequential planar 177Lu-DOTA-TATE dosimetry scans with 68Ga-DOTA-TATE PET/CT images in patients with metastasized neuroendocrine tumours undergoing peptide receptor radionuclide therapy

International Nuclear Information System (INIS)

Sainz-Esteban, Aurora; Carril, Jose Manuel; Prasad, Vikas; Schuchardt, Christiane; Zachert, Carolin; Baum, Richard P.

2012-01-01

The aim of the study was to compare sequential 177 Lu-DOTA-TATE planar scans ( 177 Lu-DOTA-TATE) in patients with metastasized neuroendocrine tumours (NET) acquired during peptide receptor radionuclide therapy (PRRT) for dosimetry purposes with the pre-therapeutic 68 Ga-DOTA-TATE positron emission tomography (PET)/CT ( 68 Ga-DOTA-TATE) maximum intensity projection (MIP) images obtained in the same patients concerning the sensitivity of the different methods. A total of 44 patients (59 ± 11 years old) with biopsy-proven NET underwent 68 Ga-DOTA-TATE and 177 Lu-DOTA-TATE imaging within 7.9 ± 7.5 days between the two examinations. 177 Lu-DOTA-TATE planar images were acquired at 0.5, 2, 24, 48 and 72 h post-injection; lesions were given a score from 0 to 4 depending on the uptake of the radiopharmaceutical (0 being lowest and 4 highest). The number of tumour lesions which were identified on 177 Lu-DOTA-TATE scans (in relation to the acquisition time after injection of the therapeutic dose as well as with regard to the body region) was compared to those detected on 68 Ga-DOTA-TATE studies obtained before PRRT. A total of 318 lesions were detected; 280 (88%) lesions were concordant. Among the discordant lesions, 29 were 68 Ga-DOTA-TATE positive and 177 Lu-DOTA-TATE negative, whereas 9 were 68 Ga-DOTA-TATE negative and 177 Lu-DOTA-TATE positive. The sensitivity, positive predictive value and accuracy for 177 Lu-DOTA-TATE as compared to 68 Ga-DOTA-TATE were 91, 97 and 88%, respectively. Significantly more lesions were seen on the delayed (72 h) 177 Lu-DOTA-TATE images (91%) as compared to the immediate (30 min) images (68%). The highest concordance was observed for bone metastases (97%) and the lowest for head/neck lesions (75%). Concordant lesions (n = 77; mean size 3.8 cm) were significantly larger than discordant lesions (n = 38; mean size 1.6 cm) (p max ). However, concordant liver lesions with a score from 1 to 3 in the 72-h 177 Lu-DOTA-TATE scan had a lower SUV max

Single vial kit formulation for preparation of PET radiopharmaceutical. 68Ga-DOTA-TOC

International Nuclear Information System (INIS)

Archana Mukherjee; Usha Pandey; Rubel Chakravarty; Ashutosh Dash; Haladhar Dev Sarma

2014-01-01

This paper describes the development of a lyophilized cold kit of DOTA-[Tyr 3 ]-Octreotide (DOTA-TOC) for instant compounding of 68 Ga-DOTA-TOC, suitable for diagnosis of neuroendocrine tumors. The work involved formulation of DOTA-TOC kits, optimization of radiolabeling, quality control of 68 Ga-DOTA-TOC and animal biodistribution studies. The prepared kits enable a reliable method for preparation of 68 Ga-DOTA-TOC of high radiochemical purity and excellent stability. Availability of such kits along with 68 Ge/ 68 Ga generators is expected to stimulate the widespread use of 68 Ga-DOTA-TOC in nuclear medicine practice in developing countries. (author)

Radiolabelled of c-DOTA-RGD and c-DOTA-RGDf with {sup 177}Lu and evaluation in vitro and in vivo stability; Radiomarcado del peptido c-DOTA-RGD y c-DOTA-RGDf con {sup 177}Lu y evaluacion de su estabilidad in vitro e in vivo

Energy Technology Data Exchange (ETDEWEB)

Vilchis J, A.

2010-07-01

Integrin {alpha}v{beta}3 has a critical role in tumor angio genesis and metastasis. Radiolabelled peptides based on the Arg-Gly-Asp (RGD) sequence have been reported as radiopharmaceuticals with high affinity and selectivity for the {alpha}v{beta}3 integrin. The aim of this study was to label c-DOTA-RGD and c-DOTA-RGDf peptides with {sup 177}Lu and to evaluate their in vitro and in vivo stability as potential specific therapeutic radiopharmaceuticals. Labelled was carried out by direct reaction of {sup 177}LuCl{sub 3} with c-DOTA-RGD peptides in 1 M acetate buffer ph 5.5 at 90{sup o} C for 30 min. Radiochemical purity and stability studies were realized by reversed phase HPLC and I TLC-Sg analyses in human serum and saline solution. Biological recognition was performed using MCF7 tumor cells (positive {alpha}v{beta}3) and in athymic mice with induced MCF7 tumors. Molecular mechanics and quantum mechanics calculations were performed to explain experimental results associated with the molecular recognition. {sup 177}Lu-DOTA-RGD and {sup 177}Lu-DOTA-RGDf were obtained with radiochemical purities > 95%, showing adequate in vitro and in vivo stability and specific binding to {open_square}{sub v}{open_square}{sub 3} receptors. (Author)

Comparison of DOTA and NODAGA as chelators for (64)Cu-labeled immunoconjugates.

Science.gov (United States)

Ghosh, Sukhen C; Pinkston, Kenneth L; Robinson, Holly; Harvey, Barrett R; Wilganowski, Nathaniel; Gore, Karen; Sevick-Muraca, Eva M; Azhdarinia, Ali

2015-02-01

Bifunctional chelators have been shown to impact the biodistribution of monoclonal antibody (mAb)-based imaging agents. Recently, radiolabeled 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA)-peptide complexes have demonstrated improved in vivo stability and performance compared to their 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) counterparts. Here, we investigated if similar utility could be achieved with mAbs and compared (64)Cu-labeled DOTA and NODAGA-immunoconjugates for the detection of epithelial cell adhesion molecule (EpCAM) in a prostate cancer model. DOTA and NODAGA-immunoconjugates of an EpCAM targeting mAb (mAb7) were synthesized and radiolabeled with (64)Cu (DOTA: 40°C for 1hr; NODAGA: 25°C for 1hr). The average number of chelators per mAb was quantified by isotopic dilution, and the biological activity of the immunoconjugates was evaluated by flow cytometry and ELISA. Radioligand assays were performed to compare cellular uptake and determine the dissociation constant (Kd) and maximum number of binding sites (Bmax) for the immunoconjugates using DsRed-transfected PC3-cells. A PC3-DsRed xenograft tumor model was established in nude mice and used to perform biodistribution studies to compare organ uptake and pharmacokinetics. (64)Cu-DOTA-mAb7 and (64)Cu-NODAGA-mAb7 were prepared with chelator/protein ratios of 2-3 and obtained in comparable radiochemical yields ranging from 59 to 71%. Similar immunoreactivity was observed with both agents, and mock labeling studies indicated that incubation at room temperature or 40°C did not affect potency. (64)Cu-NODAGA-mAb7 demonstrated higher in vitro cellular uptake while (64)Cu-DOTA-mAb7 had higher Kd and Bmax values. From the biodistribution data, we found similar tumor uptake (13.44±1.21%ID/g and 13.24±4.86%ID/g for (64)Cu-DOTA-mAb7 and (64)Cu-NODAGA-mAb7, respectively) for both agents at 24hr, although normal prostate tissue was significantly lower for (64)Cu-NODAGA-mAb7

[Tl(III)(dota)](-): An Extraordinarily Robust Macrocyclic Complex.

Science.gov (United States)

Fodor, Tamás; Bányai, István; Bényei, Attila; Platas-Iglesias, Carlos; Purgel, Mihály; Horváth, Gábor L; Zékány, László; Tircsó, Gyula; Tóth, Imre

2015-06-01

The X-ray structure of {C(NH2)3}[Tl(dota)]·H2O shows that the Tl(3+) ion is deeply buried in the macrocyclic cavity of the dota(4-) ligand (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) with average Tl-N and Tl-O distances of 2.464 and 2.365 Å, respectively. The metal ion is directly coordinated to the eight donor atoms of the ligand, which results in a twisted square antiprismatic (TSAP') coordination around Tl(3+). A multinuclear (1)H, (13)C, and (205)Tl NMR study combined with DFT calculations confirmed the TSAP' structure of the complex in aqueous solution, which exists as the Λ(λλλλ)/Δ(δδδδ) enantiomeric pair. (205)Tl NMR spectroscopy allowed the protonation constant associated with the protonation of the complex according to [Tl(dota)](-) + H(+) ⇆ [Tl(Hdota)] to be determined, which turned out to be pK(H)Tl(dota) = 1.4 ± 0.1. [Tl(dota)](-) does not react with Br(-), even when using an excess of the anion, but it forms a weak mixed complex with cyanide, [Tl(dota)](-) + CN(-) ⇆ [Tl(dota)(CN)](2-), with an equilibrium constant of Kmix = 6.0 ± 0.8. The dissociation of the [Tl(dota)](-) complex was determined by UV-vis spectrophotometry under acidic conditions using a large excess of Br(-), and it was found to follow proton-assisted kinetics and to take place very slowly (∼10 days), even in 1 M HClO4, with the estimated half-life of the process being in the 10(9) h range at neutral pH. The solution dynamics of [Tl(dota)](-) were investigated using (13)C NMR spectroscopy and DFT calculations. The (13)C NMR spectra recorded at low temperature (272 K) point to C4 symmetry of the complex in solution, which averages to C4v as the temperature increases. This dynamic behavior was attributed to the Λ(λλλλ) ↔ Δ(δδδδ) enantiomerization process, which involves both the inversion of the macrocyclic unit and the rotation of the pendant arms. According to our calculations, the arm-rotation process limits the Λ(λλλλ) ↔ �

Quantitative PET of EGFR expression in xenograft-bearing mice using {sup 64}Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody

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Cai, Weibo; Chen, Kai; He, Lina; Cao, Qizhen; Chen, Xiaoyuan [Stanford University School of Medicine, The Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford, CA (United States); Koong, Albert [Stanford University School of Medicine, Department of Radiation Oncology, Stanford, CA (United States)

2007-06-15

Cetuximab, a chimeric monoclonal antibody targeting epidermal growth factor receptor (EGFR) on the surface of cancer cells, was approved by the FDA to treat patients with metastatic colorectal cancer. It is currently also in advanced-stage development for the treatment of several other solid tumors. Here we report for the first time the quantitative positron emission tomography (PET) imaging of EGFR expression in xenograft-bearing mice using {sup 64}Cu-labeled cetuximab. We conjugated cetuximab with macrocyclic chelating agent 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA), labeled with {sup 64}Cu, and tested the resulting {sup 64}Cu-DOTA-cetuximab in seven xenograft tumor models. The tracer uptake measured by PET was correlated with the EGFR expression quantified by western blotting. The estimated human dosimetry based on the PET data in Sprague-Dawley rats was also calculated. MicroPET imaging showed that {sup 64}Cu-DOTA-cetuximab had increasing tumor activity accumulation over time in EGFR-positive tumors but relatively low uptake in EGFR-negative tumors at all times examined (<5%ID/g). There was a good correlation (R {sup 2} = 0.80) between the tracer uptake (measured by PET) and the EGFR expression level (measured by western blotting). Human dosimetry estimation indicated that the tracer may be safely administered to human patients for tumor diagnosis, with the dose-limiting organ being the liver. The success of EGFR-positive tumor imaging using {sup 64}Cu-DOTA-cetuximab can be translated into the clinic to characterize the pharmacokinetics, to select the right population of patients for EGFR-targeted therapy, to monitor the therapeutic efficacy of anti-EGFR treatment, and to optimize the dosage of either cetuximab alone or cetuximab in combination with other therapeutic agents. (orig.)

Quantitative bioanalysis of antibody-conjugated payload in monkey plasma using a hybrid immuno-capture LC-MS/MS approach: Assay development, validation, and a case study.

Science.gov (United States)

Liu, Ang; Kozhich, Alexander; Passmore, David; Gu, Huidong; Wong, Richard; Zambito, Frank; Rangan, Vangipuram S; Myler, Heather; Aubry, Anne-Françoise; Arnold, Mark E; Wang, Jian

2015-10-01

Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible. The in vivo change in drug antibody ratio (DAR), due to deconjugation, biotransformation or other clearance phenomena, generates unique and additional challenges for ADC analysis in biological samples. Here, we report a novel hybrid approach with immuno-capture of the ADC, payload cleavage by specific enzyme, and LC-MS/MS of the cleaved payload to quantitatively measure the concentration of payload molecules still attached to the antibody via linker in plasma. The ADC reference material used for the calibration curve is not likely to be identical to the ADC measured in study samples due to the change in DAR distribution over the PK time course. The assay clearly demonstrated that there was no bias in the measurement of antibody-conjugated payload for ADC with varying DAR, which thus allowed accurate quantification even when the DAR distribution dynamically changes in vivo. This hybrid assay was fully validated based on a combination of requirements for both chromatographic and ligand binding methods, and was successfully applied to support a GLP safety study in monkeys. Copyright © 2015 Elsevier B.V. All rights reserved.

Antibody drug conjugates and bystander killing: is antigen-dependent internalisation required?

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Staudacher, Alexander H; Brown, Michael P

2017-12-05

Antibody drug conjugates (ADCs) employ the exquisite specificity of tumour-specific monoclonal antibodies (mAb) for the targeted delivery of highly potent cytotoxic drugs to the tumour site. The chemistry of the linker, which connects the drug to the mAb, determines how and when the drug is released from the mAb. This, as well as the chemistry of the drug, can dictate whether the drug can diffuse into surrounding cells, resulting in 'bystander killing'. Initially, any bystander killing mechanism of action of an ADC was understood to involve an essential sequence of steps beginning with surface antigen targeting, internalisation, intracellular linker cleavage, drug release, and diffusion of drug away from the targeted cell. However, recent studies indicate that, depending on the linker and drug combination, this mechanism may not be essential and ADCs can be cleaved extracellularly or via other mechanisms. In this minireview, we will examine the role of bystander killing by ADCs and explore the emerging evidence of how this can occur independently of internalisation.

Radiolabelled of c-DOTA-RGD and c-DOTA-RGDf with 177Lu and evaluation in vitro and in vivo stability

International Nuclear Information System (INIS)

Vilchis J, A.

2010-01-01

Integrin αvβ3 has a critical role in tumor angio genesis and metastasis. Radiolabelled peptides based on the Arg-Gly-Asp (RGD) sequence have been reported as radiopharmaceuticals with high affinity and selectivity for the αvβ3 integrin. The aim of this study was to label c-DOTA-RGD and c-DOTA-RGDf peptides with 177 Lu and to evaluate their in vitro and in vivo stability as potential specific therapeutic radiopharmaceuticals. Labelled was carried out by direct reaction of 177 LuCl 3 with c-DOTA-RGD peptides in 1 M acetate buffer ph 5.5 at 90 o C for 30 min. Radiochemical purity and stability studies were realized by reversed phase HPLC and I TLC-Sg analyses in human serum and saline solution. Biological recognition was performed using MCF7 tumor cells (positive αvβ3) and in athymic mice with induced MCF7 tumors. Molecular mechanics and quantum mechanics calculations were performed to explain experimental results associated with the molecular recognition. 177 Lu-DOTA-RGD and 177 Lu-DOTA-RGDf were obtained with radiochemical purities > 95%, showing adequate in vitro and in vivo stability and specific binding to □ v □ 3 receptors. (Author)

Impact of cathepsin B-sensitive triggers and hydrophilic linkers on in vitro efficacy of novel site-specific antibody-drug conjugates.

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Bryden, Francesca; Martin, Camille; Letast, Stéphanie; Lles, Eva; Viéitez-Villemin, Inmaculada; Rousseau, Anaïs; Colas, Cyril; Brachet-Botineau, Marie; Allard-Vannier, Emilie; Larbouret, Christel; Viaud-Massuard, Marie-Claude; Joubert, Nicolas

2018-03-14

Herein we describe the synthesis and evaluation of four novel HER2-targeting, cathepsin B-sensitive antibody-drug conjugates bearing a monomethylauristatin E (MMAE) cytotoxic payload, constructed via the conjugation of cleavable linkers to trastuzumab using a site-specific bioconjugation methodology. These linkers vary by both cleavable trigger motif and hydrophilicity, containing one of two cathepsin B sensitive dipeptides (Val-Cit and Val-Ala), and engendered with either hydrophilic or hydrophobic character via application of a PEG 12 spacer. Through evaluation of physical properties, in vitro cytotoxicity, and receptor affinity of the resulting antibody-drug conjugates (ADCs), we have demonstrated that while both dipeptide triggers are effective, the increased hydrophobicity of the Val-Ala pair limits its utility within this type of linker. In addition, while PEGylation augments linker hydrophilicity, this change does not translate to more favourable ADC hydrophilicity or potency. While all described structures demonstrated excellent and similar in vitro cytotoxicity, the ADC with the ValCitPABMMAE linker shows the most promising combination of in vitro potency, structural homogeneity, and hydrophilicity, warranting further evaluation into its therapeutic potential.

Stimulation of protective antibodies against type Ia and Ib group B streptococci by a type Ia polysaccharide-tetanus toxoid conjugate vaccine.

OpenAIRE

Wessels, M R; Paoletti, L C; Rodewald, A K; Michon, F; DiFabio, J; Jennings, H J; Kasper, D L

1993-01-01

Antisera elicited by type Ia group B streptococci (GBS) contain antibodies that react with both type Ia and type Ib strains. Previous studies suggested that antibodies elicited by type Ia organisms recognized a carbohydrate antigen or epitope common to Ia and Ib strains. We now report the synthesis and immunogenicity testing of a type Ia polysaccharide-tetanus toxoid (Ia-TT) conjugate vaccine. Ia-TT elicited type Ia polysaccharide-specific immunoglobulin G antibodies in all three of the rabbi...

(68)Ga-DOTA-peptide: A novel molecular biomarker for nasopharyngeal carcinoma.

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Khor, Lih Kin; Loi, Hoi Yin; Sinha, Arvind Kumar; Tong, Kian Ti; Goh, Boon Cher; Loh, Kwok Seng; Lu, Suat-Jin

2016-04-01

Increased somatostatin receptor (SSTR) expression in patients with undifferentiated nasopharyngeal carcinoma (NPC) has been demonstrated with receptor autoradiography, (111) In-Octreotide scintigraphy, and (68) Ga-DOTA-TOC positron emission tomography (PET)/CT imaging. We sought to compare and correlate the uptake of fluorodeoxyglucose (FDG) and DOTA-NOC in undifferentiated NPC to ascertain the possible role of (68) Ga-DOTA-NOC PET/CT as a new imaging biomarker and to assess whether targeted peptide receptor radionuclide therapy is a feasible treatment option. After obtaining approval from our institutional review board, 4 patients with biopsy proven nonkeratinizing undifferentiated NPC who had just undergone routine staging/restaging (18) F-FDG PET/CT imaging were prospectively and consecutively recruited for (68) Ga-DOTA-NOC PET/CT imaging. Of these 4 patients, 3 were newly diagnosed with untreated NPC, whereas 1 patient was diagnosed with a case of recurrent NPC with previous treatment. These patients subsequently underwent (68) Ga-DOTA-NOC PET/CT within 10 days from the (18) F-FDG PET/CT to ensure lesion comparability. Tracer uptake in tumor lesions were assessed visually and semiquantitatively by measuring maximum standardized uptake values (SUVmax). There were 12 FDG-avid lesions of which 7 showed avid uptake of DOTA-NOC greater than liver uptake, whereas 5 showed low uptake of DOTA-NOC less than liver uptake. Subset analysis of the FDG-avid lesions at the primary and recurrent sites showed that all the FDG-avid primary tumors in the nasopharynx showed avid uptake of DOTA-NOC. On the contrary, the case of recurrent NPC showed avid FDG uptake but low DOTA-NOC uptake. Subset analysis of the suspicious FDG-avid cervical lymph nodes showed that 50% of them demonstrated avid DOTA-NOC uptake greater than liver uptake, whereas the remaining demonstrated low-grade DOTA-NOC uptake less than liver uptake. The 2 subcentimeter cervical lymph nodes that showed low

Method of stably radiolabeling antibodies with technetium and rhenium

International Nuclear Information System (INIS)

Paik, C.H.; Reba, R.C.; Eckelman, W.C.

1987-01-01

A method is described for labeling antibodies or antibody fragments with radionuclides of technetium or rhenium to obtain stable labeling, comprising: reacting a reduced radioisotope of technetium or rhenium with an antibody or antibody fragment, or a diethylenetriaminepentaacetic acid conjugated antibody or antibody fragment, in the presence of free or carrier-bound diethylenetriaminepentaacetic acid (DTPA). The amount of DTPA is sufficient to substantially completely inhibit binding of the reduced technetium or rhenium to nonstable binding sites of the antibody or antibody fragment, or the DTPA-conjugated antibody or antibody fragment. The resultant stably labeled antibody or antibody fragment, or DTPA[conjugated antibody or antibody fragment is recovered

Preliminary Therapy Evaluation of 225Ac-DOTA-c(RGDyK) Demonstrates that Cerenkov Radiation Derived from 225Ac Daughter Decay Can Be Detected by Optical Imaging for In Vivo Tumor Visualization

Science.gov (United States)

Pandya, Darpan N.; Hantgan, Roy; Budzevich, Mikalai M.; Kock, Nancy D.; Morse, David L.; Batista, Izadora; Mintz, Akiva; Li, King C.; Wadas, Thaddeus J.

2016-01-01

The theranostic potential of 225Ac-based radiopharmaceuticals continues to increase as researchers seek innovative ways to harness the nuclear decay of this radioisotope for therapeutic and imaging applications. This communication describes the evaluation of 225Ac-DOTA-c(RGDyK) in both biodistribution and Cerenkov luminescence imaging (CLI) studies. Initially, La-DOTA-c(RGDyK) was prepared as a non-radioactive surrogate to evaluate methodologies that would contribute to an optimized radiochemical synthetic strategy and estimate the radioactive conjugate's affinity for αvβ3, using surface plasmon resonance spectroscopy. Surface plasmon resonance spectroscopy studies revealed the IC50 and Ki of La-DOTA-c(RGDyK) to be 33 ± 13 nM and 26 ± 11 nM, respectively, and suggest that the complexation of the La3+ ion to the conjugate did not significantly alter integrin binding. Furthermore, use of this surrogate allowed optimization of radiochemical synthesis strategies to prepare 225Ac-DOTA-c(RGDyK) with high radiochemical purity and specific activity similar to other 225Ac-based radiopharmaceuticals. This radiopharmaceutical was highly stable in vitro. In vivo biodistribution studies confirmed the radiotracer's ability to target αvβ3 integrin with specificity; specificity was detected in tumor-bearing animals using Cerenkov luminescence imaging. Furthermore, tumor growth control was achieved using non-toxic doses of the radiopharmaceutical in U87mg tumor-bearing nude mice. To our knowledge, this is the first report to describe the CLI of αvβ3+ tumors in live animals using the daughter products derived from 225Ac decay in situ. This concept holds promise to further enhance development of targeted alpha particle therapy. PMID:27022417

Engineering of ultra-small diagnostic nanoprobes through oriented conjugation of single-domain antibodies and quantum dots

OpenAIRE

sprotocols

2015-01-01

Authors: Alyona Sukhanova, Klervi Even-Desrumeaux, Patrick Chames, Daniel Baty, Mikhail Artemyev, Vladimir Oleinikov & Igor Nabiev ### Abstract Nanoparticle-based biodetection commonly employs monoclonal antibodies (mAbs) for targeting. Although several types of conjugates have been used for biomarker labeling, the large size of mAbs limits the number of ligands per nanoparticle, impedes their intratumoral distribution, and limits intracellular penetration. Furthermore, the condition...

Somatostatin-based Radiopeptide Therapy with [177Lu-DOTA]-TOC versus [90Y-DOTA]-TOC in Neuroendocrine Tumors

OpenAIRE

Romer A Seiler D Marincek N Brunner P Koller MT Ng QK Maecke HR Muller-Brand J Rochlitz C B

2014-01-01

PURPOSE: Somatostatin based radiopeptide treatment is generally performed using the ß emitting radionuclides (90)Y or (177)Lu. The present study aimed at comparing benefits and harms of both therapeutic approaches. METHODS: In a comparative cohort study patients with advanced neuroendocrine tumours underwent repeated cycles of [(90)Y DOTA] TOC or [(177)Lu DOTA] TOC until progression of disease or permanent adverse events. Multivariable Cox regression and competing risks regression were emplo...

Imaging Tumor Vasculature Noninvasively with Positron Emission Tomography and RGD Peptides Labeled with Copper 64 Using the Bifunctonal Chelates DOTA, Oxo-DO3A. and PCTA

Directory of Open Access Journals (Sweden)

Donald T.T. Yapp

2013-06-01

Full Text Available Two novel bifunctional chelates, 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15,11,13-triene-3,6,9-triacetic acid (PCTA and 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (Oxo-DO3A, were found to radiolabel antibodies with copper 64 (64Cu well for positron emission tomography (PET. In this study, the same chelators were used to radiolabel peptides with 64Cu for PET imaging of angiogenesis. PCTA, Oxo-DO3A, and 1,4,7,10-tetraazacyclododecane-N,N‘,N“,N”’-tetraacetic acid (DOTA were conjugated to cyclic-(RGDyK, and their binding affinities were confirmed. Conditions for 64Cu radiolabeling were optimized for maximum yield and specific activity. The in vitro stability of the radiolabeled compounds was challenged with serum incubation. PET studies were carried out in a non-αvβ3-expressing tumor model to evaluate the compounds' specificity for proliferating tumor vasculature and their in vivo pharmacokinetics. The PCTA and Oxo-DO3A bioconjugates were labeled with 64Cu at higher effective specific activity and radiochemical yield than the DOTA bioconjugate. In the imaging studies, all the 64Cu bioconjugates could be used to visualize the tumor and the radiotracer uptake was blocked with cyclic-(RGDyK. Target uptake of each bioconjugate was similar, but differences in other tissues were observed. 64Cu-PCTA-RGD showed the best clearance from nontarget tissue and the highest tumor to nontarget ratios. PCTA was the most promising bifunctional chelate for 64Cu peptide imaging and warrants further investigation.

Evaluation of (68)Ga- and (177)Lu-DOTA-PEG4-LLP2A for VLA-4-Targeted PET Imaging and Treatment of Metastatic Melanoma.

Science.gov (United States)

Beaino, Wissam; Nedrow, Jessie R; Anderson, Carolyn J

2015-06-01

Malignant melanoma is a highly aggressive cancer, and the incidence of this disease is increasing worldwide at an alarming rate. Despite advances in the treatment of melanoma, patients with metastatic disease still have a poor prognosis and low survival rate. New strategies, including targeted radiotherapy, would provide options for patients who become resistant to therapies such as BRAF inhibitors. Very late antigen-4 (VLA-4) is expressed on melanoma tumor cells in higher levels in more aggressive and metastatic disease and may provide an ideal target for drug delivery and targeted radiotherapy. In this study, we evaluated (177)Lu- and (68)Ga-labeled DOTA-PEG4-LLP2A as a VLA-4-targeted radiotherapeutic with a companion PET agent for diagnosis and monitoring metastatic melanoma treatment. DOTA-PEG4-LLP2A was synthesized by solid-phase synthesis. The affinity of (177)Lu- and (68)Ga-labeled DOTA-PEG4-LLP2A to VLA-4 was determined in B16F10 melanoma cells by saturation binding and competitive binding assays, respectively. Biodistribution of the LLP2A conjugates was determined in C57BL/6 mice bearing B16F10 subcutaneous tumors, while PET/CT imaging was performed in subcutaneous and metastatic models. (177)Lu-DOTA-PEG4-LLP2A showed high affinity to VLA-4 with a Kd of 4.1 ± 1.5 nM and demonstrated significant accumulation in the B16F10 melanoma tumor after 4 h (31.5 ± 7.8%ID/g). The tumor/blood ratio of (177)Lu-DOTA-PEG4-LLP2A was highest at 24 h (185 ± 26). PET imaging of metastatic melanoma with (68)Ga-DOTA-PEG4-LLP2A showed high uptake in sites of metastases and correlated with bioluminescence imaging of the tumors. These data demonstrate that (177)Lu-DOTA-PEG4-LLP2A has potential as a targeted therapeutic for treating melanoma as well as other VLA-4-expressing tumors. In addition, (68)Ga-DOTA-PEG4-LLP2A is a readily translatable companion PET tracer for imaging of metastatic melanoma.

Positron emission tomography study on pancreatic somatostatin receptors in normal and diabetic rats with 68Ga-DOTA-octreotide: a potential PET tracer for beta cell mass measurement.

Science.gov (United States)

Sako, Takeo; Hasegawa, Koki; Nishimura, Mie; Kanayama, Yousuke; Wada, Yasuhiro; Hayashinaka, Emi; Cui, Yilong; Kataoka, Yosky; Senda, Michio; Watanabe, Yasuyoshi

2013-12-06

Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia, and the loss or dysfunction of pancreatic beta cells has been reported before the appearance of clinical symptoms and hyperglycemia. To evaluate beta cell mass (BCM) for improving the detection and treatment of DM at earlier stages, we focused on somatostatin receptors that are highly expressed in the pancreatic beta cells, and developed a positron emission tomography (PET) probe derived from octreotide, a metabolically stable somatostatin analog. Octreotide was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a chelating agent, and labeled with (68)Gallium ((68)Ga). After intravenous injection of (68)Ga-DOTA-octreotide, a 90-min emission scan of the abdomen was performed in normal and DM model rats. The PET studies showed that (68)Ga-DOTA-octreotide radioactivity was highly accumulated in the pancreas of normal rats and that the pancreatic accumulation was significantly reduced in the rats administered with an excess amount of unlabeled octreotide or after treatment with streptozotocin, which was used for the chemical induction of DM in rats. These results were in good agreement with the ex vivo biodistribution data. These results indicated that the pancreatic accumulation of (68)Ga-DOTA-octreotide represented specific binding to the somatostatin receptors and reflected BCM. Therefore, PET imaging with (68)Ga-DOTA-octreotide could be a potential tool for evaluating BCM. Copyright © 2013 Elsevier Inc. All rights reserved.

Considerations for the nonclinical safety evaluation of antibody drug conjugates for oncology.

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Roberts, Stanley A; Andrews, Paul A; Blanset, Diann; Flagella, Kelly M; Gorovits, Boris; Lynch, Carmel M; Martin, Pauline L; Kramer-Stickland, Kimberly; Thibault, Stephane; Warner, Garvin

2013-12-01

Antibody drug conjugates (ADCs) include monoclonal antibodies that are linked to cytotoxic small molecules. A number of these agents are currently being developed as anti-cancer agents designed to improve the therapeutic index of the cytotoxin (i.e., cytotoxic small molecule or cytotoxic agent) by specifically delivering it to tumor cells. This paper presents primary considerations for the nonclinical safety evaluation of ADCs and includes strategies for the evaluation of the entire ADC or the various individual components (i.e., antibody, linker or the cytotoxin). Considerations are presented on how to design a nonclinical safety assessment program to identify the on- and off-target toxicities to enable first-in-human (FIH) studies. Specific discussions are also included that provide details as to the need and how to conduct the studies for evaluating ADCs in genetic toxicology, tissue cross-reactivity, safety pharmacology, carcinogenicity, developmental and reproductive toxicology, biotransformation, toxicokinetic monitoring, bioanalytical assays, immunogenicity testing, test article stability and the selection of the FIH dose. Given the complexity of these molecules and our evolving understanding of their properties, there is no single all-encompassing nonclinical strategy. Instead, each ADC should be evaluated on a case-by-case scientifically-based approach that is consistent with ICH and animal research guidelines. Copyright © 2013 Elsevier Inc. All rights reserved.

(68)Ga-DOTA-Siglec-9 PET/CT imaging of peri-implant tissue responses and staphylococcal infections.

Science.gov (United States)

Ahtinen, Helena; Kulkova, Julia; Lindholm, Laura; Eerola, Erkki; Hakanen, Antti J; Moritz, Niko; Söderström, Mirva; Saanijoki, Tiina; Jalkanen, Sirpa; Roivainen, Anne; Aro, Hannu T

2014-01-01

Staphylococcus epidermidis (S. epidermidis) has emerged as one of the leading pathogens of biomaterial-related infections. Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible endothelial molecule controlling extravasation of leukocytes. Sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) is a leukocyte ligand of VAP-1. We hypothesized that (68)Ga-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated Siglec-9 motif containing peptide ((68)Ga-DOTA-Siglec-9) could detect inflammatory response due to S. epidermidis peri-implant infection by positron emission tomography (PET). Thirty Sprague-Dawley rats were randomized into three groups. A sterile catheter was implanted into the medullary canal of the left tibia. In groups 1 and 2, the implantation was followed by peri-implant injection of S. epidermidis or Staphylococcus aureus (S. aureus) with adjunct injections of aqueous sodium morrhuate. In group 3, sterile saline was injected instead of bacteria and no aqueous sodium morrhuate was used. At 2 weeks after operation, (68)Ga-DOTA-Siglec-9 PET coupled with computed tomography (CT) was performed with the measurement of the standardized uptake value (SUV). The presence of the implant-related infection was verified by microbiological analysis, imaging with fluorescence microscope, and histology. The in vivo PET results were verified by ex vivo measurements by gamma counter. In group 3, the tibias with implanted sterile catheters showed an increased local uptake of (68)Ga-DOTA-Siglec-9 compared with the intact contralateral bones (SUVratio +29.5%). (68)Ga-DOTA-Siglec-9 PET detected inflammation induced by S. epidermidis and S. aureus catheter-related bone infections (SUVratio +58.1% and +41.7%, respectively). The tracer uptake was significantly higher in the S. epidermidis group than in group 3 without bacterial inoculation, but the difference between S. epidermidis and S. aureus groups was not statistically significant. The

64Cu-DOTA as a surrogate positron analog of Gd-DOTA for cardiac fibrosis detection with PET: pharmacokinetic study in a rat model of chronic MI.

Science.gov (United States)

Kim, Heejung; Lee, Sung-Jin; Davies-Venn, Cynthia; Kim, Jin Su; Yang, Bo Yeun; Yao, Zhengsheng; Kim, Insook; Paik, Chang H; Bluemke, David A

2016-02-01

The aim of this study was to investigate the pharmacokinetics of (64)Cu-DOTA (1,4,7,10-azacyclododecane-N,N',N'',N'''-tetraacetic acid), a positron surrogate analog of the late gadolinium (Gd)-enhancement cardiac magnetic resonance agent, Gd-DOTA, in a rat model of chronic myocardial infarction (MI) and its microdistribution in the cardiac fibrosis by autoradiography. DOTA was labeled with (64)Cu-acetate. CD rats (n=5) with MI by left anterior descending coronary artery ligation and normal rats (n=6) were injected intravenously with (64)Cu-DOTA (18.5 MBq, 0.02 mmol DOTA/kg). Dynamic PET imaging was performed for 60 min after injection. (18)F-Fluorodeoxyglucose ([(18)F]-FDG) PET imaging was performed to identify the viable myocardium. For the region of interest analysis, the (64)Cu-DOTA PET image was coregistered to the [(18)F]-FDG PET image. To validate the PET images, slices of heart samples from the base to the apex were analyzed using autoradiography and by histological staining with Masson's trichrome. (64)Cu-DOTA was rapidly taken up in the infarct area. The time-activity curves demonstrated that (64)Cu-DOTA concentrations in the blood, fibrotic tissue, and perfusion-rich organs peaked within a minute post injection; thereafter, it was rapidly washed out in parallel with blood clearance and excreted through the renal system. The blood clearance curve was biphasic, with a distribution half-life of less than 3 min and an elimination half-life of ∼21.8 min. The elimination half-life of (64)Cu-DOTA from the focal fibrotic tissue (∼22.4 min) and the remote myocardium (∼20.1 min) was similar to the blood elimination half-life. Consequently, the uptake ratios of focal fibrosis-to-blood and remote myocardium-to-blood remained stable for the time period between 10 and 60 min. The corresponding ratios obtained from images acquired from 30 to 60 min were 1.09 and 0.59, respectively, indicating that the concentration of (64)Cu-DOTA in the focal

111In-DOTA-dPhe1-Tyr3-octreotide, 111In-DOTA-lanreotide and 67Ga citrate scintigraphy for visualisation of extranodal marginal zone B-cell lymphoma of the MALT type: a comparative study

International Nuclear Information System (INIS)

Li, Shuren; Kurtaran, Amir; Li, Mei; Traub-Weidinger, Tatjana; Kienast, Oskar; Schima, Wolfgang; Angelberger, Peter; Virgolini, Irene; Raderer, Markus; Dudczak, Robert

2003-01-01

Somatostatin receptor (SSTR) scintigraphy and gallium-67 citrate ( 67 Ga) scintigraphy have been used for visualisation of Hodgkin's lymphoma and non-Hodgkin's lymphoma. However, experience with B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) type is very limited. The aim of this study was to prospectively compare the 67 Ga scintigraphy results with those obtained by 111 In-DOTA-dPhe 1 -Tyr 3 -octreotide ( 111 In-DOTA-TOCT) and 111 In-DOTA-lanreotide ( 111 In-DOTA-LAN) scintigraphy in patients with proven MALT-type lymphoma. Comparative scintigraphic examinations using 67 Ga, 111 In-DOTA-TOCT and 111 In-DOTA-LAN were performed in 18 patients (11 female and 7 male, median age 64±15 years) with histologically verified MALT-type lymphomas of various origin. Planar and single-photon emission tomography imaging acquisitions were performed after injection of a mean dose of 185±26 MBq 67 Ga and 165±20 MBq 111 In-DOTA-TOCT or 111 In-DOTA-LAN. All scintigraphic results were correlated with other conventional examinations including gastroscopy, colonoscopy, endosonoscopy, ophthalmologic investigation, CT of the thorax and abdomen and bone marrow biopsy. This comparative study showed that 67 Ga scintigraphy found abnormalities in 10 of 16 patients (63%) and detected 18 of 31 clinically involved sites (58%), but was false positive in three patients. 111 In-DOTA-TOCT found abnormalities in 9 of 15 patients (60%) and detected 15 of 27 clinical lesions (56%); it was false positive in two patients. 111 In-DOTA-LAN scintigraphy showed abnormalities in 7 of 11 patients (64%) and found 12 of 22 clinical lesions (55%). False-positive 111 In-DOTA-LAN scan results were found in two patients. For supra-diaphragmatic lesions, 67 Ga scintigraphy detected 12 of 16 sites (75%). 111 In-DOTA-TOCT scintigraphy revealed 7 of 15 lesions (47%). 111 In-DOTA-LAN showed 6 of 12 positive sites (50%). For infra-diaphragmatic involvement, the sensitivities of 67 Ga, 111 In-DOTA

Synthesis and Characterization of Anti-HER2 Antibody Conjugated CdSe/CdZnS Quantum Dots for Fluorescence Imaging of Breast Cancer Cells

Directory of Open Access Journals (Sweden)

Takashi Jin

2009-11-01

Full Text Available The early detection of HER2 (human epidermal growth factor receptor 2 status in breast cancer patients is very important for the effective implementation of anti-HER2 antibody therapy. Recently, HER2 detections using antibody conjugated quantum dots (QDs have attracted much attention. QDs are a new class of fluorescent materials that have superior properties such as high brightness, high resistance to photo-bleaching, and multi-colored emission by a single-light source excitation. In this study, we synthesized three types of anti-HER2 antibody conjugated QDs (HER2Ab-QDs using different coupling agents (EDC/sulfo-NHS, iminothiolane/sulfo-SMCC, and sulfo-SMCC. As water-soluble QDs for the conjugation of antibody, we used glutathione coated CdSe/CdZnS QDs (GSH-QDs with fluorescence quantum yields of 0.23~0.39 in aqueous solution. Dispersibility, hydrodynamic size, and apparent molecular weights of the GSH-QDs and HER2Ab-QDs were characterized by using dynamic light scattering, fluorescence correlation spectroscopy, atomic force microscope, and size-exclusion HPLC. Fluorescence imaging of HER2 overexpressing cells (KPL-4 human breast cancer cell line was performed by using HER2Ab-QDs as fluorescent probes. We found that the HER2Ab-QD prepared by using SMCC coupling with partially reduced antibody is a most effective probe for the detection of HER2 expression in KPL-4 cells. We have also studied the size dependency of HER2Ab-QDs (with green, orange, and red emission on the fluorescence image of KPL-4 cells.

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

Science.gov (United States)

Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

2017-01-01

Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

Technology of DTPA and immunoglobulins conjugation and their attachment to 90Y and 177Lu radionuclides

International Nuclear Information System (INIS)

Rekova, M.; Jedinakova-Krizova, V.

2010-01-01

The aim of the study of labeling of ligand-antibody conjugates was to find optimal conditions of preparing of these conjugates and appropriate radioactivity of selected nuclide for applications in nuclear medicine. Conjugation of the γ-immunoglobulin G (human or bovine IgG, polyclonal antibodies) and bifunctional chelating agent, diethylenetriaminepentaacetic acid dianhydride (cDTPAA), was carried out. Various values of the cDTPAA/antibody ratio, the weight concentration of polyclonal or monoclonal antibodies (MEM-97) and buffers were used. Further, the labeling conditions of the DTPA-IgG conjugate by radionuclides 90 Y and 177 Lu were optimized, and the labeling yield and the conjugation ratio of prepared radionuclide-DTPA-IgG conjugates was determined. Optimal incubation time of the immunoglobulin conjugation was obtained at 30 min from mixing of individual components. The labeling yield of radionuclide-DTPA-antibody conjugate higher than 95% was achieved. Higher values of conjugation ratio of radionuclide-DTPA-antibody conjugate were achieved in 0.1 mol L -1 carbonate buffer, pH 8.5, and the 0.1 mol L -1 carbonate buffer is suitable for studied conjugation systems. This study showed that the labeling yield as well as the conjugation ratio of tested systems depend on the amount of antibody substance, bifunctional chelating agent/antibody molar ratio and pH value of the buffer used. (author)

Comparison of sequential planar {sup 177}Lu-DOTA-TATE dosimetry scans with {sup 68}Ga-DOTA-TATE PET/CT images in patients with metastasized neuroendocrine tumours undergoing peptide receptor radionuclide therapy

Energy Technology Data Exchange (ETDEWEB)

Sainz-Esteban, Aurora; Carril, Jose Manuel [Hospital Universitario Marques de Valdecilla, Department of Nuclear Medicine, Santander (Spain); Prasad, Vikas; Schuchardt, Christiane; Zachert, Carolin; Baum, Richard P. [Zentralklinik Bad Berka, Department of Nuclear Medicine and Centre for PET/CT, Bad Berka (Germany)

2012-03-15

The aim of the study was to compare sequential {sup 177}Lu-DOTA-TATE planar scans ({sup 177}Lu-DOTA-TATE) in patients with metastasized neuroendocrine tumours (NET) acquired during peptide receptor radionuclide therapy (PRRT) for dosimetry purposes with the pre-therapeutic {sup 68}Ga-DOTA-TATE positron emission tomography (PET)/CT ({sup 68}Ga-DOTA-TATE) maximum intensity projection (MIP) images obtained in the same patients concerning the sensitivity of the different methods. A total of 44 patients (59 {+-} 11 years old) with biopsy-proven NET underwent {sup 68}Ga-DOTA-TATE and {sup 177}Lu-DOTA-TATE imaging within 7.9 {+-} 7.5 days between the two examinations. {sup 177}Lu-DOTA-TATE planar images were acquired at 0.5, 2, 24, 48 and 72 h post-injection; lesions were given a score from 0 to 4 depending on the uptake of the radiopharmaceutical (0 being lowest and 4 highest). The number of tumour lesions which were identified on {sup 177}Lu-DOTA-TATE scans (in relation to the acquisition time after injection of the therapeutic dose as well as with regard to the body region) was compared to those detected on {sup 68}Ga-DOTA-TATE studies obtained before PRRT. A total of 318 lesions were detected; 280 (88%) lesions were concordant. Among the discordant lesions, 29 were {sup 68}Ga-DOTA-TATE positive and {sup 177}Lu-DOTA-TATE negative, whereas 9 were {sup 68}Ga-DOTA-TATE negative and {sup 177}Lu-DOTA-TATE positive. The sensitivity, positive predictive value and accuracy for {sup 177}Lu-DOTA-TATE as compared to {sup 68}Ga-DOTA-TATE were 91, 97 and 88%, respectively. Significantly more lesions were seen on the delayed (72 h) {sup 177}Lu-DOTA-TATE images (91%) as compared to the immediate (30 min) images (68%). The highest concordance was observed for bone metastases (97%) and the lowest for head/neck lesions (75%). Concordant lesions (n = 77; mean size 3.8 cm) were significantly larger than discordant lesions (n = 38; mean size 1.6 cm) (p < 0.05). No such significance was

Detection of early stage atherosclerotic plaques using PET and CT fusion imaging targeting P-selectin in low density lipoprotein receptor-deficient mice

Energy Technology Data Exchange (ETDEWEB)

Nakamura, Ikuko, E-mail: nakamuri@riken.jp [RIKEN Center for Molecular Imaging Science, Kobe (Japan); Department of Cardiovascular Medicine, Saga University, Saga (Japan); Hasegawa, Koki [RIKEN Center for Molecular Imaging Science, Kobe (Japan); Department of Pathology and Experimental Medicine, Kumamoto University, Kumamoto (Japan); Wada, Yasuhiro [RIKEN Center for Molecular Imaging Science, Kobe (Japan); Hirase, Tetsuaki; Node, Koichi [Department of Cardiovascular Medicine, Saga University, Saga (Japan); Watanabe, Yasuyoshi, E-mail: yywata@riken.jp [RIKEN Center for Molecular Imaging Science, Kobe (Japan)

2013-03-29

Highlights: ► P-selectin regulates leukocyte recruitment as an early stage event of atherogenesis. ► We developed an antibody-based molecular imaging probe targeting P-selectin for PET. ► This is the first report on successful PET imaging for delineation of P-selectin. ► P-selectin is a candidate target for atherosclerotic plaque imaging by clinical PET. -- Abstract: Background: Sensitive detection and qualitative analysis of atherosclerotic plaques are in high demand in cardiovascular clinical settings. The leukocyte–endothelial interaction mediated by an adhesion molecule P-selectin participates in arterial wall inflammation and atherosclerosis. Methods and results: A {sup 64}Cu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid conjugated anti-P-selectin monoclonal antibody ({sup 64}Cu-DOTA-anti-P-selectin mAb) probe was prepared by conjugating an anti-P-selectin monoclonal antibody with DOTA followed by {sup 64}Cu labeling. Thirty-six hours prior to PET and CT fusion imaging, 3 MBq of {sup 64}Cu-DOTA-anti-P-selectin mAb was intravenously injected into low density lipoprotein receptor-deficient Ldlr-/- mice. After a 180 min PET scan, autoradiography and biodistribution of {sup 64}Cu-DOTA-anti-P-selectin monoclonal antibody was examined using excised aortas. In Ldlr-/- mice fed with a high cholesterol diet for promotion of atherosclerotic plaque development, PET and CT fusion imaging revealed selective and prominent accumulation of the probe in the aortic root. Autoradiography of aortas that demonstrated probe uptake into atherosclerotic plaques was confirmed by Oil red O staining for lipid droplets. In Ldlr-/- mice fed with a chow diet to develop mild atherosclerotic plaques, probe accumulation was barely detectable in the aortic root on PET and CT fusion imaging. Probe biodistribution in aortas was 6.6-fold higher in Ldlr-/- mice fed with a high cholesterol diet than in those fed with a normal chow diet. {sup 64}Cu-DOTA-anti-P-selectin m

Positron Emission Tomography Based Analysis of Long-Circulating Cross-Linked Triblock Polymeric Micelles in a U87MG Mouse Xenograft Model and Comparison of DOTA and CB-TE2A as Chelators of Copper-64

DEFF Research Database (Denmark)

Jensen, Andreas Tue Ingemann; Binderup, Tina; Ek, Pramod Kumar

2014-01-01

Copolymers of ABC-type (PEG-PHEMA-PCMA) architecture were prepared by atom transfer radical polymerization and formulated as micelles with functionalizable primary alcohols in the shell-region (PHEMA-block) to which the metal-ion chelators DOTA or CB-TE2A were conjugated. Using this micelle system...... we compared the in vivo stabilities of DOTA and CB-TE2A as chelators of 64Cu in micelle nanoparticles. The coumarin polymer (PCMA-block) micelle core was cross-linked by UV irradiation at 2 W/cm2 for 30 min. The cross-linked micelles were labeled with 64Cu at room temperature for 2 h (DOTA) or 80 °C...... for 3 h (CB-TE2A), giving labeling efficiencies of 60–76% (DOTA) and 40–47% (CB-TE2A). 64Cu-micelles were injected into tumor-bearing mice (8 mg/kg) and PET/CT scans were carried out at 1, 22, and 46 h postinjection. The micelles showed good blood stability (T1/2: 20–26 h) and tumor uptake...

Pharmacokinetics and pharmacodynamics of DSTA4637A: A novel THIOMAB™ antibody antibiotic conjugate against Staphylococcus aureus in mice.

Science.gov (United States)

Zhou, Chenguang; Lehar, Sophie; Gutierrez, Johnny; Rosenberger, Carrie M; Ljumanovic, Nina; Dinoso, Jason; Koppada, Neelima; Hong, Kyu; Baruch, Amos; Carrasco-Triguero, Montserrat; Saad, Ola; Mariathasan, Sanjeev; Kamath, Amrita V

DSTA4637A, a novel THIOMAB™ antibody antibiotic conjugate (TAC) against Staphylococcus aureus (S. aureus), is currently being investigated as a potential therapy against S. aureus infections. Structurally, TAC is composed of an anti-S. aureus antibody linked to a potent antibiotic, dmDNA31. The goal of the current study was to characterize the pharmacokinetics (PK) of TAC in mice, assess the effect of S. aureus infection on its PK, and evaluate its pharmacodynamics (PD) by measuring the bacterial load in various organs at different timepoints following TAC treatment. Plasma concentrations of 3 analytes, total antibody (TAb), antibody-conjugated dmDNA31 (ac-dmDNA31), and unconjugated dmDNA31, were measured in these studies. In non-infected mice (target antigen absent), following intravenous (IV) administration of a single dose of TAC, systemic concentration-time profiles of both TAb and ac-dmDNA31 were bi-exponential and characterized by a short distribution phase and a long elimination phase as expected for a monoclonal antibody-based therapeutic. Systemic exposures of both TAb and ac-dmDNA31 were dose proportional over the dose range tested (5 to 50 mg/kg). In a mouse model of systemic S. aureus infection (target antigen present), a single IV dose of TAC demonstrated PK behavior similar to that in the non-infected mice, and substantially reduced bacterial load in the heart, kidney, and bones on 7 and 14 d post dosing. These findings have increased our understanding of the PK and PK/PD of this novel molecule, and have shown that at efficacious dose levels the presence of S. aureus infection had minimal effect on TAC PK.

68Ga/177Lu-labeled DOTA-TATE shows similar imaging and biodistribution in neuroendocrine tumor model.

Science.gov (United States)

Liu, Fei; Zhu, Hua; Yu, Jiangyuan; Han, Xuedi; Xie, Qinghua; Liu, Teli; Xia, Chuanqin; Li, Nan; Yang, Zhi

2017-06-01

Somatostatin receptors are overexpressed in neuroendocrine tumors, whose endogenous ligands are somatostatin. DOTA-TATE is an analogue of somatostatin, which shows high binding affinity to somatostatin receptors. We aim to evaluate the 68 Ga/ 177 Lu-labeling DOTA-TATE kit in neuroendocrine tumor model for molecular imaging and to try human-positron emission tomography/computed tomography imaging of 68 Ga-DOTA-TATE in neuroendocrine tumor patients. DOTA-TATE kits were formulated and radiolabeled with 68 Ga/ 177 Lu for 68 Ga/ 177 Lu-DOTA-TATE (M-DOTA-TATE). In vitro and in vivo stability of 177 Lu-DOTA-TATE were performed. Nude mice bearing human tumors were injected with 68 Ga-DOTA-TATE or 177 Lu-DOTA-TATE for micro-positron emission tomography and micro-single-photon emission computed tomography/computed tomography imaging separately, and clinical positron emission tomography/computed tomography images of 68 Ga-DOTA-TATE were obtained at 1 h post-intravenous injection from patients with neuroendocrine tumors. Micro-positron emission tomography and micro-single-photon emission computed tomography/computed tomography imaging of 68 Ga-DOTA-TATE and 177 Lu-DOTA-TATE both showed clear tumor uptake which could be blocked by excess DOTA-TATE. In addition, 68 Ga-DOTA-TATE-positron emission tomography/computed tomography imaging in neuroendocrine tumor patients could show primary and metastatic lesions. 68 Ga-DOTA-TATE and 177 Lu-DOTA-TATE could accumulate in tumors in animal models, paving the way for better clinical peptide receptor radionuclide therapy for neuroendocrine tumor patients in Asian population.

Production and characterization of peptide antibodies

DEFF Research Database (Denmark)

Trier, Nicole Hartwig; Hansen, Paul Robert; Houen, Gunnar

2012-01-01

Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies...... are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors......, including solid-phase peptide-carrier conjugation and peptide-carrier conjugation in solution. Upon immunization, adjuvants such as Al(OH)(3) are added together with the immunogenic peptide-carrier conjugate, which usually leads to high-titred antisera. Following immunization and peptide antibody...

Synthesis of a Fluorescently Labeled 68Ga-DOTA-TOC Analog for Somatostatin Receptor Targeting.

Science.gov (United States)

Ghosh, Sukhen C; Hernandez Vargas, Servando; Rodriguez, Melissa; Kossatz, Susanne; Voss, Julie; Carmon, Kendra S; Reiner, Thomas; Schonbrunn, Agnes; Azhdarinia, Ali

2017-07-13

Fluorescently labeled imaging agents can identify surgical margins in real-time to help achieve complete resections and minimize the likelihood of local recurrence. However, photon attenuation limits fluorescence-based imaging to superficial lesions or lesions that are a few millimeters beneath the tissue surface. Contrast agents that are dual-labeled with a radionuclide and fluorescent dye can overcome this limitation and combine quantitative, whole-body nuclear imaging with intraoperative fluorescence imaging. Using a multimodality chelation (MMC) scaffold, IRDye 800CW was conjugated to the clinically used somatostatin analog, 68 Ga-DOTA-TOC, to produce the dual-labeled analog, 68 Ga-MMC(IRDye 800CW)-TOC, with high yield and specific activity. In vitro pharmacological assays demonstrated retention of receptor-targeting properties for the dual-labeled compound with robust internalization that was somatostatin receptor (SSTR) 2-mediated. Biodistribution studies in mice identified the kidneys as the primary excretion route for 68 Ga-MMC(IRDye 800CW)-TOC, along with clearance via the reticuloendothelial system. Higher uptake was observed in most tissues compared to 68 Ga-DOTA-TOC but decreased as a function of time. The combination of excellent specificity for SSTR2-expressing cells and suitable biodistribution indicate potential application of 68 Ga-MMC(IRDye 800CW)-TOC for intraoperative detection of SSTR2-expressing tumors.

Development of a large peptoid-DOTA combinatorial library.

Science.gov (United States)

Singh, Jaspal; Lopes, Daniel; Gomika Udugamasooriya, D

2016-09-01

Conventional one-bead one-compound (OBOC) library synthesis is typically used to identify molecules with therapeutic value. The design and synthesis of OBOC libraries that contain molecules with imaging or even potentially therapeutic and diagnostic capacities (e.g. theranostic agents) has been overlooked. The development of a therapeutically active molecule with a built-in imaging component for a certain target is a daunting task, and structure-based rational design might not be the best approach. We hypothesize to develop a combinatorial library with potentially therapeutic and imaging components fused together in each molecule. Such molecules in the library can be used to screen, identify, and validate as direct theranostic candidates against targets of interest. As the first step in achieving that aim, we developed an on-bead library of 153,600 Peptoid-DOTA compounds in which the peptoids are the target-recognizing and potentially therapeutic components and the DOTA is the imaging component. We attached the DOTA scaffold to TentaGel beads using one of the four arms of DOTA, and we built a diversified 6-mer peptoid library on the remaining three arms. We evaluated both the synthesis and the mass spectrometric sequencing capacities of the test compounds and of the final library. The compounds displayed unique ionization patterns including direct breakages of the DOTA scaffold into two units, allowing clear decoding of the sequences. Our approach provides a facile synthesis method for the complete on-bead development of large peptidomimetic-DOTA libraries for screening against biological targets for the identification of potential theranostic agents in the future. © 2016 The Authors. Biopolymers Published by Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 673-684, 2016. © 2016 The Authors. Biopolymers Published by Wiley Periodicals, Inc.

Value of 111In-DOTA-lanreotide and 111In-DOTA-DPhe1-Tyr3-octreotide in differentiated thyroid cancer: results of in vitro binding studies and in vivo comparison with 18F-FDG PET

International Nuclear Information System (INIS)

Rodrigues, Margarida; Virgolini, Irene; Traub-Weidinger, Tatjana; Leimer, Maria; Li, Shuren; Dudczak, Robert; Andreae, Fritz; Angelberger, Peter

2005-01-01

Radioiodine-negative thyroid cancer presents diagnostic and therapeutic difficulties, warranting the implementation of new imaging and treatment strategies. The purpose of this study was twofold. First, we investigated in vitro the binding characteristics of 111 In-DOTA-lanreotide ( 111 In-DOTA-LAN) and 111 In-DOTA-DPhe 1 -Tyr 3 -octreotide ( 111 In-DOTA-TOC) to cells derived from differentiated thyroid cancer (DTC). Second, we evaluated the value of somatostatin receptor (SSTR) scintigraphy with these radioligands, as compared with 18 F-fluorodeoxyglucose (FDG) positron emission tomography (PET), for the detection of tumour lesions in DTC patients. Binding of 111 In-DOTA-LAN and 111 In-DOTA-TOC to cells isolated from surgically removed thyroid tissue was evaluated in vitro by performing saturation and displacement studies. Eighteen DTC patients with elevated thyroglobulin (12 radioiodine-negative, six radioiodine-positive) were investigated with 111 In-DOTA-LAN, 111 In-DOTA-TOC and 18 F-FDG PET scans. Large numbers of SSTR binding sites for 111 In-DOTA-LAN and 111 In-DOTA-TOC were found on the cells investigated. Both SSTR radioligands exhibited a high binding affinity for these SSTR binding sites. 111 In-DOTA-LAN and 111 In-DOTA-TOC scintigraphy detected 37 and 33 lesions, respectively, in 17 (94%) patients each, whereas 18 F-FDG PET revealed 30 lesions in 15 (83%) patients. Uptake of both SSTR radioligands was found in several radioiodine-negative sites. No striking differences in lesion imaging by 111 In-DOTA-LAN and 111 In-DOTA-TOC were found. In both radioiodine-negative and radioiodine-positive patients, more lesions were SSTR-positive/ 18 F-FDG-negative than were 18 F-FDG-positive/SSTR-negative. Adding a SSTR scan with these radioligands to the diagnostic work-up increases the diagnostic capacity in DTC, and should be considered particularly in radioiodine-negative patients with elevated thyroglobulin levels. (orig.)

Use of a charge reducing agent to enable intact mass analysis of cysteine-linked antibody-drug-conjugates by native mass spectrometry

Directory of Open Access Journals (Sweden)

Kamila J. Pacholarz

2016-06-01

Full Text Available Antibody-drug-conjugates (ADC are a growing class of anticancer biopharmaceuticals. Conjugation of cysteine linked ADCs, requires initial reduction of mAb inter-chain disulfide bonds, as the drugs are attached via thiol chemistry. This results in the active mAb moiety being transformed from a covalently linked tetramer to non-covalently linked complexes, which hinders precise determination of drug load with LC–MS. Here, we show how the addition of the charge reducing agent triethylammonium acetate (TEAA preserves the intact mAb structure, is well suited to the study of cysteine linked conjugates and facilitates easy drug load determination by direct infusion native MS.

Inflammation-Specific T1 Imaging Using Anti-Intercellular Adhesion Molecule 1 Antibody-Conjugated Gadolinium Diethylenetriaminepentaacetic Acid

Directory of Open Access Journals (Sweden)

Kyu-Sil Choi

2007-03-01

Full Text Available To examine inflammatory tissue, an initial and common symptom of various types of pathogenesis, we designed inflammation-targeted T1 contrast agents prepared by bioconjugation of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA with anti-intercellular adhesion molecule 1 (ICAM-1 antibody. The anti-ICAM-1 antibody was coupled with DTPA and was then conjugated with Gd. The specific binding of the Gd-DTPA-anti-ICAM-1 antibody complex to the ICAM-1-expressing cells was examined in the cultured endothelial cells where ICAM-1 expression was stimulated. Inflammation-specific T1 imaging was then assessed using a mouse abscess model with the 1.5-Tesla module. The Gd-DTPA-anti-ICAM-1 antibody displayed increased r1, which was two times higher than that of Gd-DTPA and showed predominant binding to cultured endothelial cells, which expressed a high level of ICAM-1. Moreover, the inflammation-specific T1 enhancement was imaged with the Gd-DTPA-anti-ICAM-1 antibody in the mouse acute inflammation model. The Gd-DTPA-anti-ICAM-1 antibody showed significantly increased vascular circulation time, which thereby offered a greater chance for its binding to the target cells. The Gd-DTPA-anti-ICAM-1 antibody displays a potential targeted T1 contrast agent specific to the inflammatory tissue that expresses ICAM-1.

DFT study of the interaction between DOTA chelator and competitive alkali metal ions.

Science.gov (United States)

Frimpong, E; Skelton, A A; Honarparvar, B

2017-09-01

1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetracetic acid (DOTA) is an important chelator for radiolabeling of pharmaceuticals. The ability of alkali metals found in the body to complex with DOTA and compete with radio metal ions can alter the radiolabeling process. Non-covalent interactions between DOTA complexed with alkali metals Li + , Na + , K + and Rb + , are investigated with density functional theory using B3LYP and ωB97XD functionals. Conformational possibilities of DOTA were explored with a varying number of carboxylic pendant arms of DOTA in close proximity to the ions. It is found that the case in which four arms of DOTA are interacting with ions is more stable than other conformations. The objective of this study is to explore the electronic structure properties upon complexation of alkali metals Li + Na + , K + and Rb + with a DOTA chelator. Interaction energies, relaxation energies, entropies, Gibbs free energies and enthalpies show that the stability of DOTA, complexed with alkali metals decreases down the group of the periodic table. Implicit water solvation affects the complexation of DOTA-ions leading to decreases in the stability of the complexes. NBO analysis through the natural population charges and the second order perturbation theory, revealed a charge transfer between DOTA and alkali metals. Conceptual DFT-based properties such as HOMO/LUMO energies, ΔE HOMO-LUMO and chemical hardness and softness indicated a decrease in the chemical stability of DOTA-alkali metal complexes down the alkali metal series. This study serves as a guide to researchers in the field of organometallic chelators, particularly, radiopharmaceuticals in finding the efficient optimal match between chelators and various metal ions. Copyright © 2017 Elsevier Inc. All rights reserved.

A comparison of 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging subcutaneous HER2-positive tumor xenografts in athymic mice using microSPECT/CT or microPET/CT

Science.gov (United States)

2011-01-01

Background Our objective was to compare 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging small or large s.c. tumor xenografts in athymic mice that display a wide range of human epidermal growth factor receptor-2 (HER2) expression using microSPECT/CT or microPET/CT. Methods Trastuzumab Fab were labeled with 111In or 64Cu by conjugation to 1,4,7,10-tetraazacyclododecane N, N', N'', N'''-tetraacetic acid (DOTA). The purity of 111In- and 64Cu-DOTA-trastuzumab Fab was measured by SDS-PAGE and HPLC. HER2 binding affinity was determined in saturation radioligand binding assays using SKBR-3 cells (1.3 × 106 HER2/cell). MicroSPECT/CT and microPET/CT were performed in athymic mice bearing s.c. BT-20 and MDA-MB-231 xenografts with low (0.5 to 1.6 × 105 receptors/cell), MDA-MB-361 tumors with intermediate (5.1 × 105 receptors/cell) or SKOV-3 xenografts with high HER2 expression (1.2 × 106 receptors/cell) at 24 h p.i. of 70 MBq (10 μg) of 111In-DOTA-trastuzumab Fab or 22 MBq (10 μg) of 64Cu-DOTA-trastuzumab Fab or irrelevant 111In- or 64Cu-DOTA-rituximab Fab. Tumor and normal tissue uptake were quantified in biodistribution studies. Results 111In- and 64Cu-DOTA-trastuzumab were > 98% radiochemically pure and bound HER2 with high affinity (Kd = 20.4 ± 2.5 nM and 40.8 ± 3.5 nM, respectively). MDA-MB-361 and SKOV-3 tumors were most clearly imaged using 111In- and 64Cu-DOTA-trastuzumab Fab. Significantly higher tumor/blood (T/B) ratios were found for 111In-DOTA-trastuzumab Fab than 111In-DOTA-rituximab Fab for BT-20, MDA-MB-231 and MDA-MB-361 xenografts, and there was a direct association between T/B ratios and HER2 expression. In contrast, tumor uptake of 64Cu-DOTA-trastuzumab Fab was significantly higher than 64Cu-DOTA-rituximab Fab in MDA-MB-361 tumors but no direct association with HER2 expression was found. Both 111In- and 64Cu-DOTA-trastuzumab Fab imaged small (5 to 10 mm) or larger (10 to 15 mm) MDA-MB-361 tumors. Higher blood, liver, and spleen

Molecular Evolution of the dotA Gene in Legionella pneumophila

OpenAIRE

Ko, Kwan Soo; Hong, Seong Karp; Lee, Hae Kyung; Park, Mi-Yeoun; Kook, Yoon-Hoh

2003-01-01

The molecular evolution of dotA, which is related to the virulence of Legionella pneumophila, was investigated by comparing the sequences of 15 reference strains (serogroups 1 to 15). It was found that dotA has a complex mosaic structure. The whole dotA gene of Legionella pneumophila subsp. pneumophila serogroups 2, 6, and 12 has been transferred from Legionella pneumophila subsp. fraseri. A discrepancy was found between the trees inferred from the nucleotide and deduced amino acid sequences ...

Structural Characterization of Am(III)- and Pu(III)-DOTA Complexes.

Science.gov (United States)

Audras, Matthieu; Berthon, Laurence; Berthon, Claude; Guillaumont, Dominique; Dumas, Thomas; Illy, Marie-Claire; Martin, Nicolas; Zilbermann, Israel; Moiseev, Yulia; Ben-Eliyahu, Yeshayahu; Bettelheim, Armand; Cammelli, Sebastiano; Hennig, Christoph; Moisy, Philippe

2017-10-16

The complexation of 1,4,7,10-tetrazacyclodecane-1,4,7,10-tetraacetic acid (DOTA) ligand with two trivalent actinides (Am 3+ and Pu 3+ ) was investigated by UV-visible spectrophotometry, NMR spectroscopy, and extended X-ray absorption fine structure in conjunction with computational methods. The complexation process of these two cations is similar to what has been previously observed with lanthanides(III) of similar ionic radius. The complexation takes place in different steps and ends with the formation of a (1:1) complex [(An(III)DOTA)(H 2 O)] - , where the cation is bonded to the nitrogen atoms of the ring, the four carboxylate arms, and a water molecule to complete the coordination sphere. The formation of An(III)-DOTA complexes is faster than the Ln(III)-DOTA systems of equivalent ionic radius. Furthermore, it is found that An-N distances are slightly shorter than Ln-N distances. Theoretical calculations showed that the slightly higher affinity of DOTA toward Am over Nd is correlated with slightly enhanced ligand-to-metal charge donation arising from oxygen and nitrogen atoms.

177Lu-Dendrimer Conjugated to Folate and Bombesin with Gold Nanoparticles in the Dendritic Cavity: A Potential Theranostic Radiopharmaceutical

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Héctor Mendoza-Nava

2016-01-01

Full Text Available 177Lu-labeled nanoparticles conjugated to biomolecules have been proposed as a new class of theranostic radiopharmaceuticals. The aim of this research was to synthesize 177Lu-dendrimer(PAMAM-G4-folate-bombesin with gold nanoparticles (AuNPs in the dendritic cavity and to evaluate the radiopharmaceutical potential for targeted radiotherapy and the simultaneous detection of folate receptors (FRs and gastrin-releasing peptide receptors (GRPRs overexpressed in breast cancer cells. p-SCN-Benzyl-DOTA was conjugated in aqueous-basic medium to the dendrimer. The carboxylate groups of Lys1Lys3(DOTA-bombesin and folic acid were activated with HATU and also conjugated to the dendrimer. The conjugate was mixed with 1% HAuCl4 followed by the addition of NaBH4 and purified by ultrafiltration. Elemental analysis (EDS, particle size distribution (DLS, TEM analysis, UV-Vis, and infrared and fluorescence spectroscopies were performed. The conjugate was radiolabeled using 177LuCl3 or 68GaCl3 and analyzed by radio-HPLC. Studies confirmed the dendrimer functionalization with high radiochemical purity (>95%. Fluorescence results demonstrated that the presence of AuNPs in the dendritic cavity confers useful photophysical properties to the radiopharmaceutical for optical imaging. Preliminary binding studies in T47D breast cancer cells showed a specific cell uptake (41.15±2.72%. 177Lu-dendrimer(AuNP-folate-bombesin may be useful as an optical and nuclear imaging agent for breast tumors overexpressing GRPR and FRs, as well as for targeted radiotherapy.

Functional Imaging of HER2-Positive Metastatic Breast Cancer Using 64Cu-DOTA-Trastuzumab Positron Emission Tomography

Science.gov (United States)

Mortimer, Joanne E.; Bading, James R.; Colcher, David M.; Conti, Peter S.; Frankel, Paul H.; Carroll, Mary I.; Tong, Shan; Poku, Erasmus; Miles, Joshua K.; Shively, John E.; Raubitschek, Andrew A.

2014-01-01

Women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer are candidates for treatment with the anti-HER2 antibody trastuzumab. Assessment of HER2 status in recurrent disease is usually made by core needle biopsy of a single lesion which may not be representative of the larger tumor mass or other sites of disease. Our long-range goal is to develop positron emission tomography (PET) of radiolabeled trastuzumab for systemically assessing tumor HER2 expression and identifying appropriate use of anti-HER2 therapies. The purpose of this study was to evaluate PET-CT of 64Cu-DOTA-trastuzumab for detecting and measuring tumor uptake of trastuzumab in patients with HER2-positive metastatic breast cancer. Methods Eight women with biopsy-confirmed HER2-positive metastatic breast cancer and no anti-HER2 therapy for ≥ 4 mo underwent complete staging, including 18F-fluorodeoxyglucose (FDG)/PET-CT. For 6 of the 8 patients, 64Cu-DOTA-trastuzumab injection (364-512 MBq, 5 mg trastuzumab) was preceded by trastuzumab infusion (45 mg). PET-CT (PET scan duration 1 h) was performed 21-25 (“Day 1”) and 47-49 (“Day 2”) h after 64Cu-DOTA-trastuzumab injection. Scan fields of view were chosen based on 18F-FDG/PET-CT. Lesions visualized relative to adjacent tissue on PET were considered PET-positive; analysis was limited to lesions identifiable on CT. Radiolabel uptake in prominent lesions was measured as maximum single-voxel standardized uptake value (SUVmax). Results Liver uptake of 64Cu was reduced approximately 75% with the 45 mg trastuzumab pre-dose, without significant effect on tumor uptake. The study included 89 CT-positive lesions; detection sensitivity was 77, 89 and 93% for Day 1, Day 2 and 18F-FDG, respectively. On average, tumor uptake was similar for 64Cu-DOTA-trastuzumab and 18F-FDG [SUVmax (mean, range): Day 1 (8.1, 3.0-22.5, n=48); Day 2 (8.9, 0.9-28.9, n=38); 18F-FDG (9.7, 3.3-25.4, n=56)], but the extent of same-lesion uptake was not

Functional imaging of human epidermal growth factor receptor 2-positive metastatic breast cancer using (64)Cu-DOTA-trastuzumab PET.

Science.gov (United States)

Mortimer, Joanne E; Bading, James R; Colcher, David M; Conti, Peter S; Frankel, Paul H; Carroll, Mary I; Tong, Shan; Poku, Erasmus; Miles, Joshua K; Shively, John E; Raubitschek, Andrew A

2014-01-01

Women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer are candidates for treatment with the anti-HER2 antibody trastuzumab. Assessment of HER2 status in recurrent disease is usually made by core needle biopsy of a single lesion, which may not represent the larger tumor mass or other sites of disease. Our long-range goal is to develop PET of radiolabeled trastuzumab for systemically assessing tumor HER2 expression and identifying appropriate use of anti-HER2 therapies. The purpose of this study was to evaluate PET/CT of (64)Cu-DOTA-trastuzumab for detecting and measuring tumor uptake of trastuzumab in patients with HER2-positive metastatic breast cancer. Eight women with biopsy-confirmed HER2-positive metastatic breast cancer and no anti-HER2 therapy for 4 mo or longer underwent complete staging, including (18)F-FDG PET/CT. For 6 of the 8 patients, (64)Cu-DOTA-trastuzumab injection (364-512 MBq, 5 mg of trastuzumab) was preceded by trastuzumab infusion (45 mg). PET/CT (PET scan duration 1 h) was performed 21-25 (day 1) and 47-49 (day 2) h after (64)Cu-DOTA-trastuzumab injection. Scan fields of view were chosen on the basis of (18)F-FDG PET/CT. Tumor detection sensitivity and uptake analyses were limited to lesions identifiable on CT; lesions visualized relative to adjacent tissue on PET were considered PET-positive. Radiolabel uptake in prominent lesions was measured as maximum single-voxel standardized uptake value (SUVmax). Liver uptake of (64)Cu was reduced approximately 75% with the 45-mg trastuzumab predose, without significant effect on tumor uptake. The study included 89 CT-positive lesions. Detection sensitivity was 77%, 89%, and 93% for day 1, day 2, and (18)F-FDG, respectively. On average, tumor uptake was similar for (64)Cu-DOTA-trastuzumab and (18)F-FDG (SUVmax and range, 8.1 and 3.0-22.5 for day 1 [n = 48]; 8.9 and 0.9-28.9 for day 2 [n = 38]; 9.7 and 3.3-25.4 for (18)F-FDG [n = 56]), but same-lesion SUVmax was not correlated

In vivo evaluation of an anti-PSMA antibody conjugated with varying numbers of biotin molecules in a pretargeting protocol

International Nuclear Information System (INIS)

Wilbur, D.S.; Hamlin, D.K.; Quinn, J.; Vessella, R.L.

2003-01-01

An investigation has been conducted to determine the effect of varying the number of biotin molecules conjugated with an anti-PSMA antibody (mAb) as part of our studies to optimize biotinylated antibodies and radiolabeled streptavidin in pretargeting protocols for Targeted Radionuclide Therapy of prostate cancer. In the investigation, the anti-PSMA antibody 107-1A4 was biotinylated with varying amounts of biotinamidocaproate N-hydroxysuccinimide ester. This procedure resulted in obtaining 107-1A4 with 2.3, 4.5, and 6.8 biotin conjugated as measured by the standard HABA assay. The biotinylated 107-1A4 was radioiodinated and was evaluated in a pretargeting protocol in athymic mice bearing LNCaP human tumor xenografts. In the protocol, 50 μg biotinylated [ 125 I]107-1A4 was injected, followed 48h later by 25 μg of avidin for blood clearance, and 1h after that 20 μg of radiolabeled succinylated recombinant streptavidin ([ 13 1I]sSAv) was administered. The tumor localization and tissue distribution was evaluated at 24, 48, and 72h post [ 131 I]sSAv injection. With 2.3 biotin/mAb, an approximate 1:1 molar ratio (4-5 pmol/g) of sSAv/mAb was obtained at all three time points. With 4.5 biotin/mAb, a 1:1 ratio was observed at 24h, but approx. 2: 1 was observed at 48 and 72h pi. With 6.8 biotin/mAb, sSAv/mAb ratios of approximately 1.5:1; 2:1; and 3:1 were obtained at 24, 48, and 72h pi respectively. The amount of sSAv localized in the tumor was nearly the same (4-5 pmol/g) when 107-1A4 had 2.3 or 4.5 biotin conjugated, but decreased to 3-4.5 pmol/g with 6.8 biotin conjugated. Because the highest levels of co-localized sSAv was found with the lowest number of biotin conjugates, the observed differences in ratios of sSAv/mAb may be best explained as differences in internalization, and degradation of mAb and protease resistant sSAv. In duplicate experiments, similar results were obtained with biotinylated 107-1A4 F(ab') 2 , but not with an mAb to a non-internalizing antigen

Mathematical modeling of antibody drug conjugates with the target and tubulin dynamics to predict AUC.

Science.gov (United States)

Byun, Jong Hyuk; Jung, Il Hyo

2018-04-14

Antibody drug conjugates (ADCs)are one of the most recently developed chemotherapeutics to treat some types of tumor cells. They consist of monoclonal antibodies (mAbs), linkers, and potent cytotoxic drugs. Unlike common chemotherapies, ADCs combine selectively with a target at the surface of the tumor cell, and a potent cytotoxic drug (payload) effectively prevents microtubule polymerization. In this work, we construct an ADC model that considers both the target of antibodies and the receptor (tubulin) of the cytotoxic payloads. The model is simulated with brentuximab vedotin, one of ADCs, and used to investigate the pharmacokinetic (PK) characteristics of ADCs in vivo. It also predicts area under the curve (AUC) of ADCs and the payloads by identifying the half-life. The results show that dynamical behaviors fairly coincide with the observed data and half-life and capture AUC. Thus, the model can be used for estimating some parameters, fitting experimental observations, predicting AUC, and exploring various dynamical behaviors of the target and the receptor. Copyright © 2018 Elsevier Ltd. All rights reserved.

Systemic and Mucosal Antibody Responses to Soluble and Nanoparticle-Conjugated Antigens Administered Intranasally

Directory of Open Access Journals (Sweden)

Savannah E. Howe

2016-10-01

Full Text Available Nanoparticles (NPs are increasingly being used for drug delivery, as well as antigen carriers and immunostimulants for the purpose of developing vaccines. In this work, we examined how intranasal (i.n. priming followed by i.n. or subcutaneous (s.c. boosting immunization affects the humoral immune response to chicken ovalbumin (Ova and Ova conjugated to 20 nm NPs (NP-Ova. We show that i.n. priming with 20 mg of soluble Ova, a dose known to trigger oral tolerance when administered via gastric gavage, induced substantial systemic IgG1 and IgG2c, as well as mucosal antibodies. These responses were further boosted following a s.c. immunization with Ova and complete Freund’s adjuvant (Ova+CFA. In contrast, 100 µg of Ova delivered via NPs induced an IgG1-dominated systemic response, and primed the intestinal mucosa for secretion of IgA. Following a secondary s.c. or i.n. immunization with Ova+CFA or NP-Ova, systemic IgG1 titers significantly increased, and serum IgG2c and intestinal antibodies were induced in mice primed nasally with NP-Ova. Only Ova- and NP-Ova-primed mice that were s.c.-boosted exhibited substantial systemic and mucosal titers for up to 6 months after priming, whereas the antibodies of i.n.-boosted mice declined over time. Our results indicate that although the amount of Ova delivered by NPs was 1000-fold less than Ova delivered in soluble form, the antigen-specific antibody responses, both systemic and mucosal, are essentially identical by 6 months following the initial priming immunization. Additionally, both i.n.- and s.c.-boosting strategies for NP-Ova-primed mice were capable of inducing a polarized Th1/Th2 immune response, as well as intestinal antibodies; however, it is only by using a heterogeneous prime-boost strategy that long-lasting antibody responses were initiated. These results provide valuable insight for future mucosal vaccine development, as well as furthering our understanding of mucosal antibody responses.

Application of analytic methodologies for image quantification in neuroendocrine tumor therapy with {sup 177}Lu-DOTA

Energy Technology Data Exchange (ETDEWEB)

Kubo, T.T.A.; Oliveira, S.M.V. [Instituto de Radioprotecao e Dosimetria (IRD/CNEN-RJ), Rio de Janeiro, RJ (Brazil); Marco, L.; Mamede, M., E-mail: tadeukubo@gmail.com [Instituto Nacional do Cancer, Rio de Janeiro, RJ (Brazil)

2012-07-01

Neuroendocrine tumors have annual incidence of 1 to 2 cases per one hundred thousand inhabitants. The {sup 177}Lu-DOTA-octreotate treatments in 3 or 4 cycles has been effective in controlling disease progression and, in some cases, promote tumor remission. To estimate radiation side effects in healthy organs, image quantification techniques have been broadcast for individualized patient dosimetry. In this paper, image data processing methods are presented to allowing comparisons between different image conjugate views, combined with attenuation correction and system sensitivity. Images were acquired 24, 72 and 192 h after administration of 74 GBq of {sup 177}Lu-DOTA using a dual-head gamma camera detection system and they were evaluated with ImageJ software. 4 female patients underwent to two cycles of treatment. The kidneys, liver and whole-body regions of interest were separately assessed by 4 techniques for counts method and 12 techniques for pixel intensity method, considering the main photopeak separately and aided by the attenuation correction map and adjacent windows to photopeak energy. The pixel intensity method was combined with mathematical correction for pixels with null value. The results obtained by the two methods were strongly correlated (r>0.9) (p<0.001). The paired t-test accepted the null hypothesis of compatibility between the two methods (with and without attenuation correction map) (p<0.05), but rejected it when the adjacent windows were combined. No significant tumor reduction (p>0.05) was found between the treatment cycles. In conclusion, the pixel intensity method is faster and allows macros, minimizing operator error, and may optimize dosimetry in tumor therapies with {sup 177}Lu-DOTA-octreotate. (author)

Chiral DOTA chelators as an improved platform for biomedical imaging and therapy applications.

Science.gov (United States)

Dai, Lixiong; Jones, Chloe M; Chan, Wesley Ting Kwok; Pham, Tiffany A; Ling, Xiaoxi; Gale, Eric M; Rotile, Nicholas J; Tai, William Chi-Shing; Anderson, Carolyn J; Caravan, Peter; Law, Ga-Lai

2018-02-27

Despite established clinical utilisation, there is an increasing need for safer, more inert gadolinium-based contrast agents, and for chelators that react rapidly with radiometals. Here we report the syntheses of a series of chiral DOTA chelators and their corresponding metal complexes and reveal properties that transcend the parent DOTA compound. We incorporated symmetrical chiral substituents around the tetraaza ring, imparting enhanced rigidity to the DOTA cavity, enabling control over the range of stereoisomers of the lanthanide complexes. The Gd chiral DOTA complexes are shown to be orders of magnitude more inert to Gd release than [GdDOTA] - . These compounds also exhibit very-fast water exchange rates in an optimal range for high field imaging. Radiolabeling studies with (Cu-64/Lu-177) also demonstrate faster labelling properties. These chiral DOTA chelators are alternative general platforms for the development of stable, high relaxivity contrast agents, and for radiometal complexes used for imaging and/or therapy.

Characterizing the magnetic susceptibility tensor of lanthanide-containing polymethylated-DOTA complexes

Energy Technology Data Exchange (ETDEWEB)

Strickland, Madeleine [National Institutes of Health, Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute (United States); Schwieters, Charles D. [National Institutes of Health, Office of Intramural Research, Center for Information Technology (United States); Göbl, Christoph [Technische Universität München, Department of Chemistry (Germany); Opina, Ana C. L. [National Institutes of Health, Imaging Probe Development Center, National Heart, Lung, and Blood Institute (United States); Strub, Marie-Paule [National Institutes of Health, Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute (United States); Swenson, Rolf E.; Vasalatiy, Olga [National Institutes of Health, Imaging Probe Development Center, National Heart, Lung, and Blood Institute (United States); Tjandra, Nico, E-mail: tjandran@nhlbi.nih.gov [National Institutes of Health, Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute (United States)

2016-10-15

Lanthanide complexes based on the DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) cage are commonly used as phase contrast agents in magnetic resonance imaging, but can also be utilized in structural NMR applications due to their ability to induce either paramagnetic relaxation enhancement or a pseudocontact shift (PCS) depending on the choice of the lanthanide. The size and sign of the PCS for any given atom is determined by its coordinates relative to the metal center, and the characteristics of the lanthanide’s magnetic susceptibility tensor. Using a polymethylated DOTA tag (Ln-M8-SPy) conjugated to ubiquitin, we calculated the position of the metal center and characterized the susceptibility tensor for a number of lanthanides (dysprosium, thulium, and ytterbium) under a range of pH and temperature conditions. We found that there was a difference in temperature sensitivity for each of the complexes studied, which depended on the size of the lanthanide ion as well as the isomeric state of the cage. Using {sup 17}O-NMR, we confirmed that the temperature sensitivity of the compounds was enhanced by the presence of an apically bound water molecule. Since amide-containing lanthanide complexes are known to be pH sensitive and can be used as probes of physiological pH, we also investigated the effect of pH on the Ln-M8-SPy susceptibility tensor, but we found that the changes in this pH range (5.0–7.4) were not significant.

Characterizing the magnetic susceptibility tensor of lanthanide-containing polymethylated-DOTA complexes

International Nuclear Information System (INIS)

Strickland, Madeleine; Schwieters, Charles D.; Göbl, Christoph; Opina, Ana C. L.; Strub, Marie-Paule; Swenson, Rolf E.; Vasalatiy, Olga; Tjandra, Nico

2016-01-01

Lanthanide complexes based on the DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) cage are commonly used as phase contrast agents in magnetic resonance imaging, but can also be utilized in structural NMR applications due to their ability to induce either paramagnetic relaxation enhancement or a pseudocontact shift (PCS) depending on the choice of the lanthanide. The size and sign of the PCS for any given atom is determined by its coordinates relative to the metal center, and the characteristics of the lanthanide’s magnetic susceptibility tensor. Using a polymethylated DOTA tag (Ln-M8-SPy) conjugated to ubiquitin, we calculated the position of the metal center and characterized the susceptibility tensor for a number of lanthanides (dysprosium, thulium, and ytterbium) under a range of pH and temperature conditions. We found that there was a difference in temperature sensitivity for each of the complexes studied, which depended on the size of the lanthanide ion as well as the isomeric state of the cage. Using "1"7O-NMR, we confirmed that the temperature sensitivity of the compounds was enhanced by the presence of an apically bound water molecule. Since amide-containing lanthanide complexes are known to be pH sensitive and can be used as probes of physiological pH, we also investigated the effect of pH on the Ln-M8-SPy susceptibility tensor, but we found that the changes in this pH range (5.0–7.4) were not significant.

A size exclusion-reversed phase two dimensional-liquid chromatography methodology for stability and small molecule related species in antibody drug conjugates.

Science.gov (United States)

Li, Yi; Gu, Christine; Gruenhagen, Jason; Zhang, Kelly; Yehl, Peter; Chetwyn, Nik P; Medley, Colin D

2015-05-08

Antibody drug conjugates (ADCs) are complex therapeutic agents combining the specific targeting properties of antibodies and highly potent cytotoxic small molecule drugs to selectively eliminate tumor cells while limiting the toxicity to normal healthy tissues. One unique critical quality attribute of ADCs is the content of unconjugated small molecule drug present from either incomplete conjugation or degradation of the ADC. In this work, size exclusion chromatography (SEC) was coupled with reversed-phase (RP) HPLC in an online 2-dimensional chromatography format for identification and quantitation of unconjugated small molecule drugs and related small molecule impurities in ADC samples directly without sample preparation. The SEC method in the 1st dimension not only separated the small molecule impurities from the intact ADC, but also provided information about the size variants (monomer, dimer, aggregates, etc.) of the ADC. The small molecule peak from the SEC was trapped and sent to a RP-HPLC in the 2nd dimension to further separate and quantify the different small molecule impurities present in the ADC sample. This SEC-RP 2D-LC method demonstrated excellent precision (%RSDmolecule degradation products and aggregation of the conjugate were observed in the stability samples and the degradation pathways of the ADC were investigated. This 2D-LC method offers a powerful tool for ADC characterization and provides valuable information for conjugation and formulation development. Copyright © 2015 Elsevier B.V. All rights reserved.

Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages.

OpenAIRE

Roy, C R; Isberg, R R

1997-01-01

The Legionella pneumophila dotA gene is required for intracellular growth of the bacterium in macrophages. In this study, a structure-function analysis of the DotA protein was conducted to elucidate the role of this protein in L. pneumophila pathogenesis. Translational fusions of dotA to the Escherichia coli phoA and lacZ genes indicated that DotA is an integral cytoplasmic membrane protein with eight membrane-spanning domains. DotA contains two large periplasmic domains of approximately 503 ...

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

Science.gov (United States)

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

A radiogallium-DOTA-based bivalent peptidic ligand targeting a chemokine receptor, CXCR4, for tumor imaging.

Science.gov (United States)

Sano, Kohei; Masuda, Ryo; Hisada, Hayato; Oishi, Shinya; Shimokawa, Kenta; Ono, Masahiro; Fujii, Nobutaka; Saji, Hideo; Mukai, Takahiro

2014-03-01

We have developed a novel radiogallium (Ga)-DOTA-based bivalent peptidic ligand targeting a chemokine receptor, CXCR4, for tumor imaging. A CXCR4 imaging probe with two CXCR4 antagonists (Ac-TZ14011) on Ga-DOTA core, Ga-DOTA-TZ2, was synthesized, and the affinity and binding to CXCR4 was evaluated in CXCR4 expressing cells in vitro. The affinity of Ga-DOTA-TZ2 for CXCR4 was 20-fold greater than the corresponding monovalent probe, Ga-DOTA-TZ1. (67)Ga-DOTA-TZ2 showed the significantly higher accumulation in CXCR4-expressing tumor cells compared with (67)Ga-DOTA-TZ1, suggesting the bivalent effect enhances its binding to CXCR4. The incorporation of two CXCR4 antagonists to Ga-DOTA could be effective in detecting CXCR4-expressing tumors. Copyright © 2014 Elsevier Ltd. All rights reserved.

Correlation of Somatostatin Receptor-2 Expression with Gallium-68-DOTA-TATE Uptake in Neuroblastoma Xenograft Models.

Science.gov (United States)

Zhang, Libo; Vines, Douglass C; Scollard, Deborah A; McKee, Trevor; Komal, Teesha; Ganguly, Milan; Do, Trevor; Wu, Bing; Alexander, Natasha; Vali, Reza; Shammas, Amer; Besanger, Travis; Baruchel, Sylvain

2017-01-01

Peptide-receptor imaging and therapy with radiolabeled somatostatin analogs such as 68 Ga-DOTA-TATE and 177 Lu-DOTA-TATE have become an effective treatment option for SSTR-positive neuroendocrine tumors. The purpose of this study was to evaluate the correlation of somatostatin receptor-2 (SSTR2) expression with 68 Ga-DOTA-TATE uptake and 177 Lu-DOTA-TATE therapy in neuroblastoma (NB) xenograft models. We demonstrated variable SSTR2 expression profiles in eight NB cell lines. From micro-PET imaging and autoradiography, a higher uptake of 68 Ga-DOTA-TATE was observed in SSTR2 high-expressing NB xenografts (CHLA-15) compared to SSTR2 low-expressing NB xenografts (SK-N-BE(2)). Combined autoradiography-immunohistochemistry revealed histological colocalization of SSTR2 and 68 Ga-DOTA-TATE uptake in CHLA-15 tumors. With a low dose of 177 Lu-DOTA-TATE (20 MBq/animal), tumor growth inhibition was achieved in the CHLA-15 high SSTR2 expressing xenograft model. Although, in vitro , NB cells showed variable expression levels of norepinephrine transporter (NET), a molecular target for 131 I-MIBG therapy, low 123 I-MIBG uptake was observed in all selected NB xenografts. In conclusion, SSTR2 expression levels are associated with 68 Ga-DOTA-TATE uptake and antitumor efficacy of 177 Lu-DOTA-TATE. 68 Ga-DOTA-TATE PET is superior to 123 I-MIBG SPECT imaging in detecting NB tumors in our model. Radiolabeled DOTA-TATE can be used as an agent for NB tumor imaging to potentially discriminate tumors eligible for 177 Lu-DOTA-TATE therapy.

Correlation of Somatostatin Receptor-2 Expression with Gallium-68-DOTA-TATE Uptake in Neuroblastoma Xenograft Models

Directory of Open Access Journals (Sweden)

Libo Zhang

2017-01-01

Full Text Available Peptide-receptor imaging and therapy with radiolabeled somatostatin analogs such as 68Ga-DOTA-TATE and 177Lu-DOTA-TATE have become an effective treatment option for SSTR-positive neuroendocrine tumors. The purpose of this study was to evaluate the correlation of somatostatin receptor-2 (SSTR2 expression with 68Ga-DOTA-TATE uptake and 177Lu-DOTA-TATE therapy in neuroblastoma (NB xenograft models. We demonstrated variable SSTR2 expression profiles in eight NB cell lines. From micro-PET imaging and autoradiography, a higher uptake of 68Ga-DOTA-TATE was observed in SSTR2 high-expressing NB xenografts (CHLA-15 compared to SSTR2 low-expressing NB xenografts (SK-N-BE(2. Combined autoradiography-immunohistochemistry revealed histological colocalization of SSTR2 and 68Ga-DOTA-TATE uptake in CHLA-15 tumors. With a low dose of 177Lu-DOTA-TATE (20 MBq/animal, tumor growth inhibition was achieved in the CHLA-15 high SSTR2 expressing xenograft model. Although, in vitro, NB cells showed variable expression levels of norepinephrine transporter (NET, a molecular target for 131I-MIBG therapy, low 123I-MIBG uptake was observed in all selected NB xenografts. In conclusion, SSTR2 expression levels are associated with 68Ga-DOTA-TATE uptake and antitumor efficacy of 177Lu-DOTA-TATE. 68Ga-DOTA-TATE PET is superior to 123I-MIBG SPECT imaging in detecting NB tumors in our model. Radiolabeled DOTA-TATE can be used as an agent for NB tumor imaging to potentially discriminate tumors eligible for 177Lu-DOTA-TATE therapy.

Antibody-drug conjugates for cancer therapy: The technological and regulatory challenges of developing drug-biologic hybrids.

Science.gov (United States)

Hamilton, Gregory S

2015-09-01

Antibody-drug conjugates (ADCs) are a new class of therapeutic agents that combine the targeting ability of monoclonal antibodies (mAbs) with small molecule drugs. The combination of a mAb targeting a cancer-specific antigen with a cytotoxin has tremendous promise as a new type of targeted cancer therapy. Two ADCs have been approved and many more are in clinical development, suggesting that this new class of drugs is coming to the forefront. Because of their unique nature as biologic-small drug hybrids, ADCs are challenging to develop, from both the scientific and regulatory perspectives. This review discusses both these aspects in current practice, and surveys the current state of the art of ADC drug development. Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Efficient payload delivery by a bispecific antibody-drug conjugate targeting HER2 and CD63

DEFF Research Database (Denmark)

de Goeij, Bart E.C.G.; Vink, Tom; Ten Napel, Hendrik

2016-01-01

Antibody-drug conjugates (ADC) are designed to be stable in circulation and to release potent cytotoxic drugs intracellularly following antigen-specific binding, uptake, and degradation in tumor cells. Efficient internalization and routing to lysosomes where proteolysis can take place is therefore......, for the first time, that intracellular trafficking of ADCs can be improved using a bsAb approach that targets the lysosomal membrane protein CD63 and provide a rationale for the development of novel bsADCs that combine tumor-specific targeting with targeting of rapidly internalizing antigens. © 2016 American...

SU-C-204-03: DFT Calculations of the Stability of DOTA-Based-Radiopharmaceuticals

Energy Technology Data Exchange (ETDEWEB)

Khabibullin, A.R.; Woods, L.M. [University of South Florida, Tampa, Florida (United States); Karolak, A.; Budzevich, M.M.; Martinez, M.V. [H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida (United States); McLaughlin, M.L.; Morse, D.L. [University of South Florida, Tampa, Florida (United States); H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida (United States)

2016-06-15

Purpose: Application of the density function theory (DFT) to investigate the structural stability of complexes applied in cancer therapy consisting of the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelated to Ac225, Fr221, At217, Bi213, and Gd68 radio-nuclei. Methods: The possibility to deliver a toxic payload directly to tumor cells is a highly desirable aim in targeted alpha particle therapy. The estimation of bond stability between radioactive atoms and the DOTA chelating agent is the key element in understanding the foundations of this delivery process. Thus, we adapted the Vienna Ab-initio Simulation Package (VASP) with the projector-augmented wave method and a plane-wave basis set in order to study the stability and electronic properties of DOTA ligand chelated to radioactive isotopes. In order to count for the relativistic effect of radioactive isotopes we included Spin-Orbit Coupling (SOC) in the DFT calculations. Five DOTA complex structures were represented as unit cells, each containing 58 atoms. The energy optimization was performed for all structures prior to calculations of electronic properties. Binding energies, electron localization functions as well as bond lengths between atoms were estimated. Results: Calculated binding energies for DOTA-radioactive atom systems were −17.792, −5.784, −8.872, −13.305, −18.467 eV for Ac, Fr, At, Bi and Gd complexes respectively. The displacements of isotopes in DOTA cages were estimated from the variations in bond lengths, which were within 2.32–3.75 angstroms. The detailed representation of chemical bonding in all complexes was obtained with the Electron Localization Function (ELF). Conclusion: DOTA-Gd, DOTA-Ac and DOTA-Bi were the most stable structures in the group. Inclusion of SOC had a significant role in the improvement of DFT calculation accuracy for heavy radioactive atoms. Our approach is found to be proper for the investigation of structures with DOTA

Phase 1 Evaluation of [(64)Cu]DOTA-Patritumab to Assess Dosimetry, Apparent Receptor Occupancy, and Safety in Subjects with Advanced Solid Tumors.

Science.gov (United States)

Lockhart, A Craig; Liu, Yongjian; Dehdashti, Farrokh; Laforest, Richard; Picus, Joel; Frye, Jennifer; Trull, Lauren; Belanger, Stefanie; Desai, Madhuri; Mahmood, Syed; Mendell, Jeanne; Welch, Michael J; Siegel, Barry A

2016-06-01

The purpose of this study was to evaluate the safety, dosimetry, and apparent receptor occupancy (RO) of [(64)Cu]DOTA-patritumab, a radiolabeled monoclonal antibody directed against HER3/ERBB3 in subjects with advanced solid tumors. Dosimetry subjects (n = 5) received [(64)Cu]DOTA-patritumab and underwent positron emission tomography (PET)/X-ray computed tomography (CT) at 3, 24, and 48 h. Evaluable RO subjects (n = 3 out of 6) received [(64)Cu]DOTA-patritumab at day 1 and day 8 (after 9.0 mg/kg patritumab) followed by PET/CT at 24 h post-injection. Endpoints included safety, tumor uptake, and efficacy. The tumor SUVmax (± SD) was 5.6 ± 4.5, 3.3 ± 1.7, and 3.0 ± 1.1 at 3, 24, and 48 h in dosimetry subjects. The effective dose and critical organ dose (liver) averaged 0.044 ± 0.008 mSv/MBq and 0.46 ± 0.086 mGy/MBq, respectively. In RO subjects, tumor-to-blood ratio decreased from 1.00 ± 0.32 at baseline to 0.57 ± 0.17 after stable patritumab, corresponding to a RO of 42.1 ± 3. [(64)Cu]DOTA-patritumab was safe. These limited results suggest that this PET-based method can be used to determine tumor-apparent RO.

Scintigraphic detection of metastatic melanoma using indium 111/DTPA conjugated anti-gp240 antibody (ZME-018)

International Nuclear Information System (INIS)

Kirkwood, J.M.; Neumann, R.D.; Zoghbi, S.S.; Ernstoff, M.S.; Cornelius, E.A.; Shaw, C.; Ziyadeh, T.; Fine, J.A.; Unger, M.W.

1987-01-01

We evaluated the toxicity, pharmacokinetics, and localization of a monoclonal IgG2 alpha murine anti-human melanoma (gp240) antibody (ZME-018) that recognizes a tumor-associated cell surface glycoprotein of 240,000 molecular weight present in most melanomas. The antibody was conjugated with DTPA (diethylenetriamine pentaacetic acid) and labeled by chelation of 111 In. One mg of antibody labeled with 5 mCi of 111 In was infused, together with 0 to 40 mg of cold carrier ZME-018. The blood clearance, urinary excretion, and in vivo localization were determined in 26 patients. Scintigraphic images were obtained at 24 hours and 72 hours in all patients. Mild toxicity occurred in one patient. The half-time clearance of labeled monoclonal murine antibody (MoAb) from the blood increased from 16.1 hours at an antibody dose of 1 mg to 35.9 hours at 40 mg. Males showed faster clearance from the blood than did females or a single castrated male, perhaps due to selective concentration of antibody in the testes. Nonspecific uptake in liver, spleen, bone marrow, and intestine was seen in all patients. The percentage of known metastatic foci detected increased with the total dosage of antibody, from 23% at doses less than or equal to 5 mg, to 65%, 87% and 78% for 10, 20, and 40 mg, respectively. We conclude that at doses of greater than or equal to 10 mg, ZME-018 is a safe and potentially useful agent for the scintigraphic detection of metastatic malignant melanoma

{sup 111}In-DOTA-dPhe{sup 1}-Tyr{sup 3}-octreotide, {sup 111}In-DOTA-lanreotide and {sup 67}Ga citrate scintigraphy for visualisation of extranodal marginal zone B-cell lymphoma of the MALT type: a comparative study

Energy Technology Data Exchange (ETDEWEB)

Li, Shuren; Kurtaran, Amir; Li, Mei; Traub-Weidinger, Tatjana; Kienast, Oskar [Department of Nuclear Medicine, University of Vienna, Waehringer Guertel 18-20, 1090, Vienna (Austria); Schima, Wolfgang [Department of Nuclear Medicine, University of Vienna, Waehringer Guertel 18-20, 1090, Vienna (Austria); Department of Radiodiagnostics, University of Vienna (Austria); Angelberger, Peter [Research Center Seibersdorf (Austria); Virgolini, Irene [Institute for Nuclear Medicine, Vienna Hospital Lainz (Austria); Ludwig-Boltzman Institute for Nuclear Medicine, Vienna (Austria); Raderer, Markus [Department of Internal Medicine I, Division of Oncology, University of Vienna (Austria); Dudczak, Robert [Department of Nuclear Medicine, University of Vienna, Waehringer Guertel 18-20, 1090, Vienna (Austria)

2003-08-01

Somatostatin receptor (SSTR) scintigraphy and gallium-67 citrate ({sup 67}Ga) scintigraphy have been used for visualisation of Hodgkin's lymphoma and non-Hodgkin's lymphoma. However, experience with B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) type is very limited. The aim of this study was to prospectively compare the {sup 67}Ga scintigraphy results with those obtained by {sup 111}In-DOTA-dPhe{sup 1}-Tyr{sup 3}-octreotide ({sup 111}In-DOTA-TOCT) and {sup 111}In-DOTA-lanreotide ({sup 111}In-DOTA-LAN) scintigraphy in patients with proven MALT-type lymphoma. Comparative scintigraphic examinations using {sup 67}Ga, {sup 111}In-DOTA-TOCT and {sup 111}In-DOTA-LAN were performed in 18 patients (11 female and 7 male, median age 64{+-}15 years) with histologically verified MALT-type lymphomas of various origin. Planar and single-photon emission tomography imaging acquisitions were performed after injection of a mean dose of 185{+-}26 MBq {sup 67}Ga and 165{+-}20 MBq {sup 111}In-DOTA-TOCT or {sup 111}In-DOTA-LAN. All scintigraphic results were correlated with other conventional examinations including gastroscopy, colonoscopy, endosonoscopy, ophthalmologic investigation, CT of the thorax and abdomen and bone marrow biopsy. This comparative study showed that {sup 67}Ga scintigraphy found abnormalities in 10 of 16 patients (63%) and detected 18 of 31 clinically involved sites (58%), but was false positive in three patients. {sup 111}In-DOTA-TOCT found abnormalities in 9 of 15 patients (60%) and detected 15 of 27 clinical lesions (56%); it was false positive in two patients. {sup 111}In-DOTA-LAN scintigraphy showed abnormalities in 7 of 11 patients (64%) and found 12 of 22 clinical lesions (55%). False-positive {sup 111}In-DOTA-LAN scan results were found in two patients. For supra-diaphragmatic lesions, {sup 67}Ga scintigraphy detected 12 of 16 sites (75%). {sup 111}In-DOTA-TOCT scintigraphy revealed 7 of 15 lesions (47%). {sup 111}In-DOTA

HER2 monoclonal antibodies that do not interfere with receptor heterodimerization-mediated signaling induce effective internalization and represent valuable components for rational antibody-drug conjugate design.

Science.gov (United States)

de Goeij, Bart E C G; Peipp, Matthias; de Haij, Simone; van den Brink, Edward N; Kellner, Christian; Riedl, Thilo; de Jong, Rob; Vink, Tom; Strumane, Kristin; Bleeker, Wim K; Parren, Paul W H I

2014-01-01

The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (Kadcyla(TM)). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA') fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA', was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA'-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.

44Sc-DOTA-BN[2-14]NH2 in comparison to 68Ga-DOTA-BN[2-14]NH2 in pre-clinical investigation. Is 44Sc a potential radionuclide for PET?

International Nuclear Information System (INIS)

Koumarianou, E.; Loktionova, N.S.; Fellner, M.; Roesch, F.; Thews, O.; Pawlak, D.; Archimandritis, S.C.; Mikolajczak, R.

2012-01-01

Aim: In the present study we demonstrate the in vitro and in vivo comparison of the 44 Sc and 68 Ga labeled DOTA-BN[2-14]NH 2 . 44 Sc is a positron emitter with a half life of 3.92 h. Hence it could be used for PET imaging with ligands requiring longer observation time than in the case of 68 Ga. Methods: The binding affinity of nat Sc-DOTA-BN[2-14]NH 2 and nat Ga-DOTA-BN[2-14]NH 2 to GRP receptors was studied in competition to [ 125 I-Tyr 4 ]-Bombesin in the human prostate cancer cell line PC-3. A preliminary biodistribution in normal rats was performed, while first microPET images were assessed in male Copenhagen rats bearing the androgen-independent Dunning R-3327-AT-1 prostate cancer tumor. Results: The affinity to GRP receptors in the PC-3 cell line was higher for nat Ga-DOTA-BN[2-14]NH 2 (IC 50 (nM)=0.85±0.06) than that of nat Sc-DOTA-BN[2-14]NH 2 (IC 50 (nM)=6.49±0.13). The internalization rate of 68 Ga labeled DOTA-BN[2-14]NH 2 was slower than that of 44 Sc, but their final internalization percents were comparable. 68 Ga-DOTA-BN[2-14]NH 2 was externalized faster than 44 Sc-DOTA-BN[2-14]NH 2 . The biodistribution of 44 Sc-DOTA-BN[2-14]NH 2 and 68 Ga-DOTA-BN[2-14]NH 2 in normal rats revealed a higher uptake in target organs and tissues of the first one while both excreted mainly through urinary tract. In microPET images both tracers were accumulated in the tumor with similar uptake patterns. Conclusions: Despite the differences in the receptor affinity both the 68 Ga- and the 44 Sc-labeled DOTA-BN[2-14]NH 2 tracers showed comparable distribution and similar time constants of uptake and elimination. Moreover no differences in tumor accumulation (neither in the overall uptake nor in the dynamics) were observed from the microPet imaging. From that perspective the use of either 44 Sc or 68 Ga for detecting tumors with GRP receptors is equivalent. - Highlights: ► In vitro and in vivo evaluation of 44 Sc- and 68 Ga-DOTA-BN[2-14]NH 2 in reference to published

Positron emission tomography based analysis of long-circulating cross-linked triblock polymeric micelles in a U87MG mouse xenograft model and comparison of DOTA and CB-TE2A as chelators of copper-64.

Science.gov (United States)

Jensen, Andreas I; Binderup, Tina; Kumar EK, Pramod; Kjær, Andreas; Rasmussen, Palle H; Andresen, Thomas L

2014-05-12

Copolymers of ABC-type (PEG-PHEMA-PCMA) architecture were prepared by atom transfer radical polymerization and formulated as micelles with functionalizable primary alcohols in the shell-region (PHEMA-block) to which the metal-ion chelators DOTA or CB-TE2A were conjugated. Using this micelle system we compared the in vivo stabilities of DOTA and CB-TE2A as chelators of (64)Cu in micelle nanoparticles. The coumarin polymer (PCMA-block) micelle core was cross-linked by UV irradiation at 2 W/cm(2) for 30 min. The cross-linked micelles were labeled with (64)Cu at room temperature for 2 h (DOTA) or 80 °C for 3 h (CB-TE2A), giving labeling efficiencies of 60-76% (DOTA) and 40-47% (CB-TE2A). (64)Cu-micelles were injected into tumor-bearing mice (8 mg/kg) and PET/CT scans were carried out at 1, 22, and 46 h postinjection. The micelles showed good blood stability (T1/2: 20-26 h) and tumor uptake that was comparable with other nanoparticle systems. The DOTA micelles showed a biodistribution similar to the CB-TE2A micelles and the tumor uptake was comparable for both micelle types at 1 h (1.9% ID/g) and 22 h (3.9% ID/g) but diverged at 46 h with 3.6% ID/g (DOTA) and 4.9% ID/g (CB-TE2A). On the basis of our data, we conclude that cross-linked PEG-PHEMA-PCMA micelles have long circulating properties resulting in tumor accumulation and that DOTA and CB-TE2A (64)Cu-chelates show similar in vivo stability for the studied micelle system.

SGN-CD33A: a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML.

Science.gov (United States)

Kung Sutherland, May S; Walter, Roland B; Jeffrey, Scott C; Burke, Patrick J; Yu, Changpu; Kostner, Heather; Stone, Ivan; Ryan, Maureen C; Sussman, Django; Lyon, Robert P; Zeng, Weiping; Harrington, Kimberly H; Klussman, Kerry; Westendorf, Lori; Meyer, David; Bernstein, Irwin D; Senter, Peter D; Benjamin, Dennis R; Drachman, Jonathan G; McEarchern, Julie A

2013-08-22

Outcomes in acute myeloid leukemia (AML) remain unsatisfactory, and novel treatments are urgently needed. One strategy explores antibodies and their drug conjugates, particularly those targeting CD33. Emerging data with gemtuzumab ozogamicin (GO) demonstrate target validity and activity in some patients with AML, but efficacy is limited by heterogeneous drug conjugation, linker instability, and a high incidence of multidrug resistance. We describe here the development of SGN-CD33A, a humanized anti-CD33 antibody with engineered cysteines conjugated to a highly potent, synthetic DNA cross-linking pyrrolobenzodiazepine dimer via a protease-cleavable linker. The use of engineered cysteine residues at the sites of drug linker attachment results in a drug loading of approximately 2 pyrrolobenzodiazepine dimers per antibody. In preclinical testing, SGN-CD33A is more potent than GO against a panel of AML cell lines and primary AML cells in vitro and in xenotransplantation studies in mice. Unlike GO, antileukemic activity is observed with SGN-CD33A in AML models with the multidrug-resistant phenotype. Mechanistic studies indicate that the cytotoxic effects of SGN-CD33A involve DNA damage with ensuing cell cycle arrest and apoptotic cell death. Together, these data suggest that SGN-CD33A has CD33-directed antitumor activity and support clinical testing of this novel therapeutic in patients with AML.

Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity

NARCIS (Netherlands)

Kreutz, M.; Giquel, B.; Hu, Q.; Abuknesha, R.; Uematsu, S.; Akira, S.; Nestle, F.O.; Diebold, S.S.

2012-01-01

Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is

Comparison and systematic optimization of synthetic protocols for DOTA-hydrazide generation

NARCIS (Netherlands)

Fuge, F.; Weiler, M.; Gaetjens, J.; Lammers, Twan Gerardus Gertudis Maria

2013-01-01

DOTA-based organometallic complexes are extensively used in functional and molecular imaging studies, as well as in radioimmunotherapy. DOTA forms thermodynamically stable and kinetically inert complexes with various different diagnostic and therapeutic metals, such as gadolinium, gallium, yttrium

Monoclonal anti-melanoma antibodies and their possible clinical use

International Nuclear Information System (INIS)

Hellstroem, K.E.; Hellstroem, Ingegerd; Washington Univ., Seattle; Washington Univ., Seattle

1985-01-01

Cell surface antigens of human melanoma, as defined by monoclonal antibodies, are discussed and in particular the three antigens p97, a GD3 ganglioside and a proteoglycan. The potential diagnostic uses of antibodies to melanoma antigens are reviewed including in vitro diagnosis by immuno-histology, in vitro diagnosis by serum assays and in vivo diagnosis by tumour imaging using radioactively labelled antibodies. The potential therapeutic uses of monoclonal antibodies to melanoma antigens are also reviewed including targets for antibody therapy, the use of antibodies alone, radiolabelled antibodies, antibody-toxin conjugates, antibody-drug conjugates, anti-idiotypic antibodies and vaccines. (UK)

Evaluation of Ga-DOTA-(D-Asp)n as bone imaging agents: D-aspartic acid peptides as carriers to bone.

Science.gov (United States)

Ogawa, Kazuma; Ishizaki, Atsushi; Takai, Kenichiro; Kitamura, Yoji; Makino, Akira; Kozaka, Takashi; Kiyono, Yasushi; Shiba, Kazuhiro; Odani, Akira

2017-10-25

67 Ga-DOTA-(L-Asp) 11 and 67 Ga-DOTA-(L-Asp) 14 , which have been developed as bone imaging agents, showed a high accumulation in bone and a rapid blood clearance in mice. However, peptides composed of D-amino acids are more stable in vivo than those composed of their L-equivalents. In this study, 67 Ga-DOTA-(D-Asp) n (n = 2, 5, 8, 11, or 14) were synthesized using the Fmoc-based solid-phase methodology and evaluated. In hydroxyapatite binding assay, binding of 67 Ga-DOTA-(D-Asp) n tended to increase with increasing length of the amino acid chain. 67 Ga-DOTA-(D-Asp) 11 and 67 Ga-DOTA-(D-Asp) 14 caused a high accumulation of radioactivity in the bones of the mice. However, the results for 67 Ga-DOTA-(D-Asp) n and 67 Ga-DOTA-(L-Asp) n were comparable. In urine analyses, the proportion of intact complex after injection of 67 Ga-DOTA-(D-Asp) 14 was significantly higher than that of 67 Ga-DOTA-(L-Asp) 14 . Although 67 Ga-DOTA-(D-Asp) 14 was more stable than 67 Ga-DOTA-(L-Asp) 14 , the properties of 67 Ga-DOTA-(D-Asp) n and 67 Ga-DOTA-(L-Asp) n as bone imaging agents may be comparable.

64Cu-DOTA-trastuzumab PET imaging in patients with HER2-positive breast cancer.

Science.gov (United States)

Tamura, Kenji; Kurihara, Hiroaki; Yonemori, Kan; Tsuda, Hitoshi; Suzuki, Junko; Kono, Yuzuru; Honda, Natsuki; Kodaira, Makoto; Yamamoto, Harukaze; Yunokawa, Mayu; Shimizu, Chikako; Hasegawa, Koki; Kanayama, Yousuke; Nozaki, Satoshi; Kinoshita, Takayuki; Wada, Yasuhiro; Tazawa, Shusaku; Takahashi, Kazuhiro; Watanabe, Yasuyoshi; Fujiwara, Yasuhiro

2013-11-01

The purpose of this study was to determine the safety, distribution, internal dosimetry, and initial human epidermal growth factor receptor 2 (HER2)-positive tumor images of (64)Cu-DOTA-trastuzumab in humans. PET was performed on 6 patients with primary or metastatic HER2-positive breast cancer at 1, 24, and 48 h after injection of approximately 130 MBq of the probe (64)Cu-DOTA-trastuzumab. Radioactivity data were collected from the blood, urine, and normal-tissue samples of these 6 patients, and the multiorgan biodistribution and internal dosimetry of the probe were evaluated. Safety data were collected for all the patients after the administration of (64)Cu-DOTA-trastuzumab and during the 1-wk follow-up period. According to our results, the best timing for the assessment of (64)Cu-DOTA-trastuzumab uptake by the tumor was 48 h after injection. Radiation exposure during (64)Cu-DOTA-trastuzumab PET was equivalent to that during conventional (18)F-FDG PET. The radioactivity in the blood was high, but uptake of (64)Cu-DOTA-trastuzumab in normal tissues was low. In 2 patients, (64)Cu-DOTA-trastuzumab PET showed brain metastases, indicative of blood-brain barrier disruptions. In 3 patients, (64)Cu-DOTA-trastuzumab PET imaging also revealed primary breast tumors at the lesion sites initially identified by CT. The findings of this study indicated that (64)Cu-DOTA-trastuzumab PET is feasible for the identification of HER2-positive lesions in patients with primary and metastatic breast cancer. The dosimetry and pharmacologic safety results were acceptable at the dose required for adequate PET imaging.

44Sc-DOTA-BN[2-14]NH2 in comparison to 68Ga-DOTA-BN[2-14]NH2 in pre-clinical investigation. Is 44Sc a potential radionuclide for PET?

Science.gov (United States)

Koumarianou, E; Loktionova, N S; Fellner, M; Roesch, F; Thews, O; Pawlak, D; Archimandritis, S C; Mikolajczak, R

2012-12-01

In the present study we demonstrate the in vitro and in vivo comparison of the (44)Sc and (68)Ga labeled DOTA-BN[2-14]NH(2). (44)Sc is a positron emitter with a half life of 3.92 h. Hence it could be used for PET imaging with ligands requiring longer observation time than in the case of (68)Ga. The binding affinity of (nat)Sc-DOTA-BN[2-14]NH(2) and (nat)Ga-DOTA-BN[2-14]NH(2) to GRP receptors was studied in competition to [(125)I-Tyr(4)]-Bombesin in the human prostate cancer cell line PC-3. A preliminary biodistribution in normal rats was performed, while first microPET images were assessed in male Copenhagen rats bearing the androgen-independent Dunning R-3327-AT-1 prostate cancer tumor. The affinity to GRP receptors in the PC-3 cell line was higher for (nat)Ga-DOTA-BN[2-14]NH(2) (IC(50)(nM)=0.85 ± 0.06) than that of (nat)Sc-DOTA-BN[2-14]NH(2) (IC(50) (nM)=6.49 ± 0.13). The internalization rate of (68)Ga labeled DOTA-BN[2-14]NH(2) was slower than that of (44)Sc, but their final internalization percents were comparable. (68)Ga-DOTA-BN[2-14]NH(2) was externalized faster than (44)Sc-DOTA-BN[2-14]NH(2). The biodistribution of (44)Sc-DOTA-BN[2-14]NH(2) and (68)Ga-DOTA-BN[2-14]NH(2) in normal rats revealed a higher uptake in target organs and tissues of the first one while both excreted mainly through urinary tract. In microPET images both tracers were accumulated in the tumor with similar uptake patterns. Despite the differences in the receptor affinity both the (68)Ga- and the (44)Sc-labeled DOTA-BN[2-14]NH(2) tracers showed comparable distribution and similar time constants of uptake and elimination. Moreover no differences in tumor accumulation (neither in the overall uptake nor in the dynamics) were observed from the microPet imaging. From that perspective the use of either (44)Sc or (68)Ga for detecting tumors with GRP receptors is equivalent. Copyright © 2012 Elsevier Ltd. All rights reserved.

Targeted Delivery of LXR Agonist Using a Site-Specific Antibody-Drug Conjugate.

Science.gov (United States)

Lim, Reyna K V; Yu, Shan; Cheng, Bo; Li, Sijia; Kim, Nam-Jung; Cao, Yu; Chi, Victor; Kim, Ji Young; Chatterjee, Arnab K; Schultz, Peter G; Tremblay, Matthew S; Kazane, Stephanie A

2015-11-18

Liver X receptor (LXR) agonists have been explored as potential treatments for atherosclerosis and other diseases based on their ability to induce reverse cholesterol transport and suppress inflammation. However, this therapeutic potential has been hindered by on-target adverse effects in the liver mediated by excessive lipogenesis. Herein, we report a novel site-specific antibody-drug conjugate (ADC) that selectively delivers a LXR agonist to monocytes/macrophages while sparing hepatocytes. The unnatural amino acid para-acetylphenylalanine (pAcF) was site-specifically incorporated into anti-CD11a IgG, which binds the α-chain component of the lymphocyte function-associated antigen 1 (LFA-1) expressed on nearly all monocytes and macrophages. An aminooxy-modified LXR agonist was conjugated to anti-CD11a IgG through a stable, cathepsin B cleavable oxime linkage to afford a chemically defined ADC. The anti-CD11a IgG-LXR agonist ADC induced LXR activation specifically in human THP-1 monocyte/macrophage cells in vitro (EC50-27 nM), but had no significant effect in hepatocytes, indicating that payload delivery is CD11a-mediated. Moreover, the ADC exhibited higher-fold activation compared to a conventional synthetic LXR agonist T0901317 (Tularik) (3-fold). This novel ADC represents a fundamentally different strategy that uses tissue targeting to overcome the limitations of LXR agonists for potential use in treating atherosclerosis.

Radiolabeled, Antibody-Conjugated Manganese Oxide Nanoparticles for Tumor Vasculature Targeted Positron Emission Tomography and Magnetic Resonance Imaging.

Science.gov (United States)

Zhan, Yonghua; Shi, Sixiang; Ehlerding, Emily B; Graves, Stephen A; Goel, Shreya; Engle, Jonathan W; Liang, Jimin; Tian, Jie; Cai, Weibo

2017-11-08

Manganese oxide nanoparticles (Mn 3 O 4 NPs) have attracted a great deal of attention in the field of biomedical imaging because of their ability to create an enhanced imaging signal in MRI as novel potent T 1 contrast agents. In this study, we present tumor vasculature-targeted imaging in mice using Mn 3 O 4 NPs through conjugation to the anti-CD105 antibody TRC105 and radionuclide copper-64 ( 64 Cu, t 1/2 : 12.7 h). The Mn 3 O 4 conjugated NPs, 64 Cu-NOTA-Mn 3 O 4 @PEG-TRC105, exhibited sufficient stability in vitro and in vivo. Serial positron emission tomography (PET) and magnetic resonance imaging (MRI) studies evaluated the pharmacokinetics and demonstrated targeting of 64 Cu-NOTA-Mn 3 O 4 @PEG-TRC105 to 4T1 murine breast tumors in vivo, compared to 64 Cu-NOTA-Mn 3 O 4 @PEG. The specificity of 64 Cu-NOTA-Mn 3 O 4 @PEG-TRC105 for the vascular marker CD105 was confirmed through in vivo, in vitro, and ex vivo experiments. Since Mn 3 O 4 conjugated NPs exhibited desirable properties for T 1 enhanced imaging and low toxicity, the tumor-specific Mn 3 O 4 conjugated NPs reported in this study may serve as promising multifunctional nanoplatforms for precise cancer imaging and diagnosis.

Localization of Hidden Insulinomas with ⁶⁸Ga-DOTA-Exendin-4 PET/CT: A Pilot Study.

Science.gov (United States)

Antwi, Kwadwo; Fani, Melpomeni; Nicolas, Guillaume; Rottenburger, Christof; Heye, Tobias; Reubi, Jean Claude; Gloor, Beat; Christ, Emanuel; Wild, Damian

2015-07-01

(111)In-DOTA-exendin-4 SPECT/CT has been shown to be highly efficient in the detection of insulinomas. We aimed at determining whether novel PET/CT imaging with [Nle(14),Lys(40)(Ahx-DOTA-(68)Ga)NH2]exendin-4 ((68)Ga-DOTA-exendin-4) is feasible and sensitive in detecting benign insulinomas. (68)Ga-DOTA-exendin-4 PET/CT and (111)In-DOTA-exendin-4 SPECT/CT were performed in a randomized cross-over order on 5 patients with endogenous hyperinsulinemic hypoglycemia. The gold standard for comparison was the histologic diagnosis after surgery. In 4 patients histologic diagnosis confirmed a benign insulinoma, whereas one patient refused surgery despite a positive (68)Ga-DOTA-exendin-4 PET/CT scan. In 4 of 5 patients, previously performed conventional imaging (CT or MR imaging) was not able to localize the insulinoma. (68)Ga-DOTA-exendin-4 PET/CT correctly identified the insulinoma in 4 of 4 patients, whereas (111)In-DOTA-exendin-4 SPECT/CT correctly identified the insulinoma in only 2 of 4 patients. These preliminary data suggest that the use of (68)Ga-DOTA-exendin-4 PET/CT in detecting hidden insulinomas is feasible. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Antibody or Antibody Fragments : Implications for Molecular Imaging and Targeted Therapy of Solid Tumors

NARCIS (Netherlands)

Xenaki, Katerina T; Oliveira, Sabrina; van Bergen En Henegouwen, Paul M P

2017-01-01

The use of antibody-based therapeutics has proven very promising for clinical applications in cancer patients, with multiple examples of antibodies and antibody-drug conjugates successfully applied for the treatment of solid tumors and lymphomas. Given reported recurrence rates, improvements are

High resolution separations of charge variants and disulfide isomers of monoclonal antibodies and antibody drug conjugates using ultra-high voltage capillary electrophoresis with high electric field strength.

Science.gov (United States)

Henley, W Hampton; He, Yan; Mellors, J Scott; Batz, Nicholas G; Ramsey, J Michael; Jorgenson, James W

2017-11-10

Ultra-high voltage capillary electrophoresis with high electric field strength has been applied to the separation of the charge variants, drug conjugates, and disulfide isomers of monoclonal antibodies. Samples composed of many closely related species are difficult to resolve and quantify using traditional analytical instrumentation. High performance instrumentation can often save considerable time and effort otherwise spent on extensive method development. Ideally, the resolution obtained for a given CE buffer system scales with the square root of the applied voltage. Currently available commercial CE instrumentation is limited to an applied voltage of approximately 30kV and a maximum electric field strength of 1kV/cm due to design limitations. The instrumentation described here is capable of safely applying potentials of at least 120kV with electric field strengths over 2000V/cm, potentially doubling the resolution of the best conventional CE buffer/capillary systems while decreasing analysis time in some applications. Separations of these complex mixtures using this new instrumentation demonstrate the potential of ultra-high voltage CE to identify the presence of previously unresolved components and to reduce analysis time for complex mixtures of antibody variants and drug conjugates. Copyright © 2017 Elsevier B.V. All rights reserved.

Neuroendocrine tumor imaging with 68Ga-DOTA-NOC: physiologic and benign variants.

Science.gov (United States)

Kagna, Olga; Pirmisashvili, Natalia; Tshori, Sagi; Freedman, Nanette; Israel, Ora; Krausz, Yodphat

2014-12-01

Imaging with (68)Ga-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-octreotide analogs has become an important modality in patients with neuroendocrine tumors (NETs). In addition to high uptake in NET lesions, prominent physiologic radiotracer activity has been reported in the pituitary gland, pancreas, adrenal glands, liver, and spleen, and faint activity has been reported in the thyroid and gastrointestinal tract. This article describes previously unknown sites of 68Ga-DOTA-1-NaI3-octreotide (NOC) uptake unrelated to NETs. One hundred eighty-two patients (96 female and 86 male patients; age range, 4-89 years) with documented (n=156) or suspected (n=26) NETs underwent 207 68Ga-DOTA-NOC PET/CT studies. Studies were retrospectively reviewed for the presence, intensity, and localization of foci of increased uptake that were further correlated with findings on additional imaging studies and clinical follow-up for a period of 4-32 months. Uptake of 68Ga-DOTA-NOC not identified as NET or known physiologic activity was detected in 297 sites with confirmation in 149 of 207 studies (72%). The most common location of non-NET-related 68Ga-DOTA-NOC-avid sites was in small lymph nodes, followed by prostate, uterus, breasts, lungs, brown fat, musculoskeletal system, and other sites, including oropharynx, pineal body, thymus, aortic plaque, genitalia, surgical bed, and subcutaneous granuloma. Intensity of uptake in non-NET-related 68Ga-DOTA-NOC-avid sites ranged in maximum standardized uptake value from 0.8 to 10.5. Previously unreported benign sites of 68Ga-DOTA-NOC uptake were found in the majority of studies, suggesting the presence of somatostatin receptors in physiologic variants or processes with no evidence of tumor. Knowledge of increased tracer uptake in non-NET-related sites is important for accurate interpretation and for avoiding potential pitfalls of 68Ga-DOTA-NOC PET/CT.

Value of {sup 111}In-DOTA-lanreotide and {sup 111}In-DOTA-DPhe{sup 1}-Tyr{sup 3}-octreotide in differentiated thyroid cancer: results of in vitro binding studies and in vivo comparison with {sup 18}F-FDG PET

Energy Technology Data Exchange (ETDEWEB)

Rodrigues, Margarida; Virgolini, Irene [Lainz Hospital, Institute of Nuclear Medicine, Vienna (Austria); University Clinic for Nuclear Medicine, Innsbruck (Austria); Traub-Weidinger, Tatjana [University Clinic for Nuclear Medicine, Innsbruck (Austria); University Hospital, Department of Nuclear Medicine, Vienna (Austria); Leimer, Maria; Li, Shuren; Dudczak, Robert [University Hospital, Department of Nuclear Medicine, Vienna (Austria); Andreae, Fritz [University Clinic for Nuclear Medicine, Innsbruck (Austria); Angelberger, Peter [Austrian Research Center, Department of Radiochemistry, Seibersdorf (Austria)

2005-10-01

Radioiodine-negative thyroid cancer presents diagnostic and therapeutic difficulties, warranting the implementation of new imaging and treatment strategies. The purpose of this study was twofold. First, we investigated in vitro the binding characteristics of {sup 111}In-DOTA-lanreotide ({sup 111}In-DOTA-LAN) and {sup 111}In-DOTA-DPhe{sup 1}-Tyr{sup 3}-octreotide ({sup 111}In-DOTA-TOC) to cells derived from differentiated thyroid cancer (DTC). Second, we evaluated the value of somatostatin receptor (SSTR) scintigraphy with these radioligands, as compared with {sup 18}F-fluorodeoxyglucose (FDG) positron emission tomography (PET), for the detection of tumour lesions in DTC patients. Binding of {sup 111}In-DOTA-LAN and {sup 111}In-DOTA-TOC to cells isolated from surgically removed thyroid tissue was evaluated in vitro by performing saturation and displacement studies. Eighteen DTC patients with elevated thyroglobulin (12 radioiodine-negative, six radioiodine-positive) were investigated with {sup 111}In-DOTA-LAN, {sup 111}In-DOTA-TOC and {sup 18}F-FDG PET scans. Large numbers of SSTR binding sites for {sup 111}In-DOTA-LAN and {sup 111}In-DOTA-TOC were found on the cells investigated. Both SSTR radioligands exhibited a high binding affinity for these SSTR binding sites. {sup 111}In-DOTA-LAN and {sup 111}In-DOTA-TOC scintigraphy detected 37 and 33 lesions, respectively, in 17 (94%) patients each, whereas {sup 18}F-FDG PET revealed 30 lesions in 15 (83%) patients. Uptake of both SSTR radioligands was found in several radioiodine-negative sites. No striking differences in lesion imaging by {sup 111}In-DOTA-LAN and {sup 111}In-DOTA-TOC were found. In both radioiodine-negative and radioiodine-positive patients, more lesions were SSTR-positive/{sup 18}F-FDG-negative than were {sup 18}F-FDG-positive/SSTR-negative. Adding a SSTR scan with these radioligands to the diagnostic work-up increases the diagnostic capacity in DTC, and should be considered particularly in radioiodine

Gd-DOTA enhancement of cerebral and spinal tumors on MR imaging

International Nuclear Information System (INIS)

Berry, I.; Manelfe, C.; Chastin, I.; Arrue, P.; Prere, J.

1987-01-01

The use of Gd-DOTA as a contrast agent in MR imaging to improve the diagnosis of cerebral and spinal tumors was assessed in 20 patients, ten with brain tumors and ten with spinal tumors. Imaging was performed with a 0.5-T Magniscan 5000 unit. T1-weighted (spin-echo and gradient-echo) and T2-weighted (spin-echo) images were acquired before and after intravenous injection of Gd-DOTA, 0.1 mmol/kg. On T1-weighted images, Gd-DOTA enhanced sites of presumed disruption of the blood-brain barrier. This made some brain tumors more conspicuous and helped target biopsies, but did not reveal any additional lesions. On the other hand, the use of Gd-DOTA significantly improved the reliability of spinal tumor imaging compared to imaging performed without contrast agent, allowing delineation of abnormalities on T1-weighted images, which frequently contain fewer artifacts than the most sensitive T2-weighted images. Images obtained with Gd-DOTA could be used by the physician to rule out residual tumor after surgery and to assess recurrences. Additional work should be done to discover whether spinal tumor exploration with MR imaging could include solely T1-weighted sequences, performed before and after contrast agent administration, without T2-weighted sequences

Immunosuppressive drugs impairs antibody response of the polysaccharide and conjugated pneumococcal vaccines in patients with Crohn's disease

DEFF Research Database (Denmark)

Kantsø, Bjørn; Halkjær, Sofie Ingdam; Thomsen, Ole Østergaard

2015-01-01

BACKGROUND: Patients with Crohn's disease (CD) have a higher risk of infectious diseases including pneumococcal infections, and the risk increases with immunotherapy. The primary endpoint of this study was to investigate the specific antibody response to two pneumococcal vaccines in CD patients...... with and without immunosuppressive treatment four weeks post vaccination. METHODS: In a randomized trial of the 23-valent pneumococcal polysaccharide vaccine (PPV23) and the 13-valent pneumococcal conjugated vaccine (PCV13), a group of CD patients treated with immunosuppressive drugs (IS) alone or in combination...... with TNF-α antagonists were compared to a group of CD patients not treated with any of these drugs (untreated). Specific pneumococcal antibody concentrations were measured against 12 serotypes common to the two vaccines before and 4 week after vaccination. RESULTS: PCV13 induced a significantly higher...

A new immunization procedure for the obtention of anti-leucine enkephalin antibodies. Part I. Immunization procedure and physicochemical characteristics of antibodies.

Science.gov (United States)

Cupo, A; Vion-Dury, J; Jarry, T

1986-10-01

We described here a new immunization procedure to obtain high titre and high specific antibodies against Leu-enkephalin (LE). The immunogen form is composed of one part of LE conjugate and one part of LE-Arg6 conjugate. We have observed an increase of titre, affinity and specificity of the antibodies in the coimmunization procedure compared to those obtained by conventional immunization involving only the LE conjugate. The Leu-enkephalin antibodies exhibit a high affinity (KD 8 X 10(-12) M) and we are able to detect the Leu-enkephalin at the 10(-15) mole level. These LE antibodies are highly specific of the C part of LE peptide and cross-react weakly with Met-enkephalin (1%).

Tumor-specific detection of an optically targeted antibody combined with a quencher-conjugated neutravidin "quencher-chaser": a dual "quench and chase" strategy to improve target to nontarget ratios for molecular imaging of cancer.

Science.gov (United States)

Ogawa, Mikako; Kosaka, Nobuyuki; Choyke, Peter L; Kobayashi, Hisataka

2009-01-01

In vivo molecular cancer imaging with monoclonal antibodies has great potential not only for cancer detection, but also for cancer characterization. However, the prolonged retention of intravenously injected antibody in the blood causes low target tumor-to-background ratio (TBR). Avidin has been used as a "chase" to clear the unbound, circulating biotinylated antibody and decrease the background signal. Here, we utilize a combined approach of a fluorescence resonance energy transfer (FRET) quenched antibody with an "avidin chase" to increase TBR. Trastuzumab, a humanized monoclonal antibody against human epidermal growth factor receptor type 2 (HER2), was biotinylated and conjugated with the near-infrared (NIR) fluorophore Alexa680 to synthesize Tra-Alexa680-biotin. Next, the FRET quencher, QSY-21, was conjugated to avidin, neutravidin (nAv), or streptavidin (sAv), thus creating Av-QSY21, nAv-QSY21, or sAv-QSY21 as "chasers". The fluorescence was quenched in vitro by binding Tra-Alexa680-biotin to Av-QSY21, nAv-QSY21, or sAv-QSY21. To evaluate if the injection of quencher-conjugated avidin derivatives can improve target TBR by using a dual "quench and chase" strategy, both target (3T3/HER2+) and nontarget (Balb3T3/ZsGreen) tumor-bearing mice were employed. The "FRET quench" effect induced by all the QSY21 avidin-based conjugates reduced but did not totally eliminate background signal from the blood pool. The addition of nAv-QSY21 administration increased target TBR mainly because of the "chase" effect where unbound conjugated antibody was preferentially cleared to the liver. The relatively slow clearance of unbound nAv-QSY21 leads to further reductions in background signal by leaking out of the vascular space and binding to unbound antibodies in the extravascular space of tumors, resulting in decreased nontarget tumor-to-background ratios but increased target TBR due to the "FRET quench" effect, because target-bound antibodies were internalized and could not bind

Outer membrane protein complex of Meningococcus enhances the antipolysaccharide antibody response to pneumococcal polysaccharide-CRM₁₉₇ conjugate vaccine.

Science.gov (United States)

Lai, Zengzu; Schreiber, John R

2011-05-01

Bacterial polysaccharides (PS) are T cell-independent antigens that do not induce immunologic memory and are poor immunogens in infants. Conjugate vaccines in which the PS is covalently linked to a carrier protein have enhanced immunogenicity that resembles that of T cell-dependent antigens. The Haemophilus influenzae type b (Hib) conjugate vaccine, which uses the outer membrane protein complex (OMPC) from meningococcus as a carrier protein, elicits protective levels of anti-capsular PS antibody (Ab) after a single dose, in contrast to other conjugate vaccines, which require multiple doses. We have previously shown that OMPC robustly engages Toll-like receptor 2 (TLR2) and enhances the early anti-Hib PS Ab titer associated with an increase in TLR2-mediated induction of cytokines. We now show that the addition of OMPC to the 7-valent pneumococcal PS-CRM₁₉₇ conjugate vaccine during immunization significantly increases the anti-PS IgG and IgM responses to most serotypes of pneumococcus contained in the vaccine. The addition of OMPC also increased the likelihood of anti-PS IgG3 production against serotypes 4, 6B, 9V, 18C, 19F, and 23F. Splenocytes from mice who had received OMPC with the pneumococcal conjugate vaccine produced significantly more interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) than splenocytes from mice who received phosphate-buffered saline (PBS) plus the conjugate vaccine. We conclude that OMPC enhances the anti-PS Ab response to pneumococcal PS-CRM₁₉₇ conjugate vaccine, an effect associated with a distinct change in cytokine profile. It may be possible to reduce the number of conjugate vaccine doses required to achieve protective Ab levels by priming with adjuvants that are TLR2 ligands.

MR study of acute myocardial infarction with injection of Gd-DOTA (Fifteen patients)

International Nuclear Information System (INIS)

Richoz, B.; Delcour, C.; Depelchin, P.; Lenaers, A.; Jacquemin, C.; Gusella, P.; Struyven, J.; Richoz, B.

1990-01-01

We studied 15 patients 4 to 8 days after myocardial infarction by using ECG gated MR before and after administration of 0.2 mmol/kg Gd-DOTA. The diagnosis in each patient was confirmed by electrocardiographic criteria, elevated levels of fractionated creatine kinase (CK) isoenzyme, thallium scintigraphy, ventriculography and coronarography. T1-weighted, spin-echo images, were obtained before and immediately after injection of Gd-DOTA and were repeated 15 min later. The site of infarction was visualized in 10 patients as an area of high signal intensity after the injection of Gd-DOTA. Contrast between normal and infarcted myocardium was greatest 15 min after injection. Three patients were excluded because of failure to acquire adequate MR studies. In 2 other patients, the infarct were not detected. Before injection of Gd-DOTA, only 2 infarcts were detected. These results suggest that Gd-DOTA can improve MR visualization and detection of acute myocardial infarction [fr

Development of 177Lu-DOTA-anti-CD20 for radioimmunotherapy

International Nuclear Information System (INIS)

Hassan Yousefnia; Amir Reza Jalilian; Ali Bahrami-Samani; Simindokht Shirvani-Arani; Mohammad Ghannadi-Maragheh; Azim Arbabi; Edalat Radfar

2011-01-01

Rituximab was successively labeled with 177 Lu-lutetium chloride. 177 Lu chloride was obtained by thermal neutron flux (4 x 1013 n cm -2 s -1 ) of natural Lu 2 O 3 sample with a specific activity of 2.6-3 GBq/mg. The macrocyclic bifunctional chelating agent, N-succinimidyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-NHS) was prepared at 25 deg C using DOTA, N-hydroxy succinimide (NHS) in CH 2 Cl 2 . DOTA-rituximab was obtained by the addition of 1 mL of a rituximab pharmaceutical solution (5 mg/mL, in phosphate buffer, pH 7.8) to a glass tube pre-coated with DOTA-NHS (0.01-0.1 mg) at 25 deg C with continuous mild stirring for 15 h. Radiolabeling was performed at 37 deg C in 24 h. Radio-thin layer chromatography showed an overall radiochemical purity of >98% at optimized conditions (specific activity = 444 MBq/mg, labeling efficacy; 82%). The final isotonic 177 Lu-DOTA-rituximab complex was checked by gel electrophoresis for structure integrity control. Radio-TLC was performed to ensure that only one species was present after filtration through a 0.22 μm filter. Preliminary biodistribution studies in normal rats were carried out to determine complex distribution of the radioimmunoconjugate up to 168 h. The biodistribution data were in accordance with other antiCD20 radioimmunoconjugates already reported. (author)

HER2 specific delivery of methotrexate by dendrimer conjugated anti-HER2 mAb

International Nuclear Information System (INIS)

Shukla, Rameshwer; Thomas, Thommey P; Desai, Ankur M; Kotlyar, Alina; Park, Steve J; Baker, James R Jr

2008-01-01

Herceptin, a humanized monoclonal antibody that binds to human growth factor receptor-2 (HER2), was covalently attached to a fifth-generation (G5) polyamidoamine dendrimer containing the cytotoxic drug methotrexate. The specific binding and internalization of this conjugate labeled with FITC was clearly demonstrated in cell lines overexpressing HER2 by flow cytometry as well as confocal microscopic analysis. In addition, binding and uptake of antibody conjugated dendrimers was completely blocked by excess non-conjugated herceptin. The dendrimer conjugate was also shown to inhibit the dihydrofolate reductase with similar activity to methotrexate. Co-localization experiments with lysotracker red indicate that antibody conjugate, although internalized efficiently into cells, has an unusually long residence time in the lysosome. Somewhat lower cytotoxicity of the conjugate in comparison to free methotrexate was attributed to the slow release of methotrexate from the conjugate and its long retention in the lysosomal pocket

Enhanced tumor retention of a radiohalogen label for site-specific modification of antibodies.

Science.gov (United States)

Boswell, C Andrew; Marik, Jan; Elowson, Michael J; Reyes, Noe A; Ulufatu, Sheila; Bumbaca, Daniela; Yip, Victor; Mundo, Eduardo E; Majidy, Nicholas; Van Hoy, Marjie; Goriparthi, Saritha N; Trias, Anthony; Gill, Herman S; Williams, Simon P; Junutula, Jagath R; Fielder, Paul J; Khawli, Leslie A

2013-12-12

A known limitation of iodine radionuclides for labeling and biological tracking of receptor targeted proteins is the tendency of iodotyrosine to rapidly diffuse from cells following endocytosis and lysosomal degradation. In contrast, radiometal-chelate complexes such as indium-111-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (In-111-DOTA) accumulate within target cells due to the residualizing properties of the polar, charged metal-chelate-amino acid adduct. Iodine radionuclides boast a diversity of nuclear properties and chemical means for incorporation, prompting efforts to covalently link radioiodine with residualizing molecules. Herein, we describe the Ugi-assisted synthesis of [I-125]HIP-DOTA, a 4-hydroxy-3-iodophenyl (HIP) derivative of DOTA, and demonstration of its residualizing properties in a murine xenograft model. Overall, this study displays the power of multicomponent synthesis to yield a versatile radioactive probe for antibodies across multiple therapeutic areas with potential applications in both preclinical biodistribution studies and clinical radioimmunotherapies.

Electronic effects in emission of core/shell CdSe/ZnS quantum dots conjugated to anti-Interleukin 10 antibodies

Energy Technology Data Exchange (ETDEWEB)

Quintos Vazquez, A.L. [ESIME—Instituto Politécnico Nacional, México D. F. 07738, México (Mexico); Torchynska, T.V., E-mail: ttorch@esfm.ipn.mx [ESFM–Instituto Politécnico Nacional, México D. F. 07738, México (Mexico); Casas Espinola, J.L. [ESFM–Instituto Politécnico Nacional, México D. F. 07738, México (Mexico); Jaramillo Gómez, J.A.; Douda, J. [UPIITA–Instituto Politécnico Nacional, México D. F. 07320, México (Mexico)

2013-11-15

The paper presents a comparative study of the photoluminescence (PL) and Raman scattering spectra of the core–shell CdSe/ZnS quantum dots (QDs) in nonconjugated states and after the conjugation to anti-Interleukin 10 antibodies (anti-IL10). All optical measurements are performed on the dried droplets of the original solution of nonconjugated and bioconjugated QDs located on the Si substrate. CdSe/ZnS QDs with emission at 605 and 655 nm have been used. PL spectra of nonconjugated QDs are characterized by one Gaussian shape PL band related to the exciton emission in the CdSe core. PL spectra of bioconjugated QDs have changed essentially: the core PL band shifts into the high energy spectral range (“blue” sift) and becomes asymmetric. Additionally two new PL bands appear. A set of physical reasons has been proposed for the “blue” shift explanation for the core PL band in bioconjugated QDs. Then Raman scattering spectra have been studied with the aim to analyze the impact of elastic strains or the oxidation process at the QD bioconjugation. The variation of PL spectra versus excitation light intensities has been studied to analyze the exciton emission via excited states in QDs. Finally the PL spectrum transformation for the core emission in bioconjugated QDs has been attributed to the electronic quantum confined effects stimulated by the electric charges of bioconjugated antibodies. -- Highlights: • The conjugation of CdSe/ZnS QDs to anti-Interleukin 10 antibodies has been studied. • PL shift to high energy is detected in bioconjugated CdSe/ZnS QDs. • The PL energy shift in bioconjugated QDs is stimulated by antibody electric charges. • The reasons of PL energy shift in bioconjugated QDs have been discussed.

Lutetium-177 complexation of DOTA and DTPA in the presence of competing metals

International Nuclear Information System (INIS)

Watanabe, Satoshi; Ishioka, Noriko S.; Hashimoto, Kazuyuki

2013-01-01

177 Lu complexation of DOTA and DTPA is investigated by the addition of Ca(II), Fe(II) and Zn(II). The 177 Lu complexation yield of DTPA was higher than that of DOTA in the presence of Ca(II), Fe(II) and Zn(II). Therefore, it was found that the 177 Lu complexation of DTPA was more advantageous compared with DOTA in the presence of competing metals, Ca, Fe and Zn. (author)

Peptide-Carrier Conjugation

DEFF Research Database (Denmark)

Hansen, Paul Robert

2015-01-01

To produce antibodies against synthetic peptides it is necessary to couple them to a protein carrier. This chapter provides a nonspecialist overview of peptide-carrier conjugation. Furthermore, a protocol for coupling cysteine-containing peptides to bovine serum albumin is outlined....

Characterization of SGN-CD123A, A Potent CD123-Directed Antibody-Drug Conjugate for Acute Myeloid Leukemia.

Science.gov (United States)

Li, Fu; Sutherland, May Kung; Yu, Changpu; Walter, Roland B; Westendorf, Lori; Valliere-Douglass, John; Pan, Lucy; Cronkite, Ashley; Sussman, Django; Klussman, Kerry; Ulrich, Michelle; Anderson, Martha E; Stone, Ivan J; Zeng, Weiping; Jonas, Mechthild; Lewis, Timothy S; Goswami, Maitrayee; Wang, Sa A; Senter, Peter D; Law, Che-Leung; Feldman, Eric J; Benjamin, Dennis R

2018-02-01

Treatment choices for acute myelogenous leukemia (AML) patients resistant to conventional chemotherapies are limited and novel therapeutic agents are needed. IL3 receptor alpha (IL3Rα, or CD123) is expressed on the majority of AML blasts, and there is evidence that its expression is increased on leukemic relative to normal hematopoietic stem cells, which makes it an attractive target for antibody-based therapy. Here, we report the generation and preclinical characterization of SGN-CD123A, an antibody-drug conjugate using the pyrrolobenzodiazepine dimer (PBD) linker and a humanized CD123 antibody with engineered cysteines for site-specific conjugation. Mechanistically, SGN-CD123A induces activation of DNA damage response pathways, cell-cycle changes, and apoptosis in AML cells. In vitro , SGN-CD123A-mediated potent cytotoxicity of 11/12 CD123 + AML cell lines and 20/23 primary samples from AML patients, including those with unfavorable cytogenetic profiles or FLT3 mutations. In vivo , SGN-CD123A treatment led to AML eradication in a disseminated disease model, remission in a subcutaneous xenograft model, and significant growth delay in a multidrug resistance xenograft model. Moreover, SGN-CD123A also resulted in durable complete remission of a patient-derived xenograft AML model. When combined with a FLT3 inhibitor quizartinib, SGN-CD123A enhanced the activity of quizartinib against two FLT3-mutated xenograft models. Overall, these data demonstrate that SGN-CD123A is a potent antileukemic agent, supporting an ongoing trial to evaluate its safety and efficacy in AML patients (NCT02848248). Mol Cancer Ther; 17(2); 554-64. ©2017 AACR . ©2017 American Association for Cancer Research.

Anti-PDGF receptor β antibody-conjugated squarticles loaded with minoxidil for alopecia treatment by targeting hair follicles and dermal papilla cells.

Science.gov (United States)

Aljuffali, Ibrahim A; Pan, Tai-Long; Sung, Calvin T; Chang, Shu-Hao; Fang, Jia-You

2015-08-01

This study developed lipid nanocarriers, called squarticles, conjugated with anti-platelet-derived growth factor (PDGF)-receptor β antibody to determine whether targeted Minoxidil (MXD) delivery to the follicles and dermal papilla cells (DPCs) could be achieved. Squalene and hexadecyl palmitate (HP) were used as the matrix of the squarticles. The PDGF-squarticles showed a mean diameter and zeta potential of 195 nm and -46 mV, respectively. Nanoparticle encapsulation enhanced MXD porcine skin deposition from 0.11 to 0.23 μg/mg. The antibody-conjugated nanoparticles ameliorated follicular uptake of MXD by 3-fold compared to that of the control solution in the in vivo mouse model. Both vertical and horizontal skin sections exhibited a wide distribution of nanoparticles in the follicles, epidermis, and deeper skin strata. The encapsulated MXD moderately elicited proliferation of DPCs and vascular endothelial growth factor (VEGF) expression. The active targeting of PDGF-squarticles may be advantageous to improving the limited success of alopecia therapy. Topical use of minoxidil is only one of the very few treatment options for alopecia. Nonetheless, the current delivery method is far from ideal. In this article, the authors developed lipid nanocarriers with anti-platelet-derived growth factor receptor ? antibody to target dermal papilla cells, and showed enhanced uptake of minoxidil. Copyright © 2015 Elsevier Inc. All rights reserved.

68Ga-DOTA-RGD peptide: biodistribution and binding into atherosclerotic plaques in mice

International Nuclear Information System (INIS)

Haukkala, Johanna; Laitinen, Iina; Luoto, Pauliina; Knuuti, Juhani; Iveson, Peter; Wilson, Ian; Karlsen, Hege; Cuthbertson, Alan; Laine, Jukka; Leppaenen, Pia; Ylae-Herttula, Seppo; Roivainen, Anne

2009-01-01

Increased expression of αvβ3/αvβ5 integrin is involved in angiogenesis and the inflammatory process in atherosclerotic plaques. The novel 68 Ga-DOTA-RGD peptide binds with high affinity to αvβ3/αvβ5 integrin. The aim of this study was to investigate the uptake of the 68 Ga-DOTA-RGD peptide in atherosclerotic plaques. Uptake of intravenously administered 68 Ga-DOTA-RGD peptide was studied ex vivo in excised tissue samples and aortic sections of LDLR -/- ApoB 100/100 atherosclerotic mice. The uptake of the tracer in aortic cryosections was examined by using digital autoradiography. Subsequently, the autoradiographs were combined with histological and immunohistological analysis of the sections. DOTA-RGD peptide was successfully labelled with the generator-produced 68 Ga. The tracer had reasonably good specific radioactivity (8.7 ± 1.1 GBq/μmol) and was quite stable in vivo. According to ex vivo biodistribution results, 68 Ga-DOTA-RGD was cleared rapidly from the blood circulation and excreted through the kidneys to the urine with high radioactivity in the intestine, lungs, spleen and liver. Autoradiography results showed significantly higher uptake of 68 Ga-DOTA-RGD peptide in the atherosclerotic plaques compared to healthy vessel wall (mean ratio ± SD 1.4 ± 0.1, p = 0.0004). We observed that 68 Ga-DOTA-RGD is accumulated into the plaques of atherosclerotic mice. However, this data only shows the feasibility of the approach, while the clinical significance still remains to be proven. Further studies are warranted to assess the uptake of this tracer into human atherosclerotic plaques. (orig.)

Heavy-chain isotype patterns of human antibody-secreting cells induced by Haemophilus influenzae type b conjugate vaccines in relation to age and preimmunity

DEFF Research Database (Denmark)

Barington, T; Juul, Lars; Gyhrs, A

1994-01-01

The influence of preexisting immunity on the heavy-chain isotypes of circulating antibody-secreting cells (AbSC) induced by vaccination with Haemophilus influenzae type b (Hib) capsular polysaccharide (HibCP) coupled to tetanus toxoid (TT) or diphtheria toxoid (DT) and by vaccination with TT or D...... of natural HibCP antibodies (r = 0.59; P = 0.00002). A possible role of natural exposure for Hib or cross-reactive bacteria on the mucosal surfaces in the shaping of the isotype response to HibCP conjugate vaccines is discussed....

Correlation of Somatostatin Receptor-2 Expression with Gallium-68-DOTA-TATE Uptake in Neuroblastoma Xenograft Models

OpenAIRE

Zhang, Libo; Vines, Douglass C.; Scollard, Deborah A.; McKee, Trevor; Komal, Teesha; Ganguly, Milan; Do, Trevor; Wu, Bing; Alexander, Natasha; Vali, Reza; Shammas, Amer; Besanger, Travis; Baruchel, Sylvain

2017-01-01

Peptide-receptor imaging and therapy with radiolabeled somatostatin analogs such as 68Ga-DOTA-TATE and 177Lu-DOTA-TATE have become an effective treatment option for SSTR-positive neuroendocrine tumors. The purpose of this study was to evaluate the correlation of somatostatin receptor-2 (SSTR2) expression with 68Ga-DOTA-TATE uptake and 177Lu-DOTA-TATE therapy in neuroblastoma (NB) xenograft models. We demonstrated variable SSTR2 expression profiles in eight NB cell lines. From micro-PET imagin...

Emission variation in infrared (CdSeTe)/ZnS quantum dots conjugated to antibodies

Energy Technology Data Exchange (ETDEWEB)

Jaramillo Gómez, J.A. [UPIITA – Instituto Politécnico Nacional, México D. F. 07320, México (Mexico); Casas Espinola, J.L., E-mail: jlcasas@esfm.ipn.mx [ESFM – Instituto Politécnico Nacional, México D. F. 07738, México (Mexico); Douda, J. [UPIITA – Instituto Politécnico Nacional, México D. F. 07320, México (Mexico)

2014-11-15

The paper presents the photoluminescence (PL) and Raman scattering investigations of infrared CdSeTe/ZnS quantum dots (QDs) with emission at 800 nm (1.60 eV) in nonconjugated states and after the conjugation to the anti-papilloma virus antibodies (Ab). The Raman scattering study has shown that the CdSeTe core includes two layers with different material compositions such as: CdSe{sub 0.5}Te{sub 0.5} and CdSe{sub 0.7}Te{sub 0.3}. PL spectra of nonconjugated CdSeTe/ZnS QDs are characterized by two Gaussian shape PL bands related to exciton emission in the CdSeTe core and in intermediate layer at the core/shell interface. PL spectra of bioconjugated QDs have changed essentially: the main PL band related to the core emission shifts into high energy and become asymmetric. The energy diagram of double core/shell CdSeTe/ZnS QDs has been analyzed to explain the PL spectrum of nonconjugated QDs and its transformation at the bioconjugation to the papiloma virus antibodies. It is shown that the PL spectrum transformation in bioconjugated QDs can be a powerful technique for biology and medicine.

Dosimetry of 64Cu-DOTA-AE105, a PET tracer for uPAR imaging

International Nuclear Information System (INIS)

Persson, Morten; El Ali, Henrik H.; Binderup, Tina; Pfeifer, Andreas; Madsen, Jacob; Rasmussen, Palle; Kjaer, Andreas

2014-01-01

64 Cu-DOTA-AE105 is a novel positron emission tomography (PET) tracer specific to the human urokinase-type plasminogen activator receptor (uPAR). In preparation of using this tracer in humans, as a new promising method to distinguish between indolent and aggressive cancers, we have performed PET studies in mice to evaluate the in vivo biodistribution and estimate human dosimetry of 64 Cu-DOTA-AE105. Methods: Five mice received iv tail injection of 64 Cu-DOTA-AE105 and were PET/CT scanned 1, 4.5 and 22 h post injection. Volume-of-interest (VOI) were manually drawn on the following organs: heart, lung, liver, kidney, spleen, intestine, muscle, bone and bladder. The activity concentrations in the mentioned organs [%ID/g] were used for the dosimetry calculation. The %ID/g of each organ at 1, 4.5 and 22 h was scaled to human value based on a difference between organ and body weights. The scaled values were then exported to OLINDA software for computation of the human absorbed doses. The residence times as well as effective dose equivalent for male and female could be obtained for each organ. To validate this approach, of human projection using mouse data, five mice received iv tail injection of another 64 Cu-DOTA peptide-based tracer, 64 Cu-DOTA-TATE, and underwent same procedure as just described. The human dosimetry estimates were then compared with observed human dosimetry estimate recently found in a first-in-man study using 64 Cu-DOTA-TATE. Results: Human estimates of 64 Cu-DOTA-AE105 revealed the heart wall to receive the highest dose (0.0918 mSv/MBq) followed by the liver (0.0815 mSv/MBq), All other organs/tissue were estimated to receive doses in the range of 0.02–0.04 mSv/MBq. The mean effective whole-body dose of 64 Cu-DOTA-AE105 was estimated to be 0.0317 mSv/MBq. Relatively good correlation between human predicted and observed dosimetry estimates for 64 Cu-DOTA-TATE was found. Importantly, the effective whole body dose was predicted with very high

Dosimetry of 64Cu-DOTA-AE105, a PET tracer for uPAR imaging.

Science.gov (United States)

Persson, Morten; El Ali, Henrik H; Binderup, Tina; Pfeifer, Andreas; Madsen, Jacob; Rasmussen, Palle; Kjaer, Andreas

2014-03-01

(64)Cu-DOTA-AE105 is a novel positron emission tomography (PET) tracer specific to the human urokinase-type plasminogen activator receptor (uPAR). In preparation of using this tracer in humans, as a new promising method to distinguish between indolent and aggressive cancers, we have performed PET studies in mice to evaluate the in vivo biodistribution and estimate human dosimetry of (64)Cu-DOTA-AE105. Five mice received iv tail injection of (64)Cu-DOTA-AE105 and were PET/CT scanned 1, 4.5 and 22 h post injection. Volume-of-interest (VOI) were manually drawn on the following organs: heart, lung, liver, kidney, spleen, intestine, muscle, bone and bladder. The activity concentrations in the mentioned organs [%ID/g] were used for the dosimetry calculation. The %ID/g of each organ at 1, 4.5 and 22 h was scaled to human value based on a difference between organ and body weights. The scaled values were then exported to OLINDA software for computation of the human absorbed doses. The residence times as well as effective dose equivalent for male and female could be obtained for each organ. To validate this approach, of human projection using mouse data, five mice received iv tail injection of another (64)Cu-DOTA peptide-based tracer, (64)Cu-DOTA-TATE, and underwent same procedure as just described. The human dosimetry estimates were then compared with observed human dosimetry estimate recently found in a first-in-man study using (64)Cu-DOTA-TATE. Human estimates of (64)Cu-DOTA-AE105 revealed the heart wall to receive the highest dose (0.0918 mSv/MBq) followed by the liver (0.0815 mSv/MBq), All other organs/tissue were estimated to receive doses in the range of 0.02-0.04 mSv/MBq. The mean effective whole-body dose of (64)Cu-DOTA-AE105 was estimated to be 0.0317 mSv/MBq. Relatively good correlation between human predicted and observed dosimetry estimates for (64)Cu-DOTA-TATE was found. Importantly, the effective whole body dose was predicted with very high precision

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

Directory of Open Access Journals (Sweden)

Isabel Correa

2018-03-01

Full Text Available Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1 specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

Science.gov (United States)

Correa, Isabel; Ilieva, Kristina M; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F; Tutt, Andrew N J; Nestle, Frank O; Karagiannis, Panagiotis; Lacy, Katie E; Karagiannis, Sophia N

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

Science.gov (United States)

Correa, Isabel; Ilieva, Kristina M.; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F.; Tutt, Andrew N. J.; Nestle, Frank O.; Karagiannis, Panagiotis; Lacy, Katie E.; Karagiannis, Sophia N.

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires. PMID:29628923

Anti-L1CAM radioimmunotherapy is more effective with the radiolanthanide terbium-161 compared to lutetium-177 in an ovarian cancer model

International Nuclear Information System (INIS)

Gruenberg, Juergen; Lindenblatt, Dennis; Cohrs, Susan; Fischer, Eliane; Dorrer, Holger; Zhernosekov, Konstantin; Koester, Ulli; Tuerler, Andreas; Schibli, Roger

2014-01-01

The L1 cell adhesion molecule (L1CAM) is considered a valuable target for therapeutic intervention in different types of cancer. Recent studies have shown that anti-L1CAM radioimmunotherapy (RIT) with 67 Cu- and 177 Lu-labelled internalising monoclonal antibody (mAb) chCE7 was effective in the treatment of human ovarian cancer xenografts. In this study, we directly compared the therapeutic efficacy of anti-L1CAM RIT against human ovarian cancer under equitoxic conditions with the radiolanthanide 177 Lu and the potential alternative 161 Tb in an ovarian cancer therapy model. Tb was produced by neutron bombardment of enriched 160 Gd targets. 161 Tb and 177 Lu were used for radiolabelling of DOTA-conjugated antibodies. The in vivo behaviour of the radioimmunoconjugates (RICs) was assessed in IGROV1 tumour-bearing nude mice using biodistribution experiments and SPECT/CT imaging. After ascertaining the maximal tolerated doses (MTD) the therapeutic impact of 50 % MTD of 177 Lu- and 161 Tb-DOTA-chCE7 was evaluated in groups of ten mice by monitoring the tumour size of subcutaneous IGROV1 tumours. The average number of DOTA ligands per antibody was 2.5 and maximum specific activities of 600 MBq/mg were achieved under identical radiolabelling conditions. RICs were stable in human plasma for at least 48 h. 177 Lu- and 161 Tb-DOTA-chCE7 showed high tumour uptake (37.8-39.0 %IA/g, 144 h p.i.) with low levels in off-target organs. SPECT/CT images confirmed the biodistribution data. 161 Tb-labelled chCE7 revealed a higher radiotoxicity in nude mice (MTD: 10 MBq) than the 177 Lu-labelled counterpart (MTD: 12 MBq). In a comparative therapy study with equitoxic doses, tumour growth inhibition was better by 82.6 % for the 161 Tb-DOTA-chCE7 than the 177 Lu-DOTA-chCE7 RIT. Our study is the first to show that anti-L1CAM 161 Tb RIT is more effective compared to 177 Lu RIT in ovarian cancer xenografts. These results suggest that 161 Tb is a promising candidate for future clinical

CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

Science.gov (United States)

Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor

2011-07-01

Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ~ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ~ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ~ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ~ 12 nm retained bright fluorescence over an extended duration of ~ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ~ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ~ 8.2% in human peripheral blood cells (PBMCs) which are CD33low. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

International Nuclear Information System (INIS)

Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor

2011-01-01

Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ∼ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ∼ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ∼ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ∼ 12 nm retained bright fluorescence over an extended duration of ∼ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ∼ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ∼ 8.2% in human peripheral blood cells (PBMCs) which are CD33 low . The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

Energy Technology Data Exchange (ETDEWEB)

Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor, E-mail: manzoork@aims.amrita.edu, E-mail: ullasmony@aims.amrita.edu [Amrita Centre for Nanoscience and Molecular Medicine, Amrita Institute of Medical Science, Cochin 682 041 (India)

2011-07-15

Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with {approx} 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of {approx} 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in {approx} 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of {approx} 12 nm retained bright fluorescence over an extended duration of {approx} a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of {approx} 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of {approx} 8.2% in human peripheral blood cells (PBMCs) which are CD33{sup low}. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

Thermodynamic and kinetic study of scandium(III) complexes of DTPA and DOTA: a step toward scandium radiopharmaceuticals.

Science.gov (United States)

Pniok, Miroslav; Kubíček, Vojtěch; Havlíčková, Jana; Kotek, Jan; Sabatie-Gogová, Andrea; Plutnar, Jan; Huclier-Markai, Sandrine; Hermann, Petr

2014-06-23

Diethylenetriamine-N,N,N',N'',N''-pentaacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) scandium(III) complexes were investigated in the solution and solid state. Three (45)Sc NMR spectroscopic references suitable for aqueous solutions were suggested: 0.1 M Sc(ClO4)3 in 1 M aq. HClO4 (δSc =0.0 ppm), 0.1 M ScCl3 in 1 M aq. HCl (δSc =1.75 ppm) and 0.01 M [Sc(ox)4](5-) (ox(2-) = oxalato) in 1 M aq. K2C2O4 (δSc =8.31 ppm). In solution, [Sc(dtpa)](2-) complex (δSc = 83 ppm, Δν = 770 Hz) has a rather symmetric ligand field unlike highly unsymmetrical donor atom arrangement in [Sc(dota)](-) anion (δSc = 100 ppm, Δν = 4300 Hz). The solid-state structure of K8[Sc2(ox)7]⋅13 H2O contains two [Sc(ox)3](3-) units bridged by twice "side-on" coordinated oxalate anion with Sc(3+) ion in a dodecahedral O8 arrangement. Structures of [Sc(dtpa)](2-) and [Sc(dota)](-) in [(Hguanidine)]2[Sc(dtpa)]⋅3 H2O and K[Sc(dota)][H6 dota]Cl2⋅4 H2O, respectively, are analogous to those of trivalent lanthanide complexes with the same ligands. The [Sc(dota)](-) unit exhibits twisted square-antiprismatic arrangement without an axial ligand (TSA' isomer) and [Sc(dota)](-) and (H6 dota)(2+) units are bridged by a K(+) cation. A surprisingly high value of the last DOTA dissociation constant (pKa =12.9) was determined by potentiometry and confirmed by using NMR spectroscopy. Stability constants of scandium(III) complexes (log KScL 27.43 and 30.79 for DTPA and DOTA, respectively) were determined from potentiometric and (45)Sc NMR spectroscopic data. Both complexes are fully formed even below pH 2. Complexation of DOTA with the Sc(3+) ion is much faster than with trivalent lanthanides. Proton-assisted decomplexation of the [Sc(dota)](-) complex (τ1/2 =45 h; 1 M aq. HCl, 25 °C) is much slower than that for [Ln(dota)](-) complexes. Therefore, DOTA and its derivatives seem to be very suitable ligands for scandium

Dosimetry of 64Cu-DOTA-AE105, a PET tracer for uPAR imaging

DEFF Research Database (Denmark)

Persson, Morten; El Ali, Henrik H.; Binderup, Tina

2014-01-01

64Cu-DOTA-AE105 is a novel positron emission tomography (PET) tracer specific to the human urokinase-type plasminogen activator receptor (uPAR). In preparation of using this tracer in humans, as a new promising method to distinguish between indolent and aggressive cancers, we have performed PET...... studies in mice to evaluate the in vivo biodistribution and estimate human dosimetry of 64Cu-DOTA-AE105. MethodsFive mice received iv tail injection of 64Cu-DOTA-AE105 and were PET/CT scanned 1, 4.5 and 22h post injection. Volume-of-interest (VOI) were manually drawn on the following organs: heart, lung......Favorable dosimetry estimates together with previously reported uPAR PET data fully support human testing of 64Cu-DOTA-AE105....

Oriented conjugates of monoclonal and single-domain antibodies with quantum dots for flow cytometry and immunohistochemistry diagnostic applications

Science.gov (United States)

Sukhanova, Alyona; Even-Desrumeaux, Klervi; Millot, Jean-Marc; Chames, Patrick; Baty, Daniel; Artemyev, Mikhail; Oleinikov, Vladimir; Cohen, Jacques H. M.; Nabiev, Igor

2012-03-01

Ideal diagnostic nanoprobes should not exceed 15 nm in size and should contain high-affinity homogeneously oriented capture molecules on their surface. An advanced procedure for antibody (Ab) reduction was used to cleave each Ab into two functional half-Abs, 75-kDa heavy-light chain fragments, each containing an intact antigen-binding site. Affinity purification of half-Abs followed by their linkage to quantum dots (QDs) yielded oriented QD-Ab conjugates whose functionality was considerably improved compared to those obtained using the standard protocols. Ultrasmall diagnostic nanoprobes were engineered through oriented conjugation of QDs with 13-kDa single-domain Abs (sdAbs) derived from llama IgG. sdAbs were tagged with QDs via an additional cysteine residue specifically integrated into the C-terminal region of sdAb using genetic engineering. This approach made it possible to obtain sdAb-QD nanoprobes <12 nm in diameter comprising four copies of sdAbs linked to the same QD in an oriented manner. sdAb-QD conjugates against carcinoembryonic antigen (CEA) and HER2 exhibited an extremely high specificity in flow cytometry; the quality of immunohistochemical labeling of biopsy samples was found to be superior to that of labeling according to the current "gold standard" protocols of anatomo-pathological practice. The nano-bioengineering approaches developed can be extended to oriented conjugation of Abs and sdAbs with different semiconductor, noble metal, or magnetic nanoparticles.

Development of novel radiogallium-labeled bone imaging agents using oligo-aspartic acid peptides as carriers.

Directory of Open Access Journals (Sweden)

Kazuma Ogawa

Full Text Available (68Ga (T 1/2 = 68 min, a generator-produced nuclide has great potential as a radionuclide for clinical positron emission tomography (PET. Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting (68Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Aspn (n = 2, 5, 8, 11, or 14 with easy-to-handle (67Ga, with the previously described (67Ga-DOTA complex conjugated bisphosphonate, (67Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Aspn by a Fmoc-based solid-phase method, complexes were formed with (67Ga, resulting in (67Ga-DOTA-(Aspn with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of (67Ga-DOTA-(Aspn increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, (67Ga-DOTA-(Asp8, (67Ga-DOTA-(Asp11, and (67Ga-DOTA-(Asp14 showed high accumulation in bone (10.5 ± 1.5, 15.1 ± 2.6, and 12.8 ± 1.7% ID/g, respectively but were barely observed in other tissues at 60 min after injection. Although bone accumulation of (67Ga-DOTA-(Aspn was lower than that of (67Ga-DOTA-Bn-SCN-HBP, blood clearance of (67Ga-DOTA-(Aspn was more rapid. Accordingly, the bone/blood ratios of (67Ga-DOTA-(Asp11 and (67Ga-DOTA-(Asp14 were comparable with those of (67Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of (68Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases.

Boronated monoclonal antibody conjugates for neutron capture therapy

International Nuclear Information System (INIS)

Borg, D.C.; Elmore, J.J. Jr.; Ferrone, S.

1986-01-01

This paper describes the effectiveness of 10 B-labeled monoclonal antibodies against Colo-38 human melanoma in vitro. The authors obtained high boron to antibody ratios while maintaining antibody activity by using dextran intermediate carriers to link 10 B to the antibody. They developed a double cell quasi-competitive binding bioassay to minimize the effects of nonspecific binding of boronated complexes to cells. 1 fig., 2 tabs

Neodymium-140 DOTA-LM3

DEFF Research Database (Denmark)

Severin, Gregory; Kristensen, Lotte K.; Nielsen, Carsten H.

2017-01-01

analogue, DOTA-LM3 (1,4,7,10- tetraazacyclododecane, 1,4,7- tri acetic acid, 10- acetamide N - p-Cl-Phecyclo(D-Cys-Tyr-d-4-amino-Phe(carbamoyl)-Lys-Thr-Cys)D-Tyr-NH2) and injected into H727 xenograft bearing mice. Comparative pre- and post-mortem PET imaging at 16 h postinjection was used to quantify......140Nd (t1/2 = 3.4 days), owing to its short-lived positron emitting daughter 140Pr (t1/2 = 3.4 min), has promise as an in vivo generator for positron emission tomography (PET). However, the electron capture decay of 140Nd is chemically disruptive to macrocycle-based radiolabeling, meaning...... the in vivo redistribution of 140Pr following 140Nd decay. The somatostatin receptor-positive pancreas exhibited the highest tissue accumulation of 140Nd-DOTA-LM3 (13% ID/g at 16 h) coupled with the largest observed redistribution rate, where 56 ± 7% (n = 4, mean ± SD) of the in situ produced 140Pr washed out...

{sup 68}Ga-DOTA-RGD peptide: biodistribution and binding into atherosclerotic plaques in mice

Energy Technology Data Exchange (ETDEWEB)

Haukkala, Johanna; Laitinen, Iina; Luoto, Pauliina; Knuuti, Juhani [University of Turku, Turku PET Centre, Turku (Finland); Iveson, Peter; Wilson, Ian [Medical Diagnostics, GE Healthcare Biosciences, London (United Kingdom); Karlsen, Hege; Cuthbertson, Alan [GE Healthcare MDx Research, Oslo (Norway); Laine, Jukka [Turku University Hospital, Department of Pathology, Turku (Finland); Leppaenen, Pia; Ylae-Herttula, Seppo [University of Kuopio, A.I. Virtanen Institute, Kuopio (Finland); Roivainen, Anne [University of Turku, Turku PET Centre, Turku (Finland); University of Turku, Turku Centre for Disease Modelling, Turku (Finland)

2009-12-15

Increased expression of {alpha}v{beta}3/{alpha}v{beta}5 integrin is involved in angiogenesis and the inflammatory process in atherosclerotic plaques. The novel {sup 68}Ga-DOTA-RGD peptide binds with high affinity to {alpha}v{beta}3/{alpha}v{beta}5 integrin. The aim of this study was to investigate the uptake of the {sup 68}Ga-DOTA-RGD peptide in atherosclerotic plaques. Uptake of intravenously administered {sup 68}Ga-DOTA-RGD peptide was studied ex vivo in excised tissue samples and aortic sections of LDLR{sup -/-}ApoB{sup 100/100} atherosclerotic mice. The uptake of the tracer in aortic cryosections was examined by using digital autoradiography. Subsequently, the autoradiographs were combined with histological and immunohistological analysis of the sections. DOTA-RGD peptide was successfully labelled with the generator-produced {sup 68}Ga. The tracer had reasonably good specific radioactivity (8.7 {+-} 1.1 GBq/{mu}mol) and was quite stable in vivo. According to ex vivo biodistribution results, {sup 68}Ga-DOTA-RGD was cleared rapidly from the blood circulation and excreted through the kidneys to the urine with high radioactivity in the intestine, lungs, spleen and liver. Autoradiography results showed significantly higher uptake of {sup 68}Ga-DOTA-RGD peptide in the atherosclerotic plaques compared to healthy vessel wall (mean ratio {+-} SD 1.4 {+-} 0.1, p = 0.0004). We observed that {sup 68}Ga-DOTA-RGD is accumulated into the plaques of atherosclerotic mice. However, this data only shows the feasibility of the approach, while the clinical significance still remains to be proven. Further studies are warranted to assess the uptake of this tracer into human atherosclerotic plaques. (orig.)

Radiolabeling of DOTA-like conjugated peptides with generator-produced 68Ga and using NaCl-based cationic elution method

Science.gov (United States)

Mueller, Dirk; Breeman, Wouter A P; Klette, Ingo; Gottschaldt, Michael; Odparlik, Andreas; Baehre, Manfred; Tworowska, Izabela; Schultz, Michael K

2017-01-01

Gallium-68 (68Ga) is a generator-produced radionuclide with a short half-life (t½ = 68 min) that is particularly well suited for molecular imaging by positron emission tomography (PET). Methods have been developed to synthesize 68Ga-labeled imaging agents possessing certain drawbacks, such as longer synthesis time because of a required final purification step, the use of organic solvents or concentrated hydrochloric acid (HCl). In our manuscript, we provide a detailed protocol for the use of an advantageous sodium chloride (NaCl)-based method for radiolabeling of chelator-modified peptides for molecular imaging. By working in a lead-shielded hot-cell system, 68Ga3+ of the generator eluate is trapped on a cation exchanger cartridge (100 mg, ∼8 mm long and 5 mm diameter) and then eluted with acidified 5 M NaCl solution directly into a sodium acetate-buffered solution containing a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or DOTA-like chelator-modified peptide. The main advantages of this procedure are the high efficiency and the absence of organic solvents. It can be applied to a variety of peptides, which are stable in 1 M NaCl solution at a pH value of 3–4 during reaction. After labeling, neutralization, sterile filtration and quality control (instant thin-layer chromatography (iTLC), HPLC and pH), the radiopharmaceutical can be directly administered to patients, without determination of organic solvents, which reduces the overall synthesis-to-release time. This procedure has been adapted easily to automated synthesis modules, which leads to a rapid preparation of 68Ga radiopharmaceuticals (12–16 min). PMID:27172166

In vivo and in vitro evaluation of dota-lanreotide radiolabelled with gallium-67; Avaliacao in vivo e in vitro do dota-lanreotideo radiomarcado com galio-67

Energy Technology Data Exchange (ETDEWEB)

Aldegheri, Eliane Bernardes

2005-07-01

One of the refinements of modern Nuclear Medicine is the capacity of providing dynamic and kinetics images of the administered radiopharmaceutical, reproducing its transport mechanism, action sites, receptor binding and excretion route. With the continues technological advances new radiopharmaceuticals have been developed in order to express higher specificity and with higher characters of affinity between receptor/complex. One radiopharmaceutical is formed by a reagent or bio molecule that has in its structure a radioisotope, that has the objectives of carrying it to the organs of affinity or to benign or malign tumoral process. Somatostatin inhibits the growing and proliferation of several tumoral cells. Somatostatin analogs bind to somatostatic receptors that are expressed in different kind of neoplasia DOTA-LANREOTIDE (DOTALAN) is an octapeptide analog to somatostatin. The interest of labeling the bio conjugate with gallium-67 in Nuclear Medicine comes from its physical, chemical and biological properties. Besides its gamma radiation, useful in the diagnosis of inflammation and infection foci, {sup 67}Ga emits Auger electrons (0.1 - 8 keV) and conversion electrons (80 - 90 keV), making it attractive to internal radiotherapy if the vectors used to lead the radionuclide to the tumoral cell are internalized. The objective of this work was to develop a methodology of labelling DOTA-LANREOTIDE with {sup 67}Ga, optimizing the labelling variables and its quality control. The novelty aspect was the comparison between labeling with national and imported {sup 67}Ga, in its original and purified forms. The purification of {sup 67}Ga was essential to reach yields higher than 90%. The radiolabelled bio conjugate peptides must be free of metallic contaminants that could compete in the labeling process. The following procedure was established after studying the labeling parameters: mass of peptide of 10 {mu}g (6nmol), pH5, final reaction volume of 170 {mu}L of the labelled

68Ga-DOTA0-Tyr3-octreotide positron emission tomography in nasopharyngeal carcinoma

International Nuclear Information System (INIS)

Schartinger, Volker H.; Dudas, Jozsef; Url, Christoph; Riechelmann, Herbert; Reinold, Susanne; Virgolini, Irene J.; Kroiss, Alexander; Uprimny, Christian

2015-01-01

PET/CT with 68 Ga-labelled [DOTA 0 ,Tyr 3 ]-octreotide ( 68 Ga-DOTA-TOC PET/CT) is a routinely used imaging modality for neuroendocrine tumours expressing somatostatin receptors (SSTR). Recent studies have shown SSTR expression in head and neck squamous cell carcinoma, albeit lower than in highly differentiated neuroendocrine tumours. We sought to determine whether nasopharyngeal carcinoma (NPC) positive for Epstein-Barr virus (EBV), a rare subtype of head and neck cancer, shows increased 68 Ga-DOTA-TOC uptake indicating expression of SSTR. Five patients with untreated, histologically proven EBV-positive NPC were referred for 68 Ga-DOTA-TOC PET/CT. Tracer uptake in tumour lesions was assessed visually and semiquantitatively measuring maximum standardized uptake values (SUVmax) and tumour to background ratios. Increased tumour-specific uptake was detected in all five patients with a median SUVmax of 10.6 (range 3.6 - 17.1) in the primary tumour and 13.2 (range 6.1 - 14.5) in cervical lymph node metastases. 68 Ga-DOTA-TOC PET/CT demonstrated tracer uptake in EBV-positive NPC comparable to that in highly differentiated neuroendocrine tumours. This observation is consistent with increased SSTR expression in EBV-positive NPC and may open new diagnostic and therapeutic windows in NPC. (orig.)

(68)Ga-DOTA (0)-Tyr (3)-octreotide positron emission tomography in nasopharyngeal carcinoma.

Science.gov (United States)

Schartinger, Volker H; Dudás, József; Url, Christoph; Reinold, Susanne; Virgolini, Irene J; Kroiss, Alexander; Riechelmann, Herbert; Uprimny, Christian

2015-01-01

PET/CT with (68)Ga-labelled [DOTA(0),Tyr(3)]-octreotide ((68)Ga-DOTA-TOC PET/CT) is a routinely used imaging modality for neuroendocrine tumours expressing somatostatin receptors (SSTR). Recent studies have shown SSTR expression in head and neck squamous cell carcinoma, albeit lower than in highly differentiated neuroendocrine tumours. We sought to determine whether nasopharyngeal carcinoma (NPC) positive for Epstein-Barr virus (EBV), a rare subtype of head and neck cancer, shows increased (68)Ga-DOTA-TOC uptake indicating expression of SSTR. Five patients with untreated, histologically proven EBV-positive NPC were referred for (68)Ga-DOTA-TOC PET/CT. Tracer uptake in tumour lesions was assessed visually and semiquantitatively measuring maximum standardized uptake values (SUVmax) and tumour to background ratios. Increased tumour-specific uptake was detected in all five patients with a median SUVmax of 10.6 (range 3.6 - 17.1) in the primary tumour and 13.2 (range 6.1 - 14.5) in cervical lymph node metastases. (68)Ga-DOTA-TOC PET/CT demonstrated tracer uptake in EBV-positive NPC comparable to that in highly differentiated neuroendocrine tumours. This observation is consistent with increased SSTR expression in EBV-positive NPC and may open new diagnostic and therapeutic windows in NPC.

Co-association of methotrexate and SPIONs into anti-CD64 antibody-conjugated PLGA nanoparticles for theranostic application.

Science.gov (United States)

Moura, Catarina Costa; Segundo, Marcela A; Neves, José das; Reis, Salette; Sarmento, Bruno

2014-01-01

Rheumatoid arthritis (RA) is an autoimmune disease with severe consequences for the quality of life of sufferers. Regrettably, the inflammatory process involved remains unclear, and finding successful therapies as well as new means for its early diagnosis have proved to be daunting tasks. As macrophages are strongly associated with RA inflammation, effective diagnosis and therapy may encompass the ability to target these cells. In this work, a new approach for targeted therapy and imaging of RA was developed based on the use of multifunctional polymeric nanoparticles. Poly(lactic-co-glycolic acid) nanoparticles were prepared using a single emulsion-evaporation method and comprisaed the co-association of superparamagnetic iron oxide nanoparticles (SPIONs) and methotrexate. The nanoparticles were further functionalized with an antibody against the macrophage-specific receptor, CD64, which is overexpressed at sites of RA. The devised nanoparticles were characterized for mean particle size, polydispersity index, zeta potential, and morphology, as well as the association of SPIONs, methotrexate, and the anti-CD64 antibody. Lastly, the cytotoxicity of the developed nanoparticles was assessed in RAW 264.7 cells using standard MTT and LDH assays. The nanoparticles had a mean diameter in the range of 130-200 nm and zeta potential values ranging from -32 mV to -16 mV. Association with either methotrexate or SPIONs did not significantly affect the properties of the nanoparticles. Conjugation with the anti-CD64 antibody, in turn, caused a slight increase in size and surface charge. Transmission electron microscopy confirmed the association of SPIONs within the poly(lactic-co-glycolic acid) matrix. Both anti-CD64 and methotrexate association were confirmed by Fourier transform infrared spectroscopy, and quantified yielding values as high as 36% and 79%, respectively. In vitro toxicity studies confirmed the methotrexate-loaded nanosystem to be more effective than the free drug

The effect of varied pH on the luminescence characteristics of antibody-mercaptoacetic acid conjugated ZnS nanowires

Science.gov (United States)

Chaudhry, Madeeha; Rehman, Malik Abdul; Gul, Asghari; Qamar, Raheel; Bhatti, Arshad Saleem

2017-11-01

We demonstrate here that the effect of varied pH of the media on the photoluminescence (PL) properties of mercaptoacetic acid (MAA) and digoxin antibody (Ab) conjugated zinc sulphide (ZnS) nanowires. The charge-transfer kinetics from MAA to ZnS and vice versa showed a profound effect on the luminescence of ZnS defect states. The PL intensity of the ZnS defect states showed strong dependence on the value of pH with respect to the pKa of MAA. The carboxyl and thiol group of MAA in the protonated (pH pKa) states resulted in the quenched PL intensity. While for pH ∼ pKa, the PL intensity was regained as there was equal probability of both protonated and deprotonated carboxyl and thiol groups. These findings indicated that pH of the environment is a key parameter for the use of MAA-Ab conjugated ZnS nanowires as an optical biomarker.