Sakahara, Harumi; Endo, Keigo; Nakashima, Tetsuo; Koizumi, Mitsuru; Ohta, Hitoya; Torizuka, Kanji; Okada, Kenichiro; Yoshida, Osamu; Nishi, Shinzo.
Using monoclonal antibodies against human α-fetoprotein (AFP), radioiodinated F(ab') 2 fragments were compared with whole IgG as a radiotracer for radioimmunoimaging of cancer. F(ab') 2 fragments were obtained by pepsin digestion of whole IgG (IgGl). IgG and F(ab') 2 were labeled with 125 I or 131 I by the chloramine-T method with almost full retention of antibody activity. F(ab') 2 fragments were cleared more rapidly from the circulation in normal mice with a half life of 6.3 hours than whole IgG with a half life of 5.5 days. Radioactivity of F(ab') 2 in various organs also decreased faster than IgG. In nude mice transplanted with AFP-producing human testicular tumor, F(ab') 2 fragments demonstrated superior scintigrams to whole IgG at 2 days after the injection, because of the fast disappearance of background radioactivity. Although absolute accumulation of 131 I labeled F(ab') 2 in the tumor was less than that of 131 I labeled IgG, tumor to other organ ratios were much higher with F(ab') 2 than those of IgG. The tumor to blood ratio of 131 I labeled F(ab') 2 was 1.04 at day 2, whereas tumor to blood ratio of 131 I labeled IgG was 0.55 at day 2 and 0.92 at day 4, respectively. These results indicated that for the radiolabeling of monoclonal antibodies, F(ab') 2 fragments would be superior to whole IgG in the radioimmunoimaging of cancer. (author)
Syvänen, Stina; Edén, Desireé; Sehlin, Dag
Antibodies and fragments thereof are, because of high selectivity for their targets, considered as potential therapeutics and biomarkers for several neurological disorders. However, due to their large molecular size, antibodies/fragments do not easily penetrate into the brain. The aim of the present study was to improve the brain distribution via adsorptive-mediated transcytosis of an amyloid-beta (A beta) protofibril selective F(ab')2 fragment (F(ab')2-h158). F(ab')2-h158 was cationized to d...
Wahl, R.L.; Parker, C.W.; Philpott, G.W.
Monoclonal anti-tumor antibodies have great promise for radioimmunodetection and localization of tumors. Fab and F(ab')2 fragments, which lack the Fc fragment of antibody (Ab), are cleared more rapidly from the circulation and may have less nonspecific tissue binding than intact Ab. In radioimaging studies using a murine monoclonal antibody to carcinoembryonic antigen in a human colon carcinoma xenografted into hamsters, F(ab')2 fragments were shown superior to Fab fragments and intact antibody for scintiscanning. In double-label experiments with anti-CEA antibody and control monoclonal IgG, F(ab')2 fragments were found to give better and more rapid specific tumor localization than intact antibody or Fab fragments. F(ab')2 fragments offer significant promise for tumor imaging and possibly therapy
Valedkarimi, Zahra; Nasiri, Hadi; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Esparvarinha, Mojghan; Majidi, Jafar
In present study an optimized protocol for the separation of antibodies into antigen-binding fragments F(ab')2 using pepsin digestion was investigated. The production of these fragments is a consequential step in the development of medical research, treatment and diagnosis. For production of polyclonal antibody rabbit received antigen in four steps. The rabbit serum at 1/128000 dilution showed high absorbance in reaction with human IgG at the designed ELISA method. Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method. Purity was assessed by SDS-PAGE method. In non-reduced condition only one band was seen in about 150 kDa MW position and in reduced form, two bands were seen in 50 and 25 kDa MW positions. Rabbit IgG was digested by pepsin enzyme. The antibody fragments solution was applied to Gel filtration column to isolate the F(ab')2. Non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment resulted in one band in 100 kDa corresponds to F(ab')2 fragment and a band in 150 kDa MW position corresponds to undigested IgG antibodies. The activities of FITC conjugated F(ab')2 fragment and commercial ones were compared using flowcytometry method. The activity results implied that the FITC conjugated- anti human F(ab')2 fragment worked as efficiently as the commercial one.
Bush, Sean P; Ruha, Anne-Michelle; Seifert, Steven A; Morgan, David L; Lewis, Brandon J; Arnold, Thomas C; Clark, Richard F; Meggs, William J; Toschlog, Eric A; Borron, Stephen W; Figge, Gary R; Sollee, Dawn R; Shirazi, Farshad M; Wolk, Robert; de Chazal, Ives; Quan, Dan; García-Ubbelohde, Walter; Alagón, Alejandro; Gerkin, Richard D; Boyer, Leslie V
Crotalidae Polyvalent Immune Fab (Ovine) has been the only antivenom commercially available in the US since 2007 for treatment of Crotalinae envenomation. Late coagulopathy can occur or recur after clearance of Fab antivenom, often after hospital discharge, lasting in some cases more than 2 weeks. There have been serious, even fatal, bleeding complications associated with recurrence phenomena. Frequent follow-up is required, and additional intervention or hospitalization is often necessary. F(ab')2 immunoglobulin derivatives have longer plasma half life than do Fab. We hypothesized that F(ab')2 antivenom would be superior to Fab in the prevention of late coagulopathy following treatment of patients with Crotalinae envenomation. We conducted a prospective, double-blind, randomized clinical trial, comparing late coagulopathy in snakebitten patients treated with F(ab')2 with maintenance doses [F(ab')2/F(ab')2], or F(ab')2 with placebo maintenance doses [F(ab')2/placebo], versus Fab with maintenance doses [Fab/Fab]. The primary efficacy endpoint was coagulopathy (platelet count Fab/Fab cohort experienced late coagulopathy versus 4/39 (10.3%, p < 0.05) in the F(ab')2/F(ab')2 cohort and 2/38 (5.3%, p < 0.05) in the F(ab')2/placebo cohort. The lowest heterologous protein exposure was with F(ab')2/placebo. No serious adverse events were related to study drug. In each study arm, one patient experienced an acute serum reaction and one experienced serum sickness. In this study, management of coagulopathic Crotalinae envenomation with longer-half-life F(ab')2 antivenom, with or without maintenance dosing, reduced the risk of subacute coagulopathy and bleeding following treatment of envenomation.
Monoclonal antibody against lung cancer was digested into F(ab') 2 fragment by pepsin and papain digestion. The yields of pure F(ab') 2 were 32.3 ± 5.5% and 52.3 ± 12.0% respectively. The immunoreactivity of F(ab') 2 was based on the ELISA assay and the cell binding studies was retained well. In the localization experiments, radioiodinated F(ab') 2 was injected intraperitoneally into the nude mice bearing human xenografts of lung cancer. The highest radioactivity in tumors, 1.37% of injected dose per gram, was reached on the first day after injection; its T/NT ratios were higher than those of the intact IgG in all tissues except kidney. The localization index (LI) in tumors was 4.95, while the average LI value of normal tissues was 1.25. After the injection of 131 I-F(ab') 2 intraperitoneally into lung tumor-bearing nude mice, photo scintigraphy was performed at intervals of 12 hrs. The xenografts were visualized distinctly during 36 ∼ 48 hr, and the nonspecific background was very low at 48 hr
Squaiella-Baptistão, Carla Cristina; Marcelino, José Roberto; Ribeiro da Cunha, Luiz Eduardo; Gutiérrez, José María; Tambourgi, Denise V.
2094-01 Embargo por política editorial Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several b...
Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Kazemi, Tohid; Esparvarinha, Mojghan; Majidi, Jafar
Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')2 can be used for animal’s immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme a...
Janssen, R. A. J.; Kroesen, B. J.; Mesander, G.; Sleijfer, D. T.; The, T. Hauw; Mulder, N. H.; de Leij, L
We report the immunomodulatory effects of an intravenous treatment with F(ab')(2) fragments of the bispecific monoclonal antibody BIS-1 during subcutaneous recombinant interleukin 2 (rIL-2) therapy of renal cell cancer (RCC) patients. BIS-1 is directed against both the CD3 antigen on T cells and the
The ability of horse antivenoms, consisting of immunoglobulin G (IgG) and its fragments F(ab')2 and Fab were comparatively studied in mice to neutralize several effects of Cerastes cerastes venom. The three antivenoms were produced from the same batch of hyperimmune horse plasma. Neutralization was only partial ...
Fleuren, Emmy D. G.; Versleijen-Jonkers, Yvonne M. H.; Heskamp, Sandra; Roeffen, Melissa H. S.; Bouwman, Wilbert H.; Molkenboer-Kuenen, Janneke D. M.; van Laarhoven, Hanneke W. M.; Oyen, Wim J. G.; Boerman, Otto C.; van der Graaf, Winette T. A.
To investigate whether F(ab')2-fragments of the monoclonal Insulin-like Growth Factor-1 Receptor (IGF-1R) antibody R1507 (F(ab')2-R1507) can successfully target IGF-1R in Ewing sarcomas (ES). BALB/c nude mice were subcutaneously implanted with IGF-1R-expressing human ES xenografts (EW-5 and EW-8)
Li Ling; Xu Huiyun; Mi Li; Bian Huijie; Qin Jun; Xiong Hua; Feng Qiang; Wen Ning; Tian Rong; Xu Liqing; Shen Xiaomei; Tang Hao; Chen Zhinan
Purpose: Therapeutic efficacy, suitable dose, and administration times of 131 I-CAb 1 F(ab') 2 , a new monoclonal antibody therapeutics specifically directed against a cell surface-associated glycoprotein of colon cancer, were investigated in this article. Methods and Materials: In human colon cancer xenografts, 131 I-CAb 1 F(ab') 2 at the dose of 125 μCi, 375 μCi, and 1125 μCi were administrated intraperitoneally on Days 6 and 18 after implantation of HR8348 cells with CAb 1 high reactivity. Survival time and tumor growth inhibition rate were used to evaluate the efficacy and safety of 131 I-CAb 1 F(ab') 2 in treatment of colon cancer xenografts. Results: Treatment of 125, 375, and 1125 μCi 131 I-CAb1 F(ab') 2 did not significantly decrease the mean survival time of nude mice when compared with nontreated groups (p = 0.276, 0.865, 0.582, respectively). Moreover, the mean survival times of nude mice receiving 375 μCi and 1125 μCi 131 I-CAb1 F(ab') 2 were significantly longer than that of 5-FU-treated groups (p 0.018 and 0.042). Tumor growth inhibition rates of the first therapy were 35.67% and 41.37%, with corresponding 131 I-labeled antibody dosage of 375 μCi and 1125 μCi. After single attack dosage, second reinforcement therapy may rise efficacy significantly. Tumor growth inhibition rates of 125 μCi, 375 μCi, and 1125 μCi 131 I-labeled antibody on Day 20 posttherapy were 42.65%, 56.56%, and 84.41%, respectively. Histopathology examination revealed that tissue necrosis of various degrees was found in 131 I-CAb1 F(ab') 2 -treated groups. Conclusion: 131 I-CAb 1 F(ab') 2 is safe and effective for colon cancer. It may be a novel and potentially adjuvant therapeutics for colon cancer
131 I-labeled monoclonal anti-CEA antibody and its F(ab') 2 fragments were injected into nude mice bearing human colon carcinoma xenografts for tumor localization and radioimmunoimaging studies. Transplanted tumors were visualized in 12 hours after injection of the labeled anti-CEA or its F(ab') 2 by gamma camera. Biodistribution data indicated that F(ab') 2 fragments were cleared more rapidly from blood (T 1/2 = 13.3 h for F(ab') 2 , T 1/2 = 21.1 h for intact antibody) over 6-24 h and had higher tumor blood ratios. The intact antibody was concentrated in the tumor better than F(ab') 2 . In double-label experiments, a nonspecific localization of the control ( 125 I-labeled anti-HCG) in the tumor was also observed
Doherty, P.; Schwinger, R.; King, M.; Gionet, M.
The purpose of this study was to obtain quantitative biodistribution data in patients injected with the indium-111 labeled F(ab') 2 fragments of mouse monoclonal antibody. From this data dosimetric calculations were made for the individual organs. The authors also evaluated the quantitative properties of SPECT in this application and compared it with the more conventional two view planar technique in both phantom and patient studies. For one antibody (19-9) the mean dose in rads/mCi for the organs of highest accumulation, namely, the liver and kidneys was 3.2 and 2.6 respectively. Preliminary data from another antibody (OC 125) showed much higher blood levels and a significantly lower liver dose of 2.3 indicating that antibody type is another significant determinant in dosimetry. The SPECT approach particularly in the presence of background activity, was more accurate in the phantom studies and resulted in larger estimated doses in the patient studies. Also, SPECT has the added advantage of providing an index of organ volume, which has to be balanced with the fact the planar is more rapid, and does not require special hardware. 24 references, 5 figures, 1 table
Cederkrantz, Elin; Andersson, Håkan; Bernhardt, Peter
dose associated with i.p. administration of (211)At-MX35 F(ab')2. METHODS AND MATERIALS: Patients in clinical remission after salvage chemotherapy for peritoneal recurrence of ovarian cancer underwent i.p. infusion of (211)At-MX35 F(ab')2. Potassium perchlorate was given to block unwanted accumulation...... 100 MBq/L, organ equivalent doses were less than 10% of the estimated tolerance dose. CONCLUSION: Intraperitoneal (211)At-MX35 F(ab')2 treatment is potentially a well-tolerated therapy for locally confined microscopic ovarian cancer. Absorbed doses to normal organs are low, but because the effective...
Rosebrough, S.F.; Grossman, Z.D.; McAfee, J.G.
We have previously reported successful imaging of fresh (2-4 hr old) and aged (1-5 days old) canine thrombi with 131 I-labeled intact monoclonal antibody (MAb) specific for fibrin. We now report thrombus imaging with 131 I-labeled F(ab')2 and Fab and 111 In-labeled intact MAb, F(ab')2, and Fab. Indium-111-labeled F(ab')2 proved to be the best imaging agent due to less nonspecific binding in the liver than whole IgG. Image quality was improved by the higher administered dose permissible with 111 In and its better physical characteristics for imaging, compared to 131 I. Immunofluorescence of fresh human histologic sections showed intact MAb and F(ab')2 binding to thrombi, pulmonary emboli, and atherosclerotic plaques, strengthening the feasibility of clinical thrombus imaging
Yemul, Shrishailam; Leon, J.A.; Pozniakoff, Ted; Esser, P.D.; Estabrook, Alison; Met-Path Inc., Teterboro, NJ
Radioimmunoimaging characteristics of a new monoclonal antibody EBA-1 and its F(ab') 2 fragments utilizing nu/nu mice bearing human breast carcinoma xenografts are described. 111 In-DPTA conjugates of EBA-1 localized with tumor/blood ratios of 0.99 ± 0.10 (P 2 radioconjugates at 48 h. These results suggest that EBA-1 and its F(ab') 2 might be useful reagents in radioimmunoimaging and radioimmunotherapy. (author)
Full Text Available Abstract Background Avian influenza virus H5N1 has demonstrated considerable pandemic potential. Currently, no effective vaccines for H5N1 infection are available, so passive immunotherapy may be an alternative strategy. To investigate the possible therapeutic effect of antibody against highly pathogenic H5N1 virus on a mammal host, we prepared specific equine anti-H5N1 IgGs from horses vaccinated with inactivated H5N1 virus, and then obtained the F(ab'2 fragments by pepsin digestion of IgGs. Methods The horses were vaccinated with inactivated H5N1 vaccine to prepare anti-H5N1 IgGs. The F(ab'2 fragments were purified from anti-H5N1 hyperimmune sera by a protocol for 'enhanced pepsin digestion'. The protective effect of the F(ab'2 fragments against H5N1 virus infection was determined in cultured MDCK cells by cytopathic effect (CPE assay and in a BALB/c mouse model by survival rate assay. Results By the protocol for 'enhanced pepsin digestion', total 16 g F(ab'2 fragments were finally obtained from one liter equine antisera with the purity of over 90%. The H5N1-specific F(ab'2 fragments had a HI titer of 1:1024, and the neutralization titre of F(ab'2 reached 1: 2048. The in vivo assay showed that 100 μg of the F(ab'2 fragments could protect BALB/c mice infected with a lethal dose of influenza H5N1 virus. Conclusion The availability of highly purified H5N1-specific F(ab'2 fragments may be promising for treatment of influenza H5N1 infection. Our work has provided experimental support for the application of the therapeutic equine immunoglobulin in future large primate or human trials.
León Montero, Guillermo; Stiles, Bradley G.; Alape Girón, Alberto; Rojas Céspedes, Gustavo; Gutiérrez, José María
A comparative study was performed on the ability of IgG and F(ab')2 antivenoms to neutralize lethal and myotoxic activities of Micrurus nigrocinctus venom. Both antivenoms were adjusted to a similar neutralizing potency in experiments where venom and antivenoms were preincubated prior to injection. No significant differences were observed between IgG and F(ab')2 antivenoms concerning neutralization of lethal effect in rescue experiments, i.e., when antivenom was administered intravenously aft...
Kojima, Shuji; Suzuki, Naomi; Shimura, Noriko; Kubodera, Akiko; Kubota, Kazuhiko; Yamaguchi, Toshiharu; Takahashi, Toshio; Oyamada, Hiyoshimaru
Differences of pharmacokinetics and tumor imaging ability between intact monoclonal antibody A7 (A7 MoAb) and F(ab) 2 fragments were studied in human colon cancer (LS-174T)-bearing nude mice. The authors examined the yield and the immunoreactivity of F(ab) 2 fragments after treatment with ficin as a function of time. The yield of F(ab) 2 fragments reached about 50% after ficin treatment for 8 h, and the F(ab) 2 retained about 80% of the immunoreactivity of the corresponding MoAb. Longer digestion with ficin produced smaller fragments (less than 92 kDa) with a lower yield and most of the immunoreactivity was lost. In pharmacokinetics studies, the F(ab') 2 was preferentially taken up by the tumor, cleared more rapidly from the blood circulation and seemed to have less non-specific tissue binding than intact A7 MoAb. The tumor image obtained at an early time using 131 I-F(ab') 2 was much superior in quality to that with intact 131 I-A7 MoAb. The use of F(ab') 2 fragments may be effective for tumor diagnosis and therapy. (author)
Chippaux, Jean-Philippe; Massougbodji, A.; Stock, R. P.; Alagon, A.
We report the results of a trial designed to measure the safety and efficacy of African Antivipmyn((R)), a new freeze-dried polyvalent equine F(ab')(2)-based antivenom. We tested 289 envenomations. After treatment, 19% of treated patients had undesirable events, all benign. A possible adverse effect was attributed to this antivenom in 11% of the patients. Bleeding was observed in 48% of the patients; it stopped within 2 hours after treatment with antivenom in 60% of the patients. Blood incoag...
Anderson, C.J.; Schwarz, S.W.; Connett, J.M.
Antibody fragments labeled with a radiometal using bifunctional chelates generally undergo renal clearance followed by trapping of the metabolites, leading to high radiation doses to the kidneys. Copper-64-labeled BAT-2IT-1A3-F(ab') 2 was recently reported to accumulate in colorectal tumors in an animal model, however, kidney uptake was also high. In this study, the preparation of 64 Cu-BAT-2IT-1A3-F(ab') 2 was optimized to reduce the renal uptake. The bifunctional chelate 6-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradecane-N,N',N double-prime,N'double-prime-tetraacetic acid (BAT) was conjugated to 1A3-F(ab') 2 using the linking agent 2-iminothiolane (2IT). The conjugation reaction produced 20% of a lower molecular weight impurity found to be TETA-1A3-Fab'. The conjugation procedure was optimized to include FPLC purification of the BAT-2IT-1A3-F(ab') 2 from TETA-1A3-Fab' after conjugation prior to labeling with 64 Cu. The biodistribution of 64 Cu-labeled FPLC-purified and unpurified conjugates was determined in normal Sprague-Dawley rats and tumor-bearing Golden Syrian hamsters. Human absorbed doses were calculated from rat biodistribution data and PET imaging of a baboon. Upon FPLC purification of the BAT-2IT-1A3-F(ab') 2 , the immunoreactivity of 64 Cu-labeled 1A3-F(ab') 2 was significantly improved over that of non-FPLC-purified 64 Cu-BAT-2IT-1A3-F(ab') 2 , and the kidney uptake was decreased in normal rats. The biodistribution in hamsters showed some improvement in both tumor uptake and kidney clearance with FPLC-purified 64 Cu-BAT-2IT-1A3-F(ab') 2 .The improved dosimetry of 64 Cu-labeled FPLC purified BAT-2IT-1A3-F(ab') 2 should more readily allow this agent to be investigated clinically to image colorectal cancer using PET. 33 refs., 7 figs., 3 tabs
Full Text Available Trypanosoma cruzi calreticulin (TcCRT, described in our laboratory, retains several important functional features from its vertebrate homologues. We have shown that recombinant TcCRT inhibits the human complement system when it binds to the collagenous portion of C1q. The generation of classical pathway convertases and membrane attack complexes is thus strongly inhibited. In most T. cruzi-infected individuals, TcCRT is immunogenic and mediates the generation of specific antibodies. By reverting the C1q / TcCRT interaction, a parasite immune evasion strategy, these antibodies contribute to the host / parasite equilibrium. In an in vitro correlate of this situation, we show that the C1q / TcCRT interaction is inhibited by F(ab'2 polyclonal anti-TcCRT IgG fragments. It is therefore feasible that in infected humans anti-TcCRT antibodies participate in reverting an important parasite strategy aimed at inhibiting the classical complement pathway. Thus, membrane-bound TcCRT interacts with the collagenous portion C1q, and this C1q is recognized by the CD91-bound host cell CRT, thus facilitating parasite internalization. Based on our in vitro results, it could be proposed that the in vivo interaction between TcCRT and vertebrate C1q could be inhibited by F(ab'2 fragments anti-rTcCRT or against its S functional domain, thus interfering with the internalization process
Boskovitz, Abraham; Akabani, Gamal H.; Pegram, Charles N.; Bigner, Darrell D.; Zalutsky, Michael R.
We have obtained encouraging responses in recent Phase I studies evaluating 131 I-labeled human/murine chimeric 81C6 anti-tenascin monoclonal antibody (ch81C6) administered into surgically-created tumor resection cavities in brain tumor patients. However, because the blood clearance is slow, hematologic toxicity has been higher than seen with murine 81C6 (mu81C6). In the current study, a series of paired-label experiments were performed in athymic mice bearing subcutaneous D-245 MG human glioma xenografts to compare the biodistribution of the fragment ch81C6 F(ab') 2 labeled using Iodogen to a) intact ch81C6, b) mu81C6, and c) ch81C6 F(ab') 2 labeled using N-succinimidyl 3-[ 131 I]iodobenzoate. Tumor retention of radioiodine activity for the F(ab') 2 fragment was comparable to that for intact ch81C6 for the first 24 h and to that for mu81C6 for the first 48 h; as expected, blood and other normal tissue levels declined faster for ch81C6 F(ab') 2. Radiation dosimetry calculations suggest that 131 I-labeled ch81C6 F(ab') 2 may warrant further evaluation as a targeted radiotherapeutic for the treatment of brain tumors
Back, T.; Haraldsson, B.; Hultborn, R
of the glomerular filtration rate (GFR). The renal toxicity was evaluated at levels close to the dose limit for the bone marrow and well within the range for therapeutic efficacy on tumors. Astatinated MX35-F(ab')(2) monoclonal antibodies were administered intravenously to nude mice. Both non-tumor-bearing animals...... manifested late. Examination of the kidney sections showed histologic changes that were overall subdued. Following alpha-RIT with (211)At-MX35-F(ab')(2) at levels close to the dose limit of severe myelotoxicity, the effects found on renal function were relatively small, with only minor to moderate reductions...... in GFR. These results suggest that a mean absorbed dose to the kidneys of approximately 10 Gy is acceptable, and that the kidneys would not be the primary dose-limiting organ in systemic alpha-RIT when using (211)At-MX35-F(ab')(2) Udgivelsesdato: 2009/12...
Bellaye, P-S; Moreau, M; Raguin, O; Oudot, A; Bernhard, C; Vrigneaud, J-M; Dumont, L; Vandroux, D; Denat, F; Cochet, A; Brunotte, F; Collin, B
This study aimed to investigate theranostic strategies in colorectal and skin cancer based on fragments of cetuximab, an anti-EGFR mAb, labeled with radionuclide with imaging and therapeutic properties, 111 In and 177 Lu, respectively. We designed F(ab') 2 -fragments of cetuximab radiolabeled with 111 In and 177 Lu. 111 In-F(ab') 2 -cetuximab tumor targeting and biodistribution were evaluated by SPECT in BalbC nude mice bearing primary colorectal tumors. The efficacy of 111 In-F(ab') 2 -cetuximab to assess therapy efficacy was performed on BalbC nude mice bearing colorectal tumors receiving 17-DMAG, an HSP90 inhibitor. Therapeutic efficacy of the radioimmunotherapy based on 177 Lu-F(ab') 2 -cetuximab was evaluated in SWISS nude mice bearing A431 tumors. Radiolabeling procedure did not change F(ab') 2 -cetuximab and cetuximab immunoreactivity nor affinity for HER1 in vitro. 111 In-DOTAGA-F(ab') 2 -cetuximab exhibited a peak tumor uptake at 24 h post-injection and showed a high tumor specificity determined by a significant decrease in tumor uptake after the addition of an excess of unlabeled-DOTAGA-F(ab') 2 -cetuximab. SPECT imaging of 111 In-DOTAGA-F(ab') 2 -cetuximab allowed an accurate evaluation of tumor growth and successfully predicted the decrease in tumor growth induced by 17-DMAG. Finally, 177 Lu-DOTAGA-F(ab') 2 -cetuximab radioimmunotherapy showed a significant reduction of tumor growth at 4 and 8 MBq doses. 111 In-DOTAGA-F(ab') 2 -cetuximab is a reliable and stable tool for specific in vivo tumor targeting and is suitable for therapy efficacy assessment. 177 Lu-DOTAGA-F(ab') 2 -cetuximab is an interesting theranostic tool allowing therapy and imaging.
Geoffrey K Isbister
Full Text Available There is limited information on antivenom pharmacokinetics. This study aimed to investigate the pharmacokinetics of an Indian snake antivenom in humans with Russell's viper bites.Patient data and serial blood samples were collected from patients with Russell's viper (Daboia russelii envenoming in Sri Lanka. All patients received Indian F(ab'2 snake antivenom manufactured by VINS Bioproducts Ltd. Antivenom concentrations were measured with sandwich enzyme immunoassays. Timed antivenom concentrations were analysed using MONOLIXvs4.2. One, two and three compartment models with zero order input and first order elimination kinetics were assessed. Models were parameterized with clearance (CL, intercompartmental clearance (Q, central compartment volume (V and peripheral compartment volume (VP. Between-subject-variability (BSV on relative bioavailability (F was included to account for dose variations. Covariates effects (age, sex, weight, antivenom batch, pre-antivenom concentrations were explored by visual inspection and in model building. There were 75 patients, median age 57 years (40-70 y and 64 (85% were male. 411 antivenom concentration data points were analysed. A two compartment model with zero order input, linear elimination kinetics and a combined error model best described the data. Inclusion of BSV on F and weight as a covariate on V improved the model. Inclusion of pre-antivenom concentrations or different batches on BSV of F did not. Final model parameter estimates were CL,0.078 L h(-1, V,2.2L, Q,0.178 L h(-1 and VP,8.33L. The median half-life of distribution was 4.6 h (10-90%iles:2.6-7.1 h and half-life of elimination, 140 h (10th-90th percentilesx:95-223h.Indian F(ab'2 snake antivenom displayed biexponential disposition pharmacokinetics, with a rapid distribution half-life and more prolonged elimination half-life.
Buist, M. R.; Kenemans, P.; den Hollander, W.; Vermorken, J. B.; Molthoff, C. J.; Burger, C. W.; Helmerhorst, T. J.; Baak, J. P.; Roos, J. C.
Twenty-four patients suspected of having ovarian carcinoma received i.v. injection with a combination of radiolabeled intact IgG (1 mg) and F(ab')2 fragments (1 mg) of the chimeric monoclonal antibody MOv18, each form labeled with 1.85 MBq 131I or 125I. Laparotomy was performed either 2 or 6 days
Brouwers, A.H.; Mulders, P.F.A.; Oosterwijk, E.; Buijs, W.C.A.M.; Corstens, F.H.M.; Boerman, O.C.; Oyen, W.J.G.
INTRODUCTION: Clinical and animal studies of chimeric monoclonal antibody G250 (moAb cG250) for the targeting of clear-cell renal cell carcinoma (RCC), to date, have been with the intact IgG form. To determine whether F(ab')2 fragments are more suited for radioimmunotherapy (RIT) than intact IgG,
Buchegger, F.; Pfister, C.; Fournier, K.; Prevel, F.; Schreyer, M.; Carrel, S.; Mach, J.P.
Pooled F(ab')2 fragments of three MAbs against distinct epitopes of carcinoembryonic antigen (CEA) were used for radioimmunotherapy of nude mice bearing a subcutaneous human colon carcinoma xenograft. 9-10 d after transplantation when tumor nodules were in exponential growth, 36 mice were treated by intravenous injection of different amounts of 131 I-labeled MAb F(ab')2. All 14 mice injected with a single dose of 2,200 (n = 10) or 2,800 microCi (n = 4) showed complete tumor remission. 8 of the 10 mice treated with 2,200 microCi survived in good health for 1 yr when they were killed and shown to be tumor free. Four of nine other mice treated with four fractionated doses of 400 microCi showed no tumor relapse for more than 9 mo. In contrast, all 15 mice injected with 1,600-3,000 microCi 131 I-control IgG F(ab')2 showed tumor growth retardation of only 1-4 wk, and 15 of 16 mice injected with unlabeled anti-CEA MAb F(ab')2 showed unmodified tumor progression as compared with untreated mice. From tissue radioactivity distributions it was calculated that by an injection of 2,200 microCi 131 I-MAb F(ab')2 a mean dose of 8,335 rad was selectively delivered to the tumor, while the tissue-absorbed radiation doses for the normal organs were: peripheral blood, 2,093; stomach, 1,668; kidney, 1,289; lung, 1,185; liver, 617; spleen, 501; small intestine, 427; large intestine, 367; bone, 337; and muscle, 198. These treatments were well tolerated since out of 19 mice with complete tumor remission only 4 required bone marrow transplantation and 17 were in good health for 6-12 mo of observation
Boyer, Leslie; Degan, Janice; Ruha, Anne-Michelle; Mallie, Joanne; Mangin, Emmanuelle; Alagón, Alejandro
The technology of antivenom production has gradually changed since the earliest production of antisera around the turn of the 20th century. Use of early antisera was associated with frequent acute adverse reactions and serum sickness. New F(ab')2 products, manufactured using pepsin degradation of immunoglobulin together with precipitation of unwanted protein and albumin serum fractions, should in concept cause fewer immune reactions in clinical use. A linked set of five prospective clinical trials of an equine F(ab')2 antivenom, together with one historical control study, were completed during development of the product for a Biological License Application through the US FDA. Adverse events were recorded and categorized, with particular attention to the frequency of immune reactions. A total of 1534 patients ages 0.1-90.5 years received antivenom, in Arizona and in Mexico, for treatment of scorpion envenomation. Total dosing ranged from 1 to 5 vials except for one outlier who received 10 vials. Estimated protein exposure was 12-275 mg per patient (outlier, up to 550 mg). Three patients (0.2%) had acute reactions to antivenom infusion (one urticaria, one urticaria and dyspnea, and one panic attack). Eight (0.5%) had rashes suggestive of Type 3 immune reactions, although none had the full syndrome of serum sickness. Two women were treated for envenomation during the first trimester of pregnancy, one of whom subsequently experienced a spontaneous abortion. Rates of immune reaction to this product were two orders of magnitude lower than the range (up to 75% for early and 81% for late reactions) historically reported with use of minimally refined whole immunoglobulin products against a variety of infections and envenomations. Lower protein dose, greater purity of the active component, lack of the immunogenic Fc portion of the immunoglobulin molecule, and slow intravenous infusion are likely to be the reason for this. Clinical implications of a safer product include that it can be employed in settings where antivenom was once considered too dangerous to use, such as primary care clinics and remote rural areas. Copyright © 2013 Elsevier Ltd. All rights reserved.
Munz, D.L.; Alavi, A.; Koprowski, H.; Herlyn, D.; Pennsylvania Univ., Philadelphia
F(ab') 2 fragments of MAbs GA 73-3 (IgG 2 a) and CO 29.11 (IgG 1), which detect distinct antigenic determinants on adenocarcinoma cells of the gastrointestinal tract, were labeled with 131 I using the iodogen method. 41 nude mice bearing SW-948 CRC tumores were injected either with a mixture of 100 μCi (11 μg) each (n=9) of the two 131 I-F(ab') 2 fragments or with either fragment alone at various doses (each group consisting of 8 mice): GA 73-3, 100 μCi (11 μg) and 200 μCi (25 μg); CO 29.11, 100 μCi (11 μg) and 200 μCi (26 μg). Whole-body images of the mice were obtained daily for up to six days after injection. Ratios of cpm/pixel in the tumor to those in the rest of the body (rob), representing tumor contrast, were significantly (p 1/2 biol. ) f the mixture (44.8±14.5 h) in the CRC tumors was significantly (p 1/2 biol. determined in the groups given either fragment alone. T 1/2 biol. in the rob was similar in all groups of mice examined. (orig.) [de
Buchegger, F.; Chalandon, Y.; Pelegrin, A.; Hardman, N.; Mach, J.P.
Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats
Vogt, R.; Mehnert, W.H.; Schmidt, H.E.; Altenbrunn, H.J.; Akademie der Wissenschaften der DDR, Berlin-Buch. Zentralinstitut fuer Isotopen- und Strahlenforschung)
Tumor-bearing mice injected with clostridial spores show enrichment and germination of the spores within the tumor. 131 I-labelled anti-Clostridium-antibodies and anti-Clostridium-F(ab') 2 -fragments were used for a possible localization of tumors in vivo by scintiscanning. The application of the antibody revealed increased radioactivity in the tumors of mice pretreated with spores as well as in animals without pretreatment. In using F(ab') 2 -fragments instead of total antibody neither the apparently unspecific increase of radioactivity in not pretreated mice nor the specific fixation of labelled F(ab') 2 -fragments to clostridial rods in the tumors of pretreated animals could be demonstrated. The results are discussed with respect to further investigation
Al-Abdulla, Ibrahim; Casewell, Nicholas R; Landon, John
Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab')2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab')2 and Fab fragments, respectively. A "single-reagent" method was developed for the production of F(ab')2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4h at 37°C. For ovine Fab antivenom production from whole antiserum, the "single reagent" comprised of caprylic acid, papain and l-cysteine; after incubation at 37°C for 18-20h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab')2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate. Copyright © 2013 Elsevier B.V. All rights reserved.
León Montero, Guillermo; Rojas Céspedes, Gustavo; Lomonte, Bruno; Gutiérrez, José María
The ability of whole immunoglobulin G (IgG) and F(ab')2 polyvalent (Crotalinae) antivenoms to neutralize the hemorrhagic, edema-forming and myotoxic activities of Bothrops asper venom was studied. Both antivenoms were adjusted to the same neutralizing potency against lethal and hemorrhagic activities in experiments where venom and antivenoms were incubated before injection. Thus, in these experimental conditions, differences in the neutralizing ability in experiments involving independent inj...
Chippaux, Jean-Philippe; Lang, J.; Amadi-Eddine, S.; Fagot, P.; Le Mener, V.
A clinical trail was conducted in 2 health centers in northern Cameroon to assess the safety and efficacy of a new polyvalent antivenom composed of higly purified and pasteurized F(ab')2 (FAV-Africa). Forty-six patients with objective signs of envenomation, including 67% with hemorrhage, were included in the study. Each patient received at least 20 ml of FAV-Africa by direct, slow intravenous injection ; 172 10-ml ampules were administered. All patients were clinically cured after treatment. ...
Chippaux, Jean-Philippe; Lang, J.; Amadi Eddine, S; Fagot, P.; Rage, V.; Peyrieux, J.C.; Le Mener, V.
A large-scale clinical trial was conducted, according to World Health Organization Good Clinical Practice guidelines, in 7 centres in North Cameroon to determine the safety and efficacy of a polyvalent antivenom composed of purified F(ab')2. This study included 223 patients presenting clinically with an obvious snake bite, predominantly due to #Echis ocellatus$ (viper), the most abundant species in this savannah region. Clinical surveillance was maintained for 5 d in all patients and until th...
Kalofonos, H.P.; Sivolapenko, G.B.; Courtenay-Luck, N.S.
Immunoscintigraphy using F(ab')2 fragments of tumor-associated monoclonal antibody HMFG1 was performed in 14 patients with primary and metastatic non-small cell carcinoma of lung cancer. The antibody was conjugated with diethylenetriamine pentaacetic acid and labeled with 111 In. Quality control studies showed efficient incorporation of 111 In onto antibody (5 mCi/mg), no significant loss of immunoreactivity, and in vitro and in vivo stability. The optimal time for imaging was between 48 and 72 h. Following i.v. administration, serum activity fell rapidly (t1/2a = 2.5 +/- 1.3 (SD) h; t1/2b = 42 +/- 4.5 h). The majority of the radioactivity was associated with the plasma and not with the blood cells. All patients had a significant concentration of 111 In in the liver (approximately 20% of the injected dose, 48 h postadministration). No toxicity was encountered. No human antimurine-IgG antibody was detected in any of the patients within 4 months of follow-up, even in patients receiving two administrations of F(ab')2 fragments. Localization of all primary lesions and the majority (80%) of metastatic lesions was achieved. Seven of 14 patients were also studied using a 111 In-labeled nonspecific antibody (Fab')2 fragment (4C4). In three patients the specificity index was higher than the other four (P less than 0.05). We conclude that although successful targeting of 111 In-labeled (Fab')2 fragments of HMFG1 can be achieved in patients with non-small cell carcinoma of lung, observable tumor localization can also be achieved using a nonspecific antibody
Sevcik, C; D'Suze, G; Díaz, P; Salazar, V; Hidalgo, C; Azpúrua, H; Bracho, N
Modelling Tityus scorpion venom and antivenom pharmacokinetics. Evidence of active immunoglobulin G's F(ab')(2) extrusion mechanism from blood to tissues. We measured pharmacokinetic parameters for T. discrepans venom in rams. Forty, 75 or 100 microg/kg venom were injected subcutaneously in the inner side of the thigh. Plasma venom content (venenemia) was determined by enzyme-linked immunosorbent assay (ELISA) from 0 to 300 min after injecting venom. Venenemia was fit to a three-compartment model (inoculation site, plasma and extra vascular extracellular space), it was assumed that the venom may also be irreversibly removed from plasma. Calculated time course of venom content shows that at any time no more that 30% of the venom is present in plasma. Venenemia peaks at 1h and decays afterwards. Fluorescently labelled antivenom [horse anti-TityusF(ab')(2) or fraction antigen binding, immuglobulin without Fc chain covalently bound to fluorescine or fluorescamine] pharmacokinetics was determined. Although F(ab')(2) molecular weight is >/=10 times bigger that toxin's, the rate of outflow of F(ab')(2) from blood to tissues was approximately 4 times faster than the venom's outflow. Venom content in the injection site decays exponentially for >6h, this prediction was confirmed immunohistochemically. Only approximately 5% of the venom is eliminated in 10h; approximately 80% of the venom is in the tissues after 2h and remains there for >10h.
Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies.
Welfringer, Frédéric; d'Athis, Philippe; Scherrmann, Jean-Michel; Hervé, Françoise
Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab')(2) in cell lysates and rat plasma samples, assessing the cellular uptake and plasma kinetics of these antibodies, respectively. The method used tetanus anatoxin coated on microtitre plates as capture antigen to bind sample or standard F(ab')(2), the amount of antibody binding being quantified using, first, a secondary biotinylated anti-horse antibody/streptavidin-alkaline phosphatase complex in situ and then a measurement of the substrate product. Cationization of the F(ab')(2) was performed with putrescine at pH 4.5 using soluble carbodiimide as carboxyl activator. The average substitution ratio was determined at 3 putrescine molecules per F(ab')(2) molecule. The cationized F(ab')(2) retained roughly 80% of the initial antigen binding activity and was stable over a 1 year period of storage at -20 degrees C. The ELISA validation data showed that the method was linear for both the native and cationized F(ab')(2) using Hanks' balanced saline solution with 0.2% bovine serum albumin as assay diluent for the cell lysate samples. The useful F(ab')(2) concentration range was 2.5-25 ng/ml and the limit of quantification was 2.5 ng/ml. With rat blank plasma used as assay diluent for the rat plasma samples the useful F(ab')(2) concentration range was 3.5-25 ng/ml and the limit of quantification was 3.5 ng/ml. Specific requirements for the limits of quantification were fulfilled: precision tetanus F(ab')(2) in an HL 60 cell model, and of plasma kinetics after i.v. administration to rats.
Gerardo, Charles J; Vissoci, Joao R Nickenig; Brown, Michael W J; Bush, Sean P
Coagulation derangements in copperhead envenomation are considered less severe than other crotaline envenomations, resulting in recommendations to limit both coagulation testing and antivenom treatment. A prospective, blinded, multicenter, randomized clinical trial comparing the effectiveness of F(ab') 2 versus Fab antivenom in crotaline envenomation patients was completed in 2011. We determined the difference between coagulation parameters in copperhead compared to other crotaline envenomations. We performed a post hoc analysis comparing the coagulation parameters (platelets and fibrinogen) prospectively obtained in the aforementioned trial. All the patients received antivenom in one of three treatment arms [F(ab') 2 with maintenance, F(ab') 2 with placebo maintenance, or Fab with maintenance]. Coagulation parameters were measured at pretreatment baseline, during acute hospitalization, day 5, day 8, and day 15 post-envenomation. Mean platelet count and fibrinogen levels for the copperhead and other crotaline groups were compared. The platelet and fibrinogen point estimates with distribution are presented graphically over time. 122 patients were enrolled in the study. There were 22 patients with copperhead envenomation, 93 with other crotaline envenomations, and 7 that could not be definitively determined. The mean age was 42 (SD 20) years. There was a minor pretreatment difference in mean baseline platelet count between the copperhead group (246 × 109/L 95% CI 215, 277) compared to other crotaline envenomation patients (184 × 109/L 95% CI 167, 202). There was a modest pretreatment difference in mean fibrinogen level between copperhead patients (345 mg/dL 95% CI 277, 415) and other crotaline patients (261mg/dL 95% CI 241, 281). Pretreatment coagulation parameter means were normal and converged post treatment. On average, copperhead envenomations have less severe initial coagulation derangements. However, in mild envenomations, differences in laboratory values are minimal and there is substantial variation in individual patients regardless of species. Species alone should not be used to determine the need for laboratory testing or treatment in crotaline snakebite.
Riva, P.; Paganelli, G.; Callegaro, L.
F(ab') 2 fragments of F023C5, an anti-CEA monoclonal antibody, were conjugated to diethylenetriamine pentaacetic acid (DTPA) and converted into a ready to use reagent for instant 111 In-labelling. The resulting 111 In radiopharmaceutical was administered intravenously and tested for its ability to image (at 48-72 h after administration) 31 primary and 85 metastatic carcinoma lesions in 70 adenocarcinoma patients (26 gastrointestinal, 18 breast and 26 lung tumour patients) whose serum CEA was elevated in 43 cases and normal in the other 27. (author)
Ferro-Flores, Guillermina; Pimentel-Gonzalez, Gilmara; Gonzalez-Zavala, Maria Antonia; Murphy, Consuelo Arteaga de; Melendez-Alafort, Laura; Tendilla, Jose I.; Croft, Barbara Y.
The biotinylated monoclonal antibody (MoAb) ior cea1 and its F(ab') 2 fragments were labeled with Re-188 by combination of avidin-biotin strategy. 188 Re-MoAb, 188 Re-MoAb-biotin, 188 Re-F(ab') 2 , and 188 Re-F(ab') 2 -biotin preparations were produced for these studies with specific activities of 1.30±0.18 GBq/mg and from instant freeze-dried kit formulations using ethane-1-hydroxy-1,1-diphosphonic acid (EHDP) as a weak competing ligand. There were no significant differences (p>0.05) between the biodistribution in mice of biotinylated and unbiotinylated 188 Re-labeled immunoconjugates. When avidin was injected as a chase after injection of 188 Re-MoAb-biotin or 188 Re-F(ab') 2 -biotin, the blood radioactivity level decreased approximately 75% (cumulated activity) and the effective dose decreased almost 25% with respect to that of the radioimmunoconjugates in which the chase effect was not used. Our results suggest that 188 Re-labeled biotinylated MoAb ior cea1 and its F(ab') 2 fragments prepared by this method are stable complexes in vivo
Andrew, S.M.; Pimm, M.V.; Baldwin, R.W.; Perkins, A.C.
An IgG1 mouse monoclonal antibody directed against CEA has been digested with papain to yield F(ab) 2 and Fab fragments. Following radioiodination, intact antibody and fragments showed specific binding to cells of a CEA-producing tumour, although the immune reactivities of the fragments were lower than that of intact antibody. Gamma scintigraphy of nude mice bearing CEA producing human tumour xenografts and injected with 131 I-labelled fragments showed earlier and superior imaging of tumours than did 131 I-intact antibody, and this was most marked with the Fab fragment. Sequential dissection analyses showed that this was due to earlier and higher tumour-to-blood ratios with fragments than with intact antibody, but in absolute terms the degree of localization of both fragment types was significantly lower than that of intact antibody. (orig.)
Lutje, Susanne; van Rij, Catharina M.; Franssen, Gerben M.; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J.; Colombatti, Marco; Boerman, Otto C.
D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab)(2) and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing
Lutje, S.; Rij, C.M. van; Franssen, G.M.; Fracasso, G.; Helfrich, W.; Eek, A.; Oyen, W.J.G.; Colombatti, M.; Boerman, O.C.
D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing
Tibben, J.G.; Massuger, L.F.A.G.; Boerman, O.C.; Borm, G.F.; Claessens, R.A.M.J.; Corstens, F.H.M.
The effect of the route administration on the distribution of radioiodinated OV-TL 3 F(ab') 2 was studied in Balb/c female mice with intraperitoneal or subcutaneous ovarian carcinoma xenografts. In the intraperitoneal tumour model in which both ascites and solid tumour deposits were present, intraperitoneal administration resulted in a lower estimated radiation dose to blood as compared with intravenous administration. In this model normalization to equal estimated radiation doses to blood for both routes of administration indicated that a twice as high estimated radiation dose can be guided to solid intraperitoneal tumour deposits following intraperitoneal administration. Evacuation of ascitic tumour cells prior to monoclonal antibody injection further increased the estimated radiation dose to solid intraperitoneal tumour deposits following intraperitoneal delivery. Following simultaneous intravenous and intraperitoneal injection of the monoclonal antibody, tissue uptake showed no relevant differences in the subcutaneous tumour model. Overall, the intraperitoneal route of administration was found to be the best choice for therapeutic delivery of iodine-131 labelled monoclonal antibodies. (orig.)
Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab')2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies.
Welfringer, Frédéric; D'Athis, Philippe; Scherrmann, Jean-Michel; Hervé, Françoise
International audience; Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab')(2) fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab...
Lütje, Susanne; van Rij, Catharina M; Franssen, Gerben M; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J; Colombatti, Marco; Boerman, Otto C
D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing subcutaneous prostate cancer xenografts. The optimal time point for imaging was determined in biodistribution and microSPECT imaging studies with (111)In-D2B IgG, (111)In-capromab pendetide, (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments in mice with PSMA-expressing LNCaP and PSMA-negative PC3 tumors at several time points after injection. All (111)In-labeled antibody formats specifically accumulated in the LNCaP tumors, with highest uptake of (111)In-D2B IgG and (111)In-capromab pendetide at 168 h p.i. (94.8 ± 19.2% injected dose per gram (ID/g) and 16.7 ± 2.2% ID/g, respectively), whereas uptake of (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments peaked at 24 h p.i. (12.1 ± 3.0% ID/g and 15.1 ± 2.9% ID/g, respectively). Maximum LNCaP tumor-to-blood ratios were 13.0 ± 2.3 (168 h p.i.), 6.2 ± 0.7 (24 h p.i.), 23.0 ± 4.0 (24 h p.i.) and 4.5 ± 0.6 (168 h p.i.) for (111)In-D2B IgG, (111)In-F(ab')2, (111)In-Fab and (111)In-capromab pendetide, respectively. LNCaP tumors were clearly visualized with microSPECT with all antibody formats. This study demonstrates the feasibility of D2B IgG, F(ab')2 and Fab fragments for targeting PSMA-expressing prostate cancer xenografts. Copyright © 2014 John Wiley & Sons, Ltd.
F(ab´)2 fragments were further purified by Q-Fast Flow chromatography, concentrated by molecular ultrafiltration and sterilized by filtration through 0.22 m membranes. The resulting F(ab´)2 preparations were rich in intact L and in pieces of H IgG(T) chains, as demonstrated by electrophoresis and Western blot and exhibited ...
Full Text Available Humanized monoclonal antibody U36 and its F(ab'2 fragment, radio labeled with 125I, were tested for tumor localization in nude mice bearing a squamous cell carcinoma xenograft line derived from a head and neck carcinoma. Monoclonal antibody IgG or F(ab'2 fragment were injected in parallel and at days 1, 2 and 3, mice were dissected for determination of isotope biodistribution. IgG as well as F(ab'2 showed highly specific localization in tumor tissue. The mean tumor uptake (n=3 is expressed as the percentage of the injected dose per gram of tumor tissue (%ID/g. %ID/g of IgG was 11.7% at day 1 and decreased to 10.9% at day 3 whereas %ID/g of F(ab'2 was 2.9% at day 1 and decreased on following days. Tumor to blood ratios (T/B at day 1 were 0.86 for IgG and 1.32 for F(ab'2 and reached a maximum at day 3 with values of 4.41 and 1.84 respectively. These findings suggest that the superior tumor to non-tumor ratios in the day of 1 render the F(ab'2 fragment more qualified for specific targeting radioisotopes to tumor xenografts in this exprimental setting.
Apr 19, 2010 ... African Journal of Biotechnology Vol. 9 (16), pp. ... filtered through a 30 kDa ultrafiltration membrane. F(ab´)2 .... was added to the plasma pool obtained from the horses immunized with whole ..... revision of the manuscript.
Apr 19, 2010 ... 2Laboratório de Biologia do Reconhecer, Centro de Biociencias e Biotecnologia, Universidade Estadual do Norte. Fluminense – Darcy Ribeiro, Campos dos ... Flow chromatography, concentrated by molecular ultrafiltration and sterilized by filtration through 0.22 μm membranes. The resulting F(ab´)2 ...
Isbister, Geoffrey K.; Maduwage, Kalana; Saiao, Ana; Buckley, Nicholas A.; Jayamanne, Shaluka F.; Seyed, Shahmy; Mohamed, Fahim; Chathuranga, Umesh; Alexandre, Mendes; Abeysinghe, Chandana; Karunathilake, Harinda; Gawarammana, Indika; Lalloo, David; Janaka de Silva, H.
Background\\ud \\ud There is limited information on antivenom pharmacokinetics. This study aimed to investigate the pharmacokinetics of an Indian snake antivenom in humans with Russell’s viper bites.\\ud \\ud Methods/Principal Findings\\ud \\ud Patient data and serial blood samples were collected from patients with Russell’s viper (Daboia russelii) envenoming in Sri Lanka. All patients received Indian F(ab’)2 snake antivenom manufactured by VINS Bioproducts Ltd. Antivenom concentrations were measur...
Heskamp, Sandra; van Laarhoven, Hanneke W. M.; Molkenboer-Kuenen, Janneke D. M.; Bouwman, Wilbert H.; van der Graaf, Winette T. A.; Oyen, Wim J. G.; Boerman, Otto C.
The insulin-like growth factor 1 receptor (IGF-1R) is a potential new target for the treatment of breast cancer. Patients with breast cancer lesions that express IGF-1R may benefit from treatment with anti-IGF-IR antibodies. IGF-1R expression can be visualized using radiolabeled R1507, a monoclonal
Schaijk, F.G. van; Boerman, O.C.; Soede, A.C.; McBride, W.J.; Goldenberg, D.M.; Corstens, F.H.M.; Oosterwijk, E.
PURPOSE: An effective pretargeting strategy was developed for renal cell carcinoma (RCC) based on a biologically produced bispecific monoclonal antibody: anti-RCCxanti-DTPA(In) (bsMAb: G250xDTIn-1). Tumour uptake of a (111)In-labelled bivalent peptide after pretargeting with bsMAb G250xDTIn-1 was
Beatriz P Quiambao
Full Text Available BACKGROUND: Recommended treatment for severe rabies exposure in unvaccinated individuals includes wound cleaning, administration of rabies immunoglobulins (RIG, and rabies vaccination. We conducted a survey of rabies treatment outcomes in the Philippines. METHODS: This was a case series involving 7,660 patients (4 months to 98 years of age given purified equine RIG (pERIG at the Research Institute for Tropical Medicine (Muntinlupa, Philippines from July 2003 to August 2004 following Category II or III exposures. Data on local and systemic adverse reactions (AR within 28 days and biting animal status were recorded; outcome data were obtained by telephone or home visit 6-29 months post-exposure. RESULTS: Follow-up data were collected for 6,464 patients. Of 151 patients with laboratory-confirmed rabies exposure, 143 were in good health 6-48 months later, seven could not be contacted, and one 4-year-old girl died. Of 16 deaths in total, 14 were unrelated to rabies exposure or treatment. Two deaths were considered PEP failures: the 4-year old girl, who had multiple deep lacerated wounds from a rabid dog of the nape, neck, and shoulders requiring suturing on the day of exposure, and an 8-year-old boy who only received rabies PEP on the day of exposure. CONCLUSIONS: This extensive review of outcomes in persons with Category III exposure shows the recommended treatment schedule at RITM using pERIG is well tolerated, while survival of 143 laboratory-confirmed rabies exposures confirms the intervention efficacy. Two PEP intervention failures demonstrate that sustained education and training is essential in rabies management.
Lomonte, Bruno; León Montero, Guillermo; Hanson, Lars Ake
The ability of two antivenoms to Vipera spp., consisting of Fab (Therapeutic Antibodies), or of F(ab′)2 (Zagreb Institute of Immunology) antibody fragments, to neutralize the hemorrhagic activity of Vipera berus snake venom in mice, was compared. First, the neutralizing potency was determined by in vitro preincubation of venom and antivenom, followed by intradermal injection into mice and subsequent measurement of the hemorrhagic area. Both antivenoms had the same anti-hemorrhagic potency, in...
Herrera; Maria; de O Collaco; Rita de Cassia; Villalta; Mauren; Segura; Alvaro; Vargas; Mariangela; Wright; Christine E.; Paiva; Owen K.; Matainaho; Teatulohi; Jensen; Simon D.; Leon; Guillermo; Williams; David J.; Rodrigues-Simioni; Lea; Maria Gutierrez; Jose
The neuromuscular junction activity of Oxyuranus scutellatus venom and its presynaptic neurotoxin, taipoxin, and their neutralization by two antivenoms were examined in mouse phrenic nerve-diaphragm preparations. The action of taipoxin was also studied at 21 degrees C. The efficacy of the antivenoms was also assessed in an in vivo mouse model. Both antivenoms were effective in neutralizing the neuromuscular blocking activity in preincubation-type experiments. In experiments involving independ...
Bouvier, J.F.; Charrie, A.; Fleury-Goyon, M.C.; Chauvot, P. et; Lahneche, B.E.
In 18 patients operated for malignant tumors 20 immunoscintigraphies were done with a monoclonal antibody cocktail (anti-CEA F(ab') 2 and anti-CA 19.9 F(ab') 2 ). Immediately before scintigraphy tumor marker titers in plasma were determined in all cases. Tumor marker levels corresponding to positive or doubtful scintigraphies are analysed. (Author)
Hammoudi-Triki, D.; Lefort, J.; Rougeot, C.; Robbe-Vincent, A.; Bon, C.; Laraba-Djebari, F.; Choumet, V.
This paper reports the simultaneous determination of toxicokinetic and toxicodynamic properties of Androctonus australis hector venom, in the absence and presence of antivenom (F(ab') 2 and Fab), in envenomed rats. After subcutaneous injection of the venom, toxins showed a complete absorption phase from the site of injection associated with a distribution into a large extravascular compartment. The injection of Fab and F(ab') 2 induced the neutralization of venom antigens in the blood compartment, as well as the redistribution of venom components from the extravascular compartment to the blood compartment. Interestingly, F(ab') 2 and Fab showed distinct efficiencies depending on their route of injection. F(ab') 2 induced a faster venom neutralization and redistribution than Fab when injected intravenously. Fab was more effective than F(ab') 2 by the intramuscular route. The hemodynamic effects of Aah venom were further investigated. Changes in mean arterial pressure and heart rate were observed in parallel with an upper airway obstruction. Fab was more effective than F(ab') 2 for preventing early symptoms of envenomation, whatever their route of administration. Intraperitoneal injection of F(ab') 2 and Fab was similar for the prevention of the delayed symptoms, even after a late administration. Fab was more effective than F(ab') 2 in the inhibition of airway resistance, independent of the route and time of administration. These results show that the treatment for scorpion stings might be improved by the intravascular injection of a mixture of Fab and F(ab') 2 . If antivenom cannot be administered intravenously, Fab might be an alternative as they are more effective than F(ab') 2 when injected intramuscularly
Chatal, J.F.; Fumoleau, P.; Saccavini, J.C.
In a first, retrospective study, 15 patients with known ovarian carcinoma were injected with 131 I-OC 125 F(ab')2 monoclonal antibody (MAb). The sensitivity of immunoscintigraphy based on the number of the tumor sites was 67% (12/18). In a second, prospective study, 29 patients with gynecologic carcinoma were injected with 131 I-OC 125 F(ab')2 (24) or 131 I-19-9 F(ab')2 (5) MAbs according to the histologic type. Based on the number of tested anatomic sites, sensitivity was 72% and specificity 86%. In two patients injected with both 131 I-OC-125 F(ab')2 and 125 I-NS F(ab')2 (nonspecific immunoglobulin) 1 and 4 days before tumor resection, tumor uptake of the specific antibody was 2.2 and 4.5 times greater than that of NS. Immunoscintigraphic results were complementary with those of ultrasonography and computed tomography. Finally, in one patient injected successively with 131 I-OC 125 F(ab')2 and 111 In-DTPA-OC 125 F(ab')2, the recurrent tumor was visualized with both radionuclides, with 111 In providing better abdominal tumor contrast but causing much greater liver radioactivity than 131 I
Aida, Y.; Ochiai, K.; Ito, K.; Onuma, M.; Fujimori, F.; Fujimoto, Y.; Izawa, H.
Mouse monoclonal antibody c 143 was purified and F(ab')2 fragments were generated by pepsin digestion and then radiolabeled with 125 I. The 125 I-labeled c 143 F(ab')2 fragments were injected into athymic mice bearing bovine lymphoid tumor cells. The fragments became preferentially localized in tumor tissues, but not in normal tissues, as determined by differential counting of tissue radioactivity. The fragments became localized specifically in those tumors that were reactive with c 143 in vitro, but did not become localized in unrelated tumors. Localization of labeled F(ab')2 fragments of a monoclonal antibody of the same isotype directed against Taka virus (a variant of Newcastle disease virus) was not observed in athymic mice bearing bovine lymphoid tumor cells. Tumors were detectable by radioimmunoscintigraphy, using radiolabeled c 143 F(ab')2 fragments, without background subtraction, and by use of silver-grain scattering in light microscopic autoradiography
Full Text Available Objective: Any erythrocyte transfusion among humans having type A or B blood groups is impossible due to antibodies causing fatal transfusion complications. A cross-match test is performed to prevent immune transfusion complications before transfusion. Our hypothesis is that the fragment antibody (Fab part of the antibody (incomplete antibody may be used to prevent an immune stimulus related to the complete antibody. Therefore, we designed a pilot study to evaluate the effectiveness of these incomplete antibodies using cross-match tests. Materials and Methods: Pepsin enzyme and staphylococcal protein A columns were used to cut anti-A and anti-B monoclonal antibodies and purify their Fab (2 fragments, respectively. An Rh-positive erythrocyte suspension with purified anti-A Fab (2 solution and B Rh-positive erythrocyte suspension with purified anti-B Fab (2 solution were combined correspondingly. Cross-match tests were performed by tube and gel centrifugation methods. The agglutination levels due to the anti-A and anti-B Fab (2 antibodies and their effects on the agglutination normally observed with complete antibodies were then measured. Results: No agglutination for the purified incomplete anti-A Fab (2 with A Rh+ erythrocyte and anti-B Fab (2 with B Rh+ erythrocyte combinations was observed in the tube cross-match tests. These agglutination levels were 1+ in two wells in the gel centrifugation cross-match tests. Fab (2-treated erythrocytes were also resistant to the agglutination that normally occurs with complete antibodies. Conclusion: We determined that the Fab (2 fragments of antibodies may not only be used to obtain a mild or negative reaction when compared to complete antibodies, but they might also be used for decreasing ABO incompatibility. Incomplete antibodies might be a therapeutic option in autoimmune hemolytic anemia and they may also be used in solid organ or hematopoietic stem cell transplantation. Therefore, we have planned an
Raweerith, Rutai; Ratanabanangkoon, Kavi
A combined process of caprylic acid (CA) precipitation and ion-exchange chromatography on SP-Sepharose was studied as a means to fractionate pepsin-digested horse antivenom F(ab')(2) antibody. In the CA precipitation, the optimal concentration for fractionation of F(ab')(2) from pepsin-digested horse plasma was 2%, in which 89.61% of F(ab')(2) antibody activity was recovered in the supernatant with 1.5-fold purification. A significant amount of pepsin was not precipitated and remained active under these conditions. An analytical cation exchanger Protein-Pak SP 8HR HPLC column was tested to establish optimal conditions for the effective separation of IgG, albumin, pepsin and CA from the F(ab')(2) product. From these results, the supernatant from CA precipitation of pepsin-digested plasma was subjected to a SP-Sepharose column chromatography using a linear salt gradient. With stepwise elution, a peak containing F(ab')(2) antibody could be obtained by elution with 0.25 M NaCl. The total recovery of antibody was 65.56% with 2.91-fold purification, which was higher than that achieved by ammonium sulfate precipitation. This process simultaneously and effectively removed residual pepsin, high molecular weight aggregates and CA in the final F(ab')(2) product, and should be suitable for large-scale fractionation of therapeutic equine antivenoms.
Buchegger, F.; Halpern, S.E.; Sutherland, R.M.; Schreyer, M.; Mach, J.P.; Rochester Univ., NY
Colon carcinoma multicellular spheroids were incubated in vitro with radiolabelled MAbs. The more rapid penetration of fragments as compared to intact MAbs was clearly demonstrated. For the study of antibody localization in tumors in vivo, the model of nude mice with ligated kidneys was used. Although very artificial, this model allowed to demonstrate that, without urinary excretion, Fab fragments accumulated more rapidly into the tumor than intact MAbs and disappeared faster from the blood. This difference was less striking for F(ab') 2 fragments. In the liver a decreased accumulation of both types of fragments as compared to intact MAbs was observed. Concerning radio-immunotherapy we think that Fab fragments are not useful because of their too short half-life the circulation and in tumor and because they will probably be too toxic for the kidneys. Intact MAbs and F(ab') 2 fragments have each their advantages. Intact MAbs show highest tumor accumulation in mice without ligated kidney, however, they remain mostly on the periphery of tumor nodules, as shown by autoradiography. F(ab') 2 fragments have been found to penetrate deeper into the tumor and to accumulate less in the liver. It might be therefore an advantage to combine intact MAbs with F(ab') 2 fragments, so that in the tumor two different regions could be attacked whereas in normal tissues toxicity could be distributed to different organs such as to the liver with intact MAbs and to the kidney with F(ab') 2 fragments. (orig.) [de
Møller, NPH; Christiansen, Gunna
with either rabbit anti-KLH IgG or anti-KLH F(ab')2 fragments. The Fc-Fc interactions were investigated by reacting these surface-adsorbed antibody-rich KLH immune complexes with soluble, antigen-rich ferritin-anti-ferritin complexes using either rabbit anti-ferritin IgG or the corresponding isomolar F(ab')2...... fragments as antibody. Fc-Fc interactions were indicated by the formation of clusters or ring structures of ferritin molecules, which were only seen when using KLH anti-KLH IgG and ferritin-anti-ferritin IgG complexes. When F(ab')2 fragments were used as antibody, no reaction between KLH anti-KLH complexes...
Thedrez, P.; Paineau, J.; Jacques, Y.; Chatal, J.F.; Pelegrin, A.; Bouchaud, C.; Soulillou, J.P.
Anti-interleukin-2 receptor monoclonal antibodies have been shown to prevent allograft rejection. This paper reports on the biodistribution of a mouse MoAb directed at the 55 Kd alpha chain of rat interleukin-2 receptor (IL2-R) during allograft rejection. Only a low percentage (approximately 1%) of intact 125I-labeled MoAb was detected in the rejected graft, and irrelevant control IgG1 was found at a similar level. This suggests that most of the injected intact MoAb bound to graft tissue via its monomorphic Fc segment. In contrast, OX39 F(ab')2 fragments showed a preferential localization in the rejected allograft and did not bind to the LEW-to-LEW syngeneic heart graft. Irrelevant F(ab')2 did not concentrate in the allogeneic graft. Accordingly, F(ab')2 fragments from OX39 or irrelevant MoAb were used for gamma-scintigraphy on allograft recipients together with biodistribution studies. Results show that scintigraphy was able to detect allograft accumulation of 131I OX39 F(ab')2, whereas no imaging was obtained when OX39 F(ab')2 was used in the syngeneic combination or when irrelevant 131-IgG1 F(ab')2 was given to allograft recipients. This method, applied to the clinical situation, could be of interest for detection of early graft rejection episodes by immunoscintigraphy using reagents specific for activation determinants on lymphocyte membranes, such as anti-interleukin-2 receptor MoAb
Carrel, F.; Novak-Hofer, I.; Ruch, C.; Zimmermann, K.; Amstutz, H.
The in vitro and in vivo behaviour of the monovalent single chain (scFv) and Fab-fragments derived from anti-neuroblastoma antibody chCE7 is reported. When comparing tumour uptake and -retention of radioactivity of 67 Cu-labelled monovalent chCE7 with divalent chCE7 F(ab') 2 the advantage of the radiocopper label over the radioiodine label was more pronounced with the divalent (internalising) F(ab') 2 fragments. (author) 1 fig., 1 ref
Elgqvist, Jörgen; Andersson, Håkan; Haglund, Elin
The aim of this study was to investigate the therapeutic efficacy of the alpha-radioimmunotherapy of ovarian cancer in mice, using different specific activities. This study was performed by using the monoclonal antibody, MX35 F(ab')(2), labeled with the alpha-particle-emitter, 211At.......The aim of this study was to investigate the therapeutic efficacy of the alpha-radioimmunotherapy of ovarian cancer in mice, using different specific activities. This study was performed by using the monoclonal antibody, MX35 F(ab')(2), labeled with the alpha-particle-emitter, 211At....
Moon, Dae Hyuk; Chung, June Key; Lee, Myu ng Chul; Koh, Chang Soon; Chung, Hong Keun; Park, Jae Gahb
This study was designed to evaluate a direct method of 99m Tc labeling using β-mercaptoethanol as a reducing agent, and to investigate whether 99m Tc labeled specific monoclonal antibody against carcinoembryonic antigen (CEA-92) can be used for the scintigraphic localization of human colon cancer xenograft. Purified CEA-92 IgG was fragmented into F(ab') 2 and then labeled with 99m Tc by transchelation method using glucarate as a chelator. Labeling efficiency, immunological reactivity and in vitro stability of 99m Tc CEA-92 F(ab') 2 were measured and then injected intravenously into nude mice bearing human colon cancer (SNU-C4). Scintigrams were obtained at 24 hour after injection. Then nude mice were sacrificed and the radioactivity was measured. Labeling efficiency of injected 99m Tc CEA-92 F(ab') 2 , immunoreactive fraction and in vitro stability at 24 hour of injected 99m Tc CEA-92 F(ab') 2 was 45.2%, 32.8% and 57.4%, respectively. At 24 hour after injection, %ID/g in kidney (46.77) showed high uptake, but %ID/g in tumor (1.65) was significantly higher than spleen (0.69), muscle (0.16), intestine (0.45), stomach (0.75), heart (0.48) and blood(0.45). There was no significant difference between tumor and liver (1.81). Tumor contrast as quantitated by tumor to blood ratio of 99m Tc CEA-92 F(ab') 2 was increased significantly (p 131 I-CEA-92 F(ab') 2 . The scintigram demonstrated localization of radioactivity over transplanted tumor, but significant background radioactivity was also noted over kidney and abdomen. It is concluded that CEA-92 F(ab') 2 can be labeled with 99m Tc by a direct transchelation method using β-mercaptoethanol as a reducing agent and 99m Tc labeled CEA-92 F(ab') 2 can be used for the scintigraphic localization of human colon cancer xenograft in nude mice model.
Engelberts, Patrick J.; Voorhorst, Marleen; Schuurman, Janine; van Meerten, Tom; Bakker, Joost M.; Vink, Tom; Mackus, Wendy J. M.; Breij, Esther C. W.; Derer, Stefanie; Valerius, Thomas; van de Winkel, Jan G. J.; Parren, Paul W. H. I.; Beurskens, Frank J.
Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity (CDC) of CD20(+) B cells by binding of C1 to hexamerized Fc domains. Unexpectedly, we found that type I CD20 Ab F(ab ')2 fragments, as well as C1q-binding-deficient IgG mutants,
Molema, G; Tervaert, JWC; Kroesen, BJ; Helfrich, W; Meijer, DKF; de Leij, LFMH
Bispecific antibody (BsMAb) BIS-1 has been developed to redirect the cytolytic activity of cytotoxic T lymphocytes (CTL) to epithelial glycoprotein-2 (EGP-2) expressing tumour cells; intravenous administration of BIS-1 F(ab')(2) to carcinoma patients in a phase I/II clinical trial, caused
Molthoff, C. F.; Buist, M. R.; Kenemans, P.; Pinedo, H. M.; Boven, E.
Monoclonal antibody (Mab) MOv18 preferentially reacts with gynecological carcinomas. We have analyzed the characteristics of murine MOv18 (m-MOv18) and chimeric MOv18 (c-MOv18). We found no differences in affinity and binding to IGROV1 cells between c-MOv18 as IgG and F(ab')2 fragments and m-MOv18.
Morrison, R.T.; Lyster, D.M.; Szasz, I.; Alcorn, L.N.; Rhodes, B.A.; Breslow, K.; Burchiel, S.W.
The authors have recently evaluated technetium 99 labeled antibodies specific to human chorionic gonadotropin (hCG) for the in vivo detection of a variety of human tumors. Both mouse monoclonal and sheep polyclonal antibodies were evaluated in this study. Another antibody specific to hCG studied, is an antigen-agglutinating monoclonal F(ab') 2 fragment. Some preliminary results are reported
Full Text Available Several radiolabeled monoclonal antibodies (mAbs have been used as radioimmunotherapy (RIT agents for cancer therapy. The use of mAbs as RIT agents is due to their ability to carry effectors, in the form of radionuclides which emit alpha (α particles, beta (β particles, or auger electrons, and bind specifically to cancer expressed receptor. This paper reports the preparation of radiolabelled trastuzumab in form of (177Lu-DOTAm-PAMAM G3-F(ab'2-trastuzumab, which will be expected as a potential RIT agent for therapy of breast cancer overexpressed human epidermal growth factor receptor 2 (HER2. Due to its reduced molecular weight, the use of F(ab'2-trastuzumab on the aforementioned RIT agent candidate is expected to reach its target much faster compared to the intact trastuzumab. Meanwhile, the role of PAMAM G3 is to increase the specific activity of the radiotherapeutic agent of Lu-177 due to the ability of its 32 –NH2 functional groups that are able to bind many DOTAs (£ 31 which in turn can bind a large number of 177Lu. The preparation was initiated by fragmentation of trastuzumab using pepsin enzyme in 0.02 M acetic acid buffer with a pH of 4.5 to produce F(ab'2-trastuzumab with a purity of 95 % after purification with PD-10 column. The F(ab'2-trastuzumab was then reacted with succinimidyl 4-(N-maleimidomethyl cyclohexane-1-carboxylate (SMCC to produce SMCC-F(ab'2-trastuzumab. The next reaction was to conjugate SMCC-F(ab'2-trastuzumab with DOTA-PAMAM G3.0-SH, which was prepared by reaction NHS-DOTA with PAMAM G3.0 and followed by reacting it with 2-iminothiolane to give (DOTAm-PAMAM G3.0-F(ab'2-trastuzumab. Finally, the (DOTAm-PAMAM G3.0-F(ab'2-trastuzumab was radiolabelled with 177Lu to produce (177Lu-DOTAm-PAMAM G3.0-F(ab'2-trastuzumab, resulting in a radiochemical purity of 98 % after purification with PD-10 column.Received: 31 October 2015; Revised: 30 June 2016; Accepted: 25 September 2016
Burchiel, S. W.; Crockford, D. R.; Rhodes, B. A.
F(ab') 2 or Fab fragments of antibodies to: (a) human chorionic gonadotropin (hCG), hCG alpha subunit, hCG beta subunit, or an hCG-like material; or (b) other tumor specific or tumor associated molecules, to include carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), human melanoma associated antigens, human sarcoma associated antigens or other antigens, are radiolabeled with technetium-99m (Tc-99m). When the F(ab') 2 or Fab fragments of antibody to such tumor associated antigens are injected intravenously into a patient, the radiolabeled composition accumulates at tumor sites. The accumulation of the cancer seeking radiopharmaceutical at tumor sites permits detection by external gamma scintigraphy. Thus, the composition is useful in the monitoring, localization and detection of cancer in the body. In an alternative composition, a double antibody approach to tumor localization using radiolabeled F(ab') 2 or Fab fragments is utilized. In this approach, a tumor specific antibody in the form of IgG, F(ab') 2 or Fab is first administered to a patient intravenously. Following a sufficient period of time, a second antibody in the form of F(ab') 2 or Fab is administered. The second antibody is radiolabeled with Tc-99m and has the property that it is reactive with the first antibody. This double antibody method has the advantage over a single antibody approach in that smaller tumors can be localized and detected and that the total amount of radioactive trace localized at the cancer site is increased
Chen, Zi-Ru; Kuang, Lu; Gao, Yi-Qun; Wang, Ya-Ling; Salt, David E.; Chao, Dai-Yin
Zinc (Zn) is an essential element for plant growth and development, and Zn derived from crop plants in the diet is also important for human health. Here, we report that genetic variation in Heavy Metal-ATPase 4 (HMA4) controls natural variation in leaf Zn content. Investigation of the natural variation in leaf Zn content in a world-wide collection of 349 Arabidopsis thaliana wild collected accessions identified two accessions, Van-0 and Fab-2, which accumulate significantly lower Zn when compared with Col-0. Both quantitative trait loci (QTL) analysis and bulked segregant analysis (BSA) identified HMA4 as a strong candidate accounting for this variation in leaf Zn concentration. Genetic complementation experiments confirmed this hypothesis. Sequence analysis revealed that a 1-bp deletion in the third exon of HMA4 from Fab-2 is responsible for the lose of function of HMA4 driving the low Zn observed in Fab-2. Unlike in Fab-2 polymorphisms in the promoter region were found to be responsible for the weak function of HMA4 in Van-0. This is supported by both an expression analysis of HMA4 in Van-0 and through a series of T-DNA insertion mutants which generate truncated HMA4 promoters in the Col-0 background. In addition, we also observed that Fab-2, Van-0 and the hma4-2 null mutant in the Col-0 background show enhanced resistance to a combination of high Zn and high Cd in the growth medium, raising the possibility that variation at HMA4 may play a role in environmental adaptation. PMID:29545819
Chen, Zi-Ru; Kuang, Lu; Gao, Yi-Qun; Wang, Ya-Ling; Salt, David E; Chao, Dai-Yin
Zinc (Zn) is an essential element for plant growth and development, and Zn derived from crop plants in the diet is also important for human health. Here, we report that genetic variation in Heavy Metal-ATPase 4 ( HMA4 ) controls natural variation in leaf Zn content. Investigation of the natural variation in leaf Zn content in a world-wide collection of 349 Arabidopsis thaliana wild collected accessions identified two accessions, Van-0 and Fab-2, which accumulate significantly lower Zn when compared with Col-0. Both quantitative trait loci (QTL) analysis and bulked segregant analysis (BSA) identified HMA4 as a strong candidate accounting for this variation in leaf Zn concentration. Genetic complementation experiments confirmed this hypothesis. Sequence analysis revealed that a 1-bp deletion in the third exon of HMA4 from Fab-2 is responsible for the lose of function of HMA4 driving the low Zn observed in Fab-2. Unlike in Fab-2 polymorphisms in the promoter region were found to be responsible for the weak function of HMA4 in Van-0. This is supported by both an expression analysis of HMA4 in Van-0 and through a series of T-DNA insertion mutants which generate truncated HMA4 promoters in the Col-0 background. In addition, we also observed that Fab-2, Van-0 and the hma4-2 null mutant in the Col-0 background show enhanced resistance to a combination of high Zn and high Cd in the growth medium, raising the possibility that variation at HMA4 may play a role in environmental adaptation.
Walker, K.Z.; Seymour-Munn, K.; Axiak, S.M.; Raison, R.L.; Basten, A.; Towson, J.E.; Bautovitch, G.J.; Morris, J.
Monoclonal antibody (MoAb) fragments are known to have advantages over intact immunoglobulins for radioimmunoscintigraphy. It is less clear whether they are as effective in the delivery of radioimmunotherapy. The imaging and dosimetric properties of an intact MoAb, K-1-21, reactive against human kappa light chains (LC) were compared with that of its F(ab') 2 and Fab fragments using a normal rat model system. Two days after injection of 131 I-K-1-21 into rats bearing antigen-sepharose implants, gamma camera images showed specific localization of the MoAb to the target (kappa LC) but not to the control (lambda LC) implant. Better images were obtained with K-1-21 F(ab') 2 than with Fab or intact antibody. Mean kappa implant: blood ratios were 8.6 ± 3.9 for Fab, 7.9 ± 1.8 for F(ab') 2 and 2.0 ± 0.3 for intact K-1-21. The improvement associated with the use of 131 I-K-1-21 fragments was, however, achieved at the expense of lower absolute values of activity at the target site. Thus the absorbed dose delivered to the implant by the intact K-1-21 was double that delivered with F(ab') 2 and six times that delivered with Fab. As intact K-1-21 also delivered a greater radiation dose to normal tissues, F(ab') 2 fragments may have the greatest overall advantages for therapy with radionuclide MoAb conjugates. (author)
Korimbocus, Jehanara; Dehay, Nicolas; Tordo, Noël; Cano, François; Morgeaux, Sylvie
In case of a bite by a rabies infected animal, the World Health Organisation recommends a prophylactic treatment including the administration of Human Rabies Immunoglobulins (HRIGs) or highly purified F(ab')2 fragments produced from Equine Rabies Immunoglobulin (F(ab')2 - ERIGs). According to international regulation, quality control of F(ab')2 - ERIGs lots requires potency testing by the in vivo Mouse Neutralisation Test (MNT) prior marketing. However, the strategy of the 3Rs (Reduce, Refine, Replace) for animal testing required by the European Directive encourages the replacement of the in vivo potency test by an in vitro assay. In this context, a competitive ELISA method (c-ELISA) has been developed by the Agence Nationale de Sécurité du Médicament et des Produits de Santé where F(ab')2 - ERIGs are in competition with a monoclonal antibody recognizing the trimeric native form of the rabies glycoprotein. After a full validation study, the c-ELISA has been applied to commercial batches of F(ab')2 - ERIGs. A correlation study with the MNT demonstrated a similarity between the two methods (r=0.751). Moreover, the c-ELISA method which does not need any species specific reagent has been applied to HRIGs potency testing as an alternative method to Rapid Fluorescent Focus Inhibition Test (RFFIT), thus avoiding the handling of live rabies virus in BSL3 containment. In conclusion, the c-ELISA has shown its potential to replace MNT and possibly RFFIT for the quantification of rabies immunoglobulin. After optimisation it may be used for the quantification of rabies immunoglobulin in any animal species, notably for rabies immunogenicity assay in mice. Copyright © 2016 Elsevier Ltd. All rights reserved.
Griffiths, G.L.; Jones, A.L.; Hansen, H.J.; Goldenberg, D.M.
Intact IgG and Fab' can be labeled directly with 99m Tc to give quantitative incorporation of radioactivity into the protein. With F(ab') 2 the reductive conditions yield a mixture of 99m Tc-F(ab') 2 and 99m Tc-Fab'. We now report a direct labeling method to produce only 99m Tc-F(ab') 2 in quantitative yield and contaminated with 99m Tc-Fab'. The properties, stability and biodistribution of the 99m Tc-F(ab') 2 have been compared to 99m Tc-Fab'. This new technology will allow us to compare technetium direct-labeled IgG, F(ab') 2 and Fab' derivatives of the same antibody for radioimmunodetection. (author)
Smailes, Sarah T; Engelsman, Kayleen; Dziewulski, Peter
Determining the discharge outcome of burn patients can be challenging and therefore a validated objective measure of functional independence would assist with this process. We developed the Functional Assessment for Burns (FAB) score to measure burn patients' functional independence. FAB scores were taken on discharge from ICU (FAB 1) and on discharge from inpatient burn care (FAB 2) in 56 patients meeting the American Burn Association criteria for major burn. We retrospectively analysed prospectively collected data to measure the progress of patients' physical functional outcomes and to evaluate the predictive validity of the FAB score for discharge outcome. Mean age was 38.6 years and median burn size 35%. Significant improvements were made in the physical functional outcomes between FAB 1 and FAB 2 scores (pburn patients. Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.
Baatrup, G; Svehag, S E
The binding of fibronectin to human Clq, C3b, and complement-reacted immune complexes (IC) was investigated by enzyme-linked immunosorbent assays. Microplates were coated with BSA followed by incubation with rabbit-anti-BSA IgG or F(ab')2 fragments of rabbit anti-BSA. Incubation of the solid phase...... with serum at 37 degrees C caused attachment of Clq and C3b. Addition of EDTA to the serum inhibited the binding of C3b, but not Clq, whereas substitution of the anti-BSA IgG on the solid phase with the F(ab')2 fragments abrogated the Clq, but not the C3b binding. Fibronectin binding was observed after...
Garg, P.K.; Garg, S.; Zalutsky, M.R.
N-Succinimidyl 4-methyl-3-(tri-n-butylstannyl)benzoate (MATE) was synthesized in two steps from 4-methyl-3-iodobenzoic acid. Radioiododestannylation of MATE proceeded more slowly than N-succinimidyl 3-(tri-n-butylstannyl)benzoate (ATE), but for reaction periods of 10 min, identical yields were obtained. Paired-label biodistribution studies were performed in mice with an intact monoclonal antibody and an F(ab') 2 fragment labeled using MATE, ATE and Iodogen. Thyroid uptake with MATE was low, comparable to that seen with ATE, and considerably lower than that observed when the Iodogen method was used. With the F(ab') 2 fragment, kidney uptake using MATE was 8-fold higher than that observed when either the ATE or Iodogen methods were used. (Author)
Cardoso, D.S.; Nunan, E.A.; Toledo, V.P.C.P.; Moraes-Santos, T.; Cardoso, V.N.
F(ab') 2 is the fragment involved in the immunotherapy for scorpion stings and it would be convenient to label it with 99m Tc for organ distribution and pharmacokinetics studies. The aim of the present study was to label scorpion antivenom F(ab') 2 with 99m Tc keeping its biological activity, integrity and stability. High labeling yield was obtained using stannous chloride and sodium borohydride. Stability, immunoreactivity and integrity of 99m Tc-F(ab') 2 was preserved. It was not observed any difference between potencies of unlabeled and labeled antivenom. 99m Tc-F(ab') 2 can be a useful tool for use in biodistribution and pharmacokinetics studies on the evaluation of the efficacy of the antivenom against scorpion envenomation. (author)
Mar 8, 2018 ... Sci. (2018) 127:29. Figure 3. Field photographs of different facies types in facies associations FA-B1 and FA-B2. (a) Large channel-fill cross- stratified sandstone facies (B1A). Person sitting in the photo is for scale. (b) Trough cross-stratified, coarse-grained sandstone facies (B1B). Length of the pen is 15cm.
Jang, Dae Hwan; Choi, Duck Joo; Lee, Bum Woo; Park, Won; Han, Chang Soon; Kim, Hak San; Kim, Chong Soon
The cocktails of two 131 I labeled Monoclonal antibody (MCAB) (Anti CA 19-9 F(ab') 2 +Anti CEA F(ab') 2 fragment), which react specially, with human gastrointestinal cancers, were administered to 10 patients with colorectal (7), stomach (2) and pancreas (l) cancer for scintigraphic detection. All patients were known or postoperatively recurrent cases, and serum tumor markers, CA 19-9 and CEA, were measured with immunoradiometric assay, just before immunoscintigraphy (ISG). The tumor marker's level in serum is not correlated with positive tumor uptake in ISG. The sensitivity and specificity of ISG in detection of 21 tumor sites, based on surgery, CT, ultrasonography and pathology, were 90.5% and 100%. One case of colon cancer showed gall bladder metastasis, which was neglected on CT study. Tumor/non tumor uptake ratio of radiolabelled antibody were progressively increased from day 3 to day 7 during study. We summarized as follows: 1) The use of cocktails of CEA and CA 19-9 MCAB F(ab') 2 increased sensitivity and specificity in ISG. 2) Delayed imaging (later than 5 days) increases sensitivity and specificity due to exclusion of nonspecific iodine accumulation in stomach and lung. 3) Second tracer technique is essential for anatomical landmark by use of a double isotope scan, but subtraction technique, a possible source of artifacts, is no longer necessary when delayed imaging is performed. 4) It may be possible to use two MCAB cocktails of CA 19-9 and CEA in Radioimmunodetection of stomach and pancreas cancer. In conclusion, ISG using MCAB cocktails, F(ab') 2 fragment of anti CA 19-9 and Anti CEA, provide additional opportunity for tumor localization and detection of colorectal and other G-I cancer, such as stomach and pancreas.
F(ab) 2 fragments and horseradish peroxidase (HRP)-conjugated sheep anti-M13 antibodies were 2.5. Preparation of phage displayed, soluble and...this regard, the association between HLA-DR antigen and estrogen and progesterone receptor expression in breast carcinoma lesions suggests a role for...class I and class II antigens in primary breast carcinomas and synchronous nodal metastases. Clin Exp Metastasis. 1995, 13:43-48. 33. Cabrera T
BUIST, MR; KENEMANS, P; VANKAMP, GJ; Haisma, Hidde
The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab')(2) (n = 28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18; n = 24, 3
R.D. Haryuni; A. Bahtiar; S. Soenarjo; Y. Harahap; A. Mutalib; M. Ramli; S. Hermanto; C.N. Ardiyatno; V.Y. Susilo; D. Haffid
Nimotuzumab is an anticancer agent which belongs to the inhibitor group of Epidermal Growth Factor Receptor (EGFR). Thismonoclonal antibody has a relatively high molecular weight which slowspenetration on tumor cells, making it less attractive in imaging kinetics and potentially elicits antibodies responses. Therefore, in this study nimotuzumab was fragmented to form a bivalent antibody [F(ab')2] and then labeled with 125I to form 125I-F(ab')2-nimotuzumab which can be used further as a precur...
Buist, M. R.; Kenemans, P.; van Kamp, G. J.; Haisma, H. J.
The human anti-(mouse Ig) antibody (HAMA) response was measured in serum of 52 patients suspected of having ovarian carcinoma who had received an i.v. injection of either the murine monoclonal antibody (mAb) OV-TL 3 F(ab')2 (n = 28, 1 mg) or the chimeric mouse/human mAb MOv18 (cMOv18; n = 24, 3 mg).
Levy, N.J.; Kasper, D.L.
The role of surface-bound type Ia group B Streptococcus (GBS) capsular polysaccharide in anti-body-independent binding of C1 and activation of the classic component pathway was investigated. In a radiolabeled bacterial-polymorphonuclear leukocyte (PMN) association assay, a measure of bacterial opsonization, preincubation of 3 H-type Ia GBS with purified F(ab') 2 to the organism blocked the association of the bacteria with PMN', and the inhibitory effect was dose dependent. The specificity of F(ab') 2 blocking was shown after adsorption of F(ab') 2 with type Ia polysaccharide-sensitized erythrocytes. Polysaccharide-adsorbed F(ab') 2 had a 70% decrease in ability to block the association of bacteria with PMN. Neuraminidase digestion removed 80% of the terminal sialic acid residues from the native polysaccharide. These neuraminidase-digested organisms had a 72% decrease in binding and transfer of purified C1 compared with non-enzyme-treated organisms. Type Ia capsular polysaccharide bound to sheep erythrocytes promoted classic complement pathway-mediated hemolysis of the cells. The role of C1 inhibitor (INH) in modulation of C1 activation by the organisms was investigated. The possibility existed that the C1 INH could be bound by the bacteria, allowing C1 activation to occur in the fluid phase. The inhibitor was purified from human serum, and its activity was measured before and after incubation with type Ia GBS. The organisms had no effect on C1 INH activity. Thus surface-bound capsular polysacchardie of type Ia GBS mediates C1 binding and classic pathway activation, and this does not involve the C1 INH
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been observed with Crotalidae polyvalent immune Fab therapy after crotaline snake envenomation.9 Additionally, fasciotomy, amputation, or other...purified, equine -derived F(ab)2 fragments and are able to treat a variety of the most commonly encountered vipers and elapids (including cobras) in the...antivenom therapy for severe crotaline snakebite. Ann Emerg Med. 2011;57:128–137. Heiner et al416
Full Text Available Snake bite is a common medical emergency in Papua New Guinea (PNG. The taipan, Oxyuranus scutellatus, inflicts a large number of bites that, in the absence of antivenom therapy, result in high mortality. Parenteral administration of antivenoms manufactured in Australia is the current treatment of choice for these envenomings. However, the price of these products is high and has increased over the last 25 years; consequently the country can no longer afford all the antivenom it needs. This situation prompted an international collaborative project aimed at generating a new, low-cost antivenom against O. scutellatus for PNG.A new monospecific equine whole IgG antivenom, obtained by caprylic acid fractionation of plasma, was prepared by immunising horses with the venom of O. scutellatus from PNG. This antivenom was compared with the currently used F(ab'(2 monospecific taipan antivenom manufactured by CSL Limited, Australia. The comparison included physicochemical properties and the preclinical assessment of the neutralisation of lethal neurotoxicity and the myotoxic, coagulant and phospholipase A(2 activities of the venom of O. scutellatus from PNG. The F(ab'(2 antivenom had a higher protein concentration than whole IgG antivenom. Both antivenoms effectively neutralised, and had similar potency, against the lethal neurotoxic effect (both by intraperitoneal and intravenous routes of injection, myotoxicity, and phospholipase A(2 activity of O. scutellatus venom. However, the whole IgG antivenom showed a higher potency than the F(ab'(2 antivenom in the neutralisation of the coagulant activity of O. scutellatus venom from PNG.The new whole IgG taipan antivenom described in this study compares favourably with the currently used F(ab'(2 antivenom, both in terms of physicochemical characteristics and neutralising potency. Therefore, it should be considered as a promising low-cost candidate for the treatment of envenomings by O. scutellatus in PNG, and is ready to be tested in clinical trials.
Fand, Irwin; Sharkey, R.M.; Grundy, J.P.; Goldenberg, D.M.
In this report, we have employed macroautoradiography to compare the tumor targeting of 125 I-labeled anti-carcinoembryonic antigen (CEA) MAb (NP-4) to 125 I-labeled anti-colon-specific antigen-p (CSAp) MAb (Mu-9) and their labeled F(ab') 2 and Fab' fragments, in nude mice each bearing large dorsal human colonic tumor xenografts, and small nodular tumors in the liver and lungs. Using intact MAbs (NP-4 and Mu-9), clearance of background radioactivity was delayed to 3-7 days post-treatment. Treatment with F(ab') 2 and Fab' fragments of both NP-4 and Mu-9 MAbs, however, promoted clearance of background 125 I-radioactivity which was well advanced by 6-24 h and complete by 24-48 h after injection. Localization of 125 I-radioactivity in large and micrometastatic tumor perimeters was the most characteristic uptake pattern observed for both intact and fragmented MAbs. Qualitative analysis of macroautoradiographic images and quantitative densitometry indicated that the higher tumor-to-blood ratios achieved with labeled F(ab') 2 and Fab' fragments at early time points, compared to labeled whole immunoglobulin, appeared to be more a function of rapid plasma clearance, tumor mass, location of xenografts and specific tumor growth patterns than increased tumor penetrance by lower molecular weight univalent and bivalent immune fragments. (Author)
Elgqvist, Jörgen; Andersson, Håkan; Jensen, Holger
The aim of this study was to investigate the therapeutic efficacy of alpha-radioimmunotherapy of ovarian cancer in mice using different fractionated treatment regimens. The study was performed using the monoclonal antibody MX35 F(ab')(2) labeled with the alpha-particle emitter (211)At. Methods....... Nude mice were intraperitoneally inoculated with ~1 x 10(7) cells of the cell line NIH:OVCAR-3. Four weeks later 6 groups of animals were given 400 kBq (211)At-MX35 F(ab')(2) as a single or as a repeated treatment of up to 6 times (n = 18 in each group). The fractionated treatments were given every...... seventh day. Control animals were treated with unlabeled MX35 F(ab')(2) (n = 12). Eight weeks posttreatment the animals were sacrificed and the presence of macro- and microscopic tumors and ascites was determined. Results. The tumor-free fractions (TFFs) of the animals, defined as the fraction of animals...
In this study, an IgG2a (KS1/4), a monoclonal antibody (MoAb) specific against a human lung adenocarcinoma (UCLA P-3) was successfully fragmented enzymatically to yield F(ab') 2 and Fab by using pepsin and papain, respectively. The kinetic of fragmentation of the MoAb was compared to that of human immunoglobulin G (IgG). A similar pattern of fragmentation was observed with both antibodies with a higher percentage yield of the F(ab') 2 and Fab obtained upon the fragmentation of the IgG by the enzymes. The KS1/4 and the two fragments were labeled with three different radionuclides, namely iodine-131, indium-111 and selenium-75. The radioiodination of the MoAb and the fragments was carried out by using a modified chloramine-T method. Radiometal labeling of the MoAb and the fragments with indium-111 was performed by using DTPA as a bifunctional chelating agent, while intrinsic labeling of the MoAb was done by culturing the hybridoma in the presence of 75 Se-methionine. The biodistribution of the radiolabeled MoAb, F(ab') 2 and Fab fragments were performed by injecting the preparations intravenously into nude mice bearing human lung adenocarcinoma
Cascinelli, N; Attili, A; Belli, F; Buraggi, G; Turrin, A; Gasparini, M; Terno, G; Vaglini, M
To investigate possible undesirable due to the intravenous administration of a reagent of a xenogenic nature (monoclonal antibody 225-28S) in man, a toxicologic study was carried out on 85 patients with metastatic cutaneous melanoma. Two reagents were tested in this study: purified monoclonal antybody (MoAb) 225-28S and its F(ab')2 fragment. Purified MoAb was labelled with /sup 131/I and F(ab')2 fragment with /sup 131/I, or /sup 123/I, or /sup 111/In or /sup 99/Tc. The quantity of MoAb or F(ab')2 injected ranged from 14 to 750 ..mu..g, and the specific activity from 37.0 to 2116.4 MBqmg. The total radioactivity injected varied from 25.9 to 891.7 MBqmg. In addition to a careful clinical examination, the following tests were done to monitor possible adverse effects: blood glucose, azotemia, RBC WBC, platelet count, serum creatine, creatine clearence, plasma electrolyte levels, serum proteins, albuminglobulin ratio, serum bilirubin, SGOT, SgPT, /sub ..gamma../GT, and CPK. These tests were done before the injection and on days 7 and 14. No patient experienced adverse general effects like fever, nausea, vomiting or allergic reaction. None of the afore mentioned hematometric and biochemical tests showed significant variations compared with the initial values. It is concluded that a single injection of these reagents at the dosages tested is completely atoxic.
Mladenov, B.; Peshev, N.
The results of labelled monoclonal antibodies (MoA) immunoscintigraphy in malignant tumors involving the gastrointestinal tract are presented. The obtained data have an essential practical bearing on the early diagnosis and radical treatment undertaken. Immunoscintigraphy is performed with Imacis-I ( 131 I, monoclonal antibody, 19-9 F(ab') 2 anti-CEA F(ab') 2 ) obtained from the CIS company, and Jodomab-R-2( 131 I, anti-CEA monoclonal antibody F(ab') 2 ) of the Sorin Biomedica Company, inserted at activity ranging from 11 to 185 MBq. Scanning by a planar gamma-camera is performed at 72 hours. A total of twenty-four patients are examined: 14 preoperatively (with gastric cancer - 2, pancreatic cancer - 1 and location of the neoplasm in different segments of the colon - 11), and ten postoperatively. Positive results are obtained in twenty-two (92 per cent) of the total number of patients under study. In twelve (86 per cent) of those examined preoperatively intensive accumulation of labelled autoantibodies in the cancer area is documented with a negative result recorded in two cases only. Metastases are found in two patients operated on, while in the remainder the results are negative and consistent with those of the other methods of examination. 13 refs., 4 figs. (author)
Full Text Available Antivenoms have been used successfully for more than a century and up to now constitute the only effectivetreatment for snakebites .The production of antivenin started long time ago when the calmette was preparedthe antivenom in 1894.The method currently used to prepare antivenom by most of the manufacturers areoriginated from the method of Pope which was develop in 1938. Several new approaches in the production ofantivenom have been proposed to produce IgG, F(ab2, F(ab antivenin to improve their quality .Theseimprovement include complete or partial modification in the antivenom production regarding animal,immunization protocols , new adjuvants in hyperimmunization of animals , purification processes ( caprylicacid ,chromatography , diafiltration and ulterafiltration ,enzymatic digestion of IgG (pepsin, papain andfractionation of venom .When the IgG is digested enzymatically, different fragments are obtained depending on the enzyme used, that is, if papain is used, three fragments are obtained, the crystallizing fragment (Fc and two antigen-binding fragments F(ab and, if pepsin is used, one F(ab'2 fragment is obtained, while thecrystallizing fragment is digested. Fab and F(ab2 fragments conserve their capacity to specifically bind to the antigen that gave rise to them.
Endo, K.; Kamma, H.; Ogata, T.
Monoclonal antibody (MAb) 15 and its F(ab')2 and Fab fragments were radioiodinated, and their biodistribution and imaging were compared in BALB/c nude mice bearing a xenograft of a human lung cancer (TKB-2). Association constants for 125I-labeled MAb 15 IgG, F(ab')2, and Fab were 1.9 X 10(9), 1.8 X 10(9), and 3.7 X 10(8) M-1, respectively. Immunoreactive fractions ranged from 0.59 to 0.50. Cultured TKB-2 cells expressed 1.1 X 10(4) binding sites/cell for MAb 15 IgG in vitro. The binding of a control antibody and the binding of its fragments to TKB-2 cells were less than 3% of the input doses. The mice with the TKB-2 tumors were given simultaneous injections of 10 microCi of 131I-labeled MAb 15 or its fragments and 10 microCi of 125I-labeled control IgG or its fragments. With MAb 15 IgG, the percentage of the injected dose bound per gram of tissue (ID/g) of the tumor was 3.68% at day 7, when the localization index (LI) was 4.38. At day 2 after MAb 15 F(ab')2 injection, 1.12% of the ID/g was localized in the tumor and the LI was 3.04. After MAb 15 Fab injection, the percentage of the ID/g of the tumor was 0.31% and the LI was 2.58 at day 1. MAb 15 IgG, F(ab')2, and Fab cleared from the blood early, with a half-life of 33, 16, and 9 hours, respectively. The distributions of MAb 15 and its fragments in the normal organs did not differ from those of the control. Radioimaging with 100 microCi of 131I-labeled MAb 15 and its fragments showed that 42%, 44%, and 32% of the total-body count were localized in the tumor with IgG at day 7, F(ab')2 at day 2, or Fab at day 1, respectively. Because the radioactivity remaining in the tumor with Fab was low, the image was insufficient. Throughout the period, less than 10% of the control IgG and its fragments remained in the tumor. Microautoradiography confirmed the binding of MAb 15 and its fragments to the tumor cells
Carvalho, Guilherme Luiz de Castro
In the acute myocardium infarction, the myocytes cell membrane loses its integrity, allowing the influx of extracellular macromolecules such as circulating antibody into the damaged cell. The use of the specific antibodies against cardiac myosin labeled with 99m Tc allows to determine the localization and extension of myocardial infarction. The purpose of this work was to study the viability of labeling of the antimyosin monoclonal antibody and its fragment F(ab')2 with 99m Tc. Because of the high cost of antimyosin antibody, others antibodies were used to optimize the methodology and the best condition was used for antimyosin antibody. The intact antibody was cleaved by pepsin to produce F(ab') 2 fragment. The F(ab') 2 and the intact antibody were reduced by treatment with Dithiothreitol (DTT) and 2-Mercaptoethanol (2-ME) and labeled with 99m Tc by direct method. Different concentrations of reductant, mixing conditions and incubation times were studied. In the standard condition, incubation at molar ratio 1:1000 (antibody:reducing agent) at room temperature for 30 minutes with continuous rotation (850 rpm), 13.28 - SH groups were formed per molecule. It was studied the influence of p H, of the concentration of stannous chloride (Sn 2+ ) and incubation time in the labeling condition. The better radiochemical yield (90.06 +- 1.53%) was obtained using 2.5 μg of Sn 2+ in p H 4.5 for 60 minutes. The labeling of the fragment F(ab') 2 did not present satisfactory results because of the low yield of the digestion. After purification by PD-10, the biodistribution study was performed and showed that the intact antimyosin antibody labeled with 99m Tc presented fast kinetic compatible with the biodistribution of an intact antibody labeled with 99m Tc. Scintigraphy image of the animal with myocardial infarction was obtained and compared with the image of a normal animal. The studies allow to conclude that the use of fragment F(ab') 2 are not viable, but the use of the labeled antimyosin antibody are promising. (author)
Nelson, H.; Ramsey, P.S.; Kerr, L.A.; McKean, D.J.; Donohue, J.H.
Anti-tumor antibody (317G5) covalently coupled to an anti-CD3 antibody (OKT3) produces a heteroaggregate (HA) antibody that can target PBL to lyse tumor cells expressing the appropriate tumor Ag. The i.v. and i.p. distribution of radiolabeled HA antibody 317G5 x OKT3 and of radiolabeled cultured human PBL were studied in athymic nude mice bearing solid intraperitoneal tumor established from the human colon tumor line, LS174T. Mice were injected with 125I-labeled HA antibody, 125I-labeled anti-tumor mAb, or 111In-labeled PBL, and at designated timepoints tissues were harvested and measured for radioactivity. 125I-317G5 x OKT3 localized specifically to tumor sites. Tumor radioactivity levels (percent injected dose/gram) were lower with 125I-317G5 x OKT3 HA antibody than with 125I-317G5 anti-tumor mAb, but were similar to levels reported for other anti-tumor mAb. The major difference in radioactivity levels observed between i.v. and i.p. administration of 125I-317G5 x OKT3 was an increase in hepatic radioactivity after i.v. HA antibody administration. HA antibodies produced from F(ab')2 fragments, which exhibit decreased m. w. and decreased Fc receptor-mediated binding, demonstrated improved tumor:tissue ratios as compared to intact antibody HA. 125I-317G5 F(ab')2 x OKT3 F(ab')2 antibody levels were equivalent to intact HA antibody levels in tumor, but were lower than intact HA antibody levels in the blood, bowel, and liver. Tumor:bowel ratios (20:1 at 48 h) were highest when 317G5 F(ab')2 x OKT3 F(ab')2 was injected i.p. Autoradiography confirmed that anti-tumor x anti-CD3 HA antibodies localized specifically to intraperitoneal tumor; that i.p. administered HA antibodies penetrated tumor directly; and that i.v. administered HA antibodies distributed along tumor vasculature
Fjeld, J.G.; Nustad, K.; Michaelsen, T.E.
The binding parameters of iodine-125-labelled intact monoclonal immunoglobulin G (IgG), F(ab') 2 and Fab' fragments were compared. The study was carried out with the two monoclonal antibodies (MoAbs) K13 and K16 specific for human Ig light chains κ and λ, respectively. When testing the 125 I-MoAbs against monodisperse polymer particles coated with the specific antigens, the K a for the F(ab') 2 fragments were similar to that for IgG, while the K a for the Fab' fragments were reduced to 10%-20% of that for IgG. The number N of effective target sites revealed with Fab' was higher than with F(ab') and IgG, presumably because less surface area is occupied by the small Fab' molecules. The immunoreactive fraction F ranged according to IgG>F(ab') 2 >Fab'. The explanation of the moderate difference between the K a of the monoclonal Fab' and the divalent IgG and F(ab') 2 was that the divalent molecules were not divalently attached to the particles. When testing the same antibody preparations against humanlymphoma cells producing Ig with light chains κ or λ, the binding results were less reliable than when particles were utilised, presumably due to antigen shedding. Different MoAbs vary in their loss of immunoreactivity due to enzymatic degradation and the radiolabelling procedure. The preparation of the radiolabelled fragments should therefore be optimized for each MoAb, and evaluation is necessary before injection. Artificial targets with a low leakage of antigen, like the monodisperse polymer particles here applied, are recommended for the in vitro evaluation of the immunoreactivity of labelled MoAb preparations. (orig.)
Wy Ching Ng
Full Text Available BACKGROUND: Despite the availability of specific vaccines and antiviral drugs, influenza continues to impose a heavy toll on human health worldwide. Passive transfer of specific antibody (Ab may provide a useful means of preventing or treating disease in unvaccinated individuals or those failing to adequately seroconvert, especially now that resistance to antiviral drugs is on the rise. However, preparation of appropriate Ab in large scale, quickly and on a yearly basis is viewed as a significant logistical hurdle for this approach to control seasonal influenza. METHODOLOGY/PRINCIPAL FINDINGS: In this study, bovine colostrum, which contains approximately 500 g of IgG per milking per animal, has been investigated as a source of polyclonal antibody for delivery to the respiratory tract. IgG and F(ab'2 were purified from the hyperimmune colostrum of cows vaccinated with influenza A/Puerto Rico/8/34 (PR8 vaccine and were shown to have high hemagglutination-inhibitory and virus-neutralizing titers. In BALB/c mice, a single administration of either IgG or F(ab'2 could prevent the establishment of infection with a sublethal dose of PR8 virus when given as early as 7 days prior to exposure to virus. Pre-treated mice also survived an otherwise lethal dose of virus, the IgG- but not the F(ab'2-treated mice showing no weight loss. Successful reduction of established infection with this highly virulent virus was also observed with a single treatment 24 hr after virus exposure. CONCLUSIONS/SIGNIFICANCE: These data suggest that a novel and commercially-scalable technique for preparing Ab from hyperimmune bovine colostrum could allow production of a valuable substitute for antiviral drugs to control influenza with the advantage of eliminating the need for daily administration.
Li Jun; Wang Shizhen; Yang Ziyi
Direct labelling method and conditions of monomeric antibody Fab' with 99m Tc were investigated. Polyclonal antibody IgG was digested with ficin to produce dimeric fragments F(ab') 2 , which was subsequently reduced to monomeric fragments Fab' with 2-mercaptoethylamine. Finally, Fab' was incubated with sodium gluconate (Sn(II)) kit solution and 99m TcO 4 - eluted at room temperature to form 99m Tc-Fab'. The labelling efficiency was 85%-95%. The stability of labelled products was satisfactory and the elimination rate was faster than 99m Tc-IgG
Zolfagharian H.; Mohammadpour Dounighi, N.
Antivenoms have been used successfully for more than a century and up to now constitute the only effectivetreatment for snakebites .The production of antivenin started long time ago when the calmette was preparedthe antivenom in 1894.The method currently used to prepare antivenom by most of the manufacturers areoriginated from the method of Pope which was develop in 1938. Several new approaches in the production ofantivenom have been proposed to produce IgG, F(ab)2, F(ab) antivenin to improve...
Hansen, J E; Sørensen, A M; Arendrup, M
Monoclonal mouse IgG3 antibody (ABL 364) against the carbohydrate Le(y) antigen enhanced infection in vitro with HTLV-1 and with HIV-1 when propagated in both transformed and normal lymphocytes. Enhancement was independent of complement, occurred with both lymphocytes and monocytes as target cells...... with no indication of any alternative pathway of infection, as evidenced by abrogation of enhancement by anti-CD4 MAb or soluble recombinant CD4, and also the inability of anti-Le(y) MAb to mediate HIV infection of HSB-2 cells in which HTLV-1/HIV pseudovirus infection was enhanced. While F(ab)2 fragments of ABL 364...
Benichou, G.; Dormont, D.; Herodin, F.; Pasquier, C.; Hopital Saint Antoine, 75 - Paris
In resting cells, for low radiation doses, a transient activation of chemiluminescence was observed, maximal at 3 Gy. At 10 Gy, CL returned to the control value; greater doses (above 30 Gy) induced a progressive diminution of the response which was abolished at 100 Gy. Activated alveolar macrophages showed a 30% decrease of the chemiluminescence at 10 Gy. The respiratory burst induced by opsonized zymosan was abolished at 30 Gy. IgG anti-MHC(IgGαB 1 ) activated specifically the GP S2 alveolar macrophages by alloantibody bipolar bridging; by contrast IgG which are directed against non-specific allogeneic determinants (IgG α B 3 ) or specific F(ab') 2 (F(ab') 2 αB 1 ) are unable to stimulate the cells. For all the tested doses, irradiation had no effect on this activation mechanism. The results with the three doses tested (3 Gy, 10 Gy, 30 Gy) are comparable to those using the positive control cells. The same results are obtained with the class II antigens and their specific IgG. (UK)
Liu, P C; Chen, C A; Chen, C M; Yen, C H; Lee, M H; Chuang, C K; Tu, C F; Su, B L
The clinical feasibility of passive immunotherapy has not been demonstrated in dogs naturally infected with canine distemper. In this study, porcine anti-canine distemper virus IgG and F(ab') 2 antibody fragments were used to treat infected puppies. A total of 41 naturally infected puppies (age Äsix months) exhibiting severe respiratory signs, but lacking neurological signs, were enrolled in the study. Twenty-five puppies were treated with a combination of IgG or F(ab') 2 antibody fragments (Group 1) and supportive therapy and 16 puppies received routine supportive care only (Group 2). The survival rate of dogs in Group 1 (19/25; 76%) was significantly higher than that in Group 2 (5/16; 31·3%) (Pdistemper virus antibodies improved survival in puppies affected with canine distemper with minimal adverse effects. Therefore, this therapy could be considered for treatment of endangered animal species infected with canine distemper virus. © 2016 British Small Animal Veterinary Association.
Immunization of Guinea pigs with diphtheria toxoid generated antibodies of the IgG class that were capable of neutralizing native toxin in vivo. Sera from these animals were used to affinity purify idiotypic antibodies (AB1). AB1 vaccines derived from the IgG1 class and from F(ab') 2 of IgG1 + IgG2 (IgG1/2) classes were effective in inducing a syngeneic anti-idiotype (AB2) response. Animals immunized with AB1 consisting of both IgG1/2 did not elicit a detectable AB2 response. Binding of homologous 125 I-F(ab') 2 (AB1) to the antiidiotype was inhibited 90% in the presence of DT.F(ab') 2 derived from preimmune serum or had no inhibitory effects on the idiotype-antiidiotype interactions. Two groups of outbred guinea pigs were vaccinated with alum absorbed F(ab') 2 of anti-idiotype IgG1/2 (AB2). Of the ten animals inoculated with AB2, three tested positive by RIA against 125 I-DT. Two of the RIA positive sera contained antibodies that neutralized diphtheria toxin in a rabbit intracutaneous assay. Purification of guinea pig IgG by protein A-Sepharose affinity chromatography resulted in the separation of three distinct IgG populations
Le Doussal, J.M.; Gruaz-Guyon, A.; Martin, M.; Gautherot, E.; Delaage, M.; Barbet, J.
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
Full Text Available Proton beam irradiation is a form of advanced radiotherapy providing superior distributions of a low LET radiation dose relative to that of photon therapy for the treatment of cancer. Even though this clinical treatment has been developing for several decades, the proton radiobiology critical to the optimization of proton radiotherapy is far from being understood. Proteomic changes were analyzed in human melanoma cells treated with a sublethal dose (3 Gy of proton beam irradiation. The results were compared with untreated cells. Two-dimensional electrophoresis was performed with mass spectrometry to identify the proteins. At the dose of 3 Gy a minimal slowdown in proliferation rate was seen, as well as some DNA damage. After allowing time for damage repair, the proteomic analysis was performed. In total 17 protein levels were found to significantly (more than 1.5 times change: 4 downregulated and 13 upregulated. Functionally, they represent four categories: (i DNA repair and RNA regulation (VCP, MVP, STRAP, FAB-2, Lamine A/C, GAPDH, (ii cell survival and stress response (STRAP, MCM7, Annexin 7, MVP, Caprin-1, PDCD6, VCP, HSP70, (iii cell metabolism (TIM, GAPDH, VCP, and (iv cytoskeleton and motility (Moesin, Actinin 4, FAB-2, Vimentin, Annexin 7, Lamine A/C, Lamine B. A substantial decrease (2.3 x was seen in the level of vimentin, a marker of epithelial to mesenchymal transition and the metastatic properties of melanoma.
Carrel, Francois; Amstutz, Hanspeter; Novak-Hofer, Ilse; Schubiger, P. August
Monovalent fragments of antineuroblastoma antibody mAb chCE7 were evaluated for their in vitro and in vivo tumor cell binding properties. Single chain fragments were constructed from the variable region genes cloned from hybridoma cells, expressed in E.coli and purified by metal chelate affinity chromatography. Radioiodinated CE7-scFv fragments were found to bind with high affinity (K d ∼10 -9 M) to target cells in vitro but formed aggregates at 37 deg. C, and bound to serum proteins in vitro and in vivo. Circular Dichroism spectra revealed the protein to be in a conformationally altered form and no permanent 'refolding' could be achieved. In contrast, chCE7-Fab fragments were found to bind to target tumor cells with similar affinity than the parent mAb chCE7 (K d ∼10 -10 M), showed no tendency to aggregate and were stable in serum both in vitro and in vivo. Kinetics of association and dissociation of radioiodinated scFv and Fab fragments were found to be rapid. Radioiodination with the Iodogen method led to impaired immunoreactivity which was found to further increase the off- rates of radioiodinated fragments from tumor cells. Radioiodination with the Bolton-Hunter reagent as well as labeling of chCE7-Fab fragments with 67 Cu via the macrocyclic CPTA ligand led to fully immunoreactive Fab fragments. Radioiodinated and radiocopper labeled monovalent CE7 fragments did not internalize into target tumor cells as the parent mAb and its F(ab') 2 fragment. A comparison of the biodistribution in tumor bearing nude mice of the radiocopper labeled monovalent, non internalizing Fab fragments with the internalizing divalent F(ab') 2 fragments showed in both cases high levels of radioactivity in the kidneys. Concerning tumor uptake, radioactivity from both internalizing and non internalizing fragments remained associated with tumor tissue for longer times than in case of the corresponding radioiodinated fragments. When compared with the radioiodinated forms, tumor uptake of radiocopper-labeled 67 Cu-chCE7 and its F(ab') 2 fragments was found to be higher. However in the case of the non internalizing 67 Cu-chCE7-Fab fragment no increase in the absolute amount of radioactivity in tumor tissue compared with the radioiodinated Fab was observed, indicating an advantage of using radiocopper labeling in conjunction with internalizing antibody fragments for delivering high doses of radioactivity to neuroblastoma
Welch, Nicholas G; Madiona, Robert M T; Payten, Thomas B; Easton, Christopher D; Pontes-Braz, Luisa; Brack, Narelle; Scoble, Judith A; Muir, Benjamin W; Pigram, Paul J
Antibody orientation at solid phase interfaces plays a critical role in the sensitive detection of biomolecules during immunoassays. Correctly oriented antibodies with solution-facing antigen binding regions have improved antigen capture as compared to their randomly oriented counterparts. Direct characterization of oriented proteins with surface analysis methods still remains a challenge however surface sensitive techniques such as Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) provide information-rich data that can be used to probe antibody orientation. Diethylene glycol dimethyl ether plasma polymers (DGpp) functionalized with chromium (DGpp+Cr) have improved immunoassay performance that is indicative of preferential antibody orientation. Herein, ToF-SIMS data from proteolytic fragments of anti-EGFR antibody bound to DGpp and DGpp+Cr are used to construct artificial neural network (ANN) and principal component analysis (PCA) models indicative of correctly oriented systems. Whole antibody samples (IgG) test against each of the models indicated preferential antibody orientation on DGpp+Cr. Cross-reference between ANN and PCA models yield 20 mass fragments associated with F(ab') 2 region representing correct orientation, and 23 mass fragments associated with the Fc region representing incorrect orientation. Mass fragments were then compared to amino acid fragments and amino acid composition in F(ab') 2 and Fc regions. A ratio of the sum of the ToF-SIMS ion intensities from the F(ab') 2 fragments to the Fc fragments demonstrated a 50% increase in intensity for IgG on DGpp+Cr as compared to DGpp. The systematic data analysis methodology employed herein offers a new approach for the investigation of antibody orientation applicable to a range of substrates. Controlled orientation of antibodies at solid phases is critical for maximizing antigen detection in biosensors and immunoassays. Surface-sensitive techniques (such as ToF-SIMS), capable of direct characterization of surface immobilized and oriented antibodies, are under-utilized in current practice. Selection of a small number of mass fragments for analysis, typically pertaining to amino acids, is commonplace in literature, leaving the majority of the information-rich spectra unanalyzed. The novelty of this work is the utilization of a comprehensive, unbiased mass fragment list and the employment of principal component analysis (PCA) and artificial neural network (ANN) models in a unique methodology to prove antibody orientation. This methodology is of significant and broad interest to the scientific community as it is applicable to a range of substrates and allows for direct, label-free characterization of surface bound proteins. Copyright © 2017 Acta Materialia Inc. All rights reserved.
Kate, C.I.; Kroonenburgh, M.J. van; Schipperheyn, J.J.; Doornbos, J.; Hoedemaeker, P.J.; Maes, A.; Nat, K.H. van der; Camps, J.A.; Huysmans, H.A.; Pauwels, E.K.
Indium-111 antimyosin F(ab')2 was used in a series of scintigraphic studies on experimentally induced myocardial infarctions in pigs. Antimyosin distribution recorded by planar images of in vivo pigs and by single photon emission computed tomography (SPECT) of excised hearts delineated areas of myocardial necrosis if infarct volume exceeded 3.3 cm3. Scintigraphic images were compared with magnetic resonance images (MRI) obtained from excised hearts and with photographs of slices of the hearts. Infarct size and localization determined with antimyosin were compared. The MR images, with or without gadolinium-DTPA (Gd-DTPA), of the in vivo pigs were all false-negative; some myocardial wall thinning and high bloodpool signals were visible. Results show that both the antimyosin and the MR technique are specific methods for the visualization of induced myocardial necrosis in this animal model. However, the use of antimyosin is limited to a period ranging from 24 to 72 hours after infarction
Cuff, C F; Rogers, C M; Lamb, B J; Rogers, T J
Normal splenocytes cultured with Formalin-killed Candida albicans were shown to acquire significant suppressor cell activity in a period of 3 days. These cells were found to suppress both the phytohemagglutinin-induced mitogen response as well as the anti-sheep erythrocyte antibody response. Experiments were carried out to determine the nature of the suppressor cell population. Results showed that these cells were not susceptible to treatment with anti-Thy 1 antibody and complement. Panning experiments showed that the suppressor cells were not plastic-adherent or Mac-1 antigen-positive. The suppressor cells were, however, adherent to anti-mouse immunoglobulin (F(ab')2-fragment)-coated dishes. Additional experiments showed that the suppressor cell activity was susceptible to treatment with monoclonal anti-Lyb 2.1 antibody and complement. These results suggest that the suppressor cell induced in vitro by Candida is a member of the B-lymphocyte lineage.
Kávai, M; Rasmussen, J M; Baatrup, G
, but the binding was low (2-3%) when compared to the binding of the corresponding IgG-IC (50-60%). Solid phase IC were prepared by coating microwells with heat-aggregated bovine serum albumin (BSA) followed by incubation with rabbit IgM anti-BSA antibody. The IC were reacted with human serum at 37 degrees C....... The binding of C3b-iC3b was determined by use of biotinylated F(ab')2 antibodies to C3b-C3c and avidin-coupled alkaline phosphatase. The incorporation of C3b-iC3b into solid-phase IgM-IC increased when increasing amounts of IgM antibody were reacted with the antigen. The binding reaction was slow, reaching...
Rieber, E.P.; Linke, R.P.; Riethmueller, G.; Heyden, H.W. von; Waller, H.D.
Using 125 I-labelled aggregated IgG in a quantitative assay a strong expression of Fc-receptors was found on the leukemic cells of a patient with hairy cell leukemia. The Fc-receptor activity on these cells was much higher than that on monocytes and B-lymphocytes from normal blood. Surface immunoglobulins were detected by radioautography using radioactively labelled (Fab') 2 -fragments of monospecific antibodies directed against immunoglobulin heavy chains. Prior to radioautography the cells were stained for the tartrate resistant acid phosphatase. It is found that all cells containing this enzyme bore delta-chains on their surface. On more than 90% of these cells a simultaneous expression of μ-chains was detected. γ-chains could only be demonstrated on cells which were negative for the tartrate resistant acid phosphatase; part of these cells, however, were hairy cells by morphological criteria. (orig.) [de
Janina Jamasbi, RPh
Full Text Available To enhance the antithrombotic properties of recombinant glycoprotein VI fragment crystallizable (GPVI-Fc, the authors incubated GPVI-Fc with anti-human Fc antibodies to cross-link the Fc tails of GPVI-Fc. Cross-linking potentiated the inhibition of human plaque- and collagen-induced platelet aggregation by GPVI-Fc under static and flow conditions without increasing bleeding time in vitro. Cross-linking with anti-human-Fc Fab2 was even superior to anti-human-Fc immunoglobulin G (IgG. Advanced optical imaging revealed a continuous sheath-like coverage of collagen fibers by cross-linked GPVI-Fc complexes. Cross-linking of GPVI into oligomeric complexes provides a new, highly effective, and probably safe antithrombotic treatment as it suppresses platelet GPVI-plaque interaction selectively at the site of acute atherothrombosis.
Aarden, Lucien; Ruuls, Sigrid R; Wolbink, Gertjan
To date, millions of people have been treated with therapeutic monoclonal antibodies (TmAbs) for various indications. It is becoming increasingly clear that TmAbs can be immunogenic, which may reduce efficacy or induce adverse effects. Over the years, the importance of antibody formation has been questioned and sometimes minimized, as few antibody responses to TmAbs (HACA or HAHA) were reported. However, the methods to detect and quantify such antibodies used in the past have been problematic. Only recently, methods have been developed that have adequate sensitivity and are not seriously disturbed by false-positive reactions caused by rheumatoid factors, natural antibodies to Fab or F(ab')2 fragments, or Fc interactions of IgG4. The large number of treated patients, in combination with these new assays, presents a unique opportunity to study the anti-antibody immune response in man, possibly allowing us to manipulate immunogenicity in the future.
The biodistribution and tumour uptake of radiolabelled (131 I) glioma-seeking monoclonal antibodies (14 AC1) and their F(ab') 2 fragments were investigated in nude mice having received glioma transplants. Radioimmunoimaging by external scintigraphy at 48 and 96 hours pointed to a superior tumour localisation by the fragments that was clearly related to the dose. Wholebody determinations of the biokinetic behaviour led to the following results: Faster clearance anc more ready elimination from the blood pool for the fragments, preferential uptake in the tumour; intact antibodies; binding in the liver, spleen and lungs. The study confirmed the value of fragments of monoclonal antibodies in the diagnosis of tumours and pointed to the possibility of using intact monoclonal antibodies as carriers of radioisotopes and cytotoxic drugs within the scope of therapeutic programmes. (TRV) [de
X. Li; A.M. Kriegel; T.C. Bishop; R.C. Blake; E. Figueiredo; H. Yu; D.A. Blake
The goal of our project is to continue the development of new techniques for rapid, automated identification of radionuclides, metals, and chelators that may contaminant sur face and groundwater at DOE sites. One of the four specific aims of the present project is to develop new technologies in antibody engineering that will enhance our immunosensor program. Recombinant antibodies have potential advantages over monoclonal antibodies produced by standard hybridoma technology. The cloned genes represent a stable, recoverable source for antibody production. In addition, the recombinant format offers opportunities for protein engineering that enhances antibody performance and for studies that relate antibody sequence to binding activity. In this study, a hybridoma that synthesized an antibody (12F6) that recognized a 1:1 complex between 2,9-dicarboxyl-1,10- phenanthroline (DCP) and UO 2 2+ was used as a source of RNA for the development of a recombinant (Fab) 2 fragment. RNA was isolated from the 12F6 hybridoma and the cDNA encoding the entire κ light chain and the linked VH and C1 portions of the heavy chain were amplified from total RNA. cDNA sequences were verified by comparison with the N-terminal amino acid sequences of the light and heavy chains of the native 12F6 monoclonal antibody. A leader sequence and appropriate restriction sites were added to each chain, and the fragments were ligated into a commercial dicistronic vector (pBudCE4.1, Invitrogen, Inc.). COS-1 cells were transfected with this vector and the culture supernatant was assayed for activity and the (Fab) 2 protein. Cells transfected with vector containing 12F6 cDNA synthesized and secreted recombinant (Fab) 2 fragments that bound to the UO 2 2+ -DCP complex with an affinity indistinguishable from that of a (Fab) 2 fragment prepared from the native antibody. Molecular models of the heavy and light chain variable domains were constructed according to the canonical structures method detailed by Morea
Kirley, Terence L; Greis, Kenneth D; Norman, Andrew B
Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab') 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab') 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. Copyright © 2016 Elsevier Inc. All rights reserved.
Bhattacharya, Biplab; Bhattacharjee, Joyeeta; Bandyopadhyay, Sandip; Banerjee, Sudipto; Adhikari, Kalyan
The present research is an attempt to assess the Barakar Formation of the Raniganj Gondwana Basin, India, in the frame of fluvio-marine (estuarine) depositional systems using sequence stratigraphic elements. Analysis of predominant facies associations signify deposition in three sub-environments: (i) a river-dominated bay-head delta zone in the inner estuary, with transition from braided fluvial channels (FA-B1) to tide-affected meandering fluvial channels and flood plains (FA-B2) in the basal part of the succession; (ii) a mixed energy central basin zone, which consists of transitional fluvio-tidal channels (FA-B2), tidal flats, associated with tidal channels and bars (FA-B3) in the middle-upper part of the succession; and (iii) a wave-dominated outer estuary (coastal) zone (FA-B4 with FA-B3) in the upper part of the succession. Stacked progradational (P1, P2)-retrogradational (R1, R2) successions attest to one major base level fluctuation, leading to distinct transgressive-regressive (T-R) cycles with development of initial falling stage systems tract (FSST), followed by lowstand systems tract (LST) and successive transgressive systems tracts (TST-1 and TST-2). Shift in the depositional regime from regressive to transgressive estuarine system in the early Permian Barakar Formation is attributed to change in accommodation space caused by mutual interactions of (i) base level fluctuations in response to climatic amelioration and (ii) basinal tectonisms (exhumation/sagging) related to post-glacial isostatic adjustments in the riftogenic Gondwana basins.
Harris, David A; Zhao, Yunge; LaPar, Damien J; Emaminia, Abbas; Steidle, John F; Stoler, Mark; Linden, Joel; Kron, Irving L; Lau, Christine L
Fibrocytes are integral in the development of fibroproliferative disease after lung transplantation. Undifferentiated fibrocytes (CD45+anti-collagen 1+CXCR4+) preferentially traffic by way of the CXCR4/CXCL12 axis and differentiate into smooth muscle actin-producing (CD45+CXCR4+α-smooth muscle actin+) cells. We postulated that an antibody directed against CXCL12 would attenuate fibrocyte migration and fibro-obliteration of heterotopic tracheal transplant allografts. A total alloantigenic mismatch murine heterotopic tracheal transplant model of obliterative bronchiolitis was used. The mice were treated with either goat-anti-human CXCL12 F(ab')(2) or goat IgG F(ab')(2). Buffy coat, bone marrow, and trachea allografts were collected and analyzed using flow cytometry. Tracheal luminal obliteration was assessed using hematoxylin-eosin and Direct Red 80 collagen stain. Compared with the controls, the anti-CXCL12-treated mice showed a significant decrease in tracheal allograft fibrocyte populations at 7 and 21 days after transplantation. Bone marrow and buffy coat aspirates showed the same trend at 7 days. In the anti-CXCL12-treated mice, there was a 35% decrease in luminal obliteration at 21 days (65% vs 100% obliterated; interquartile range, 38% vs 10%; P = .010) and decreased luminal collagen deposition at 21 and 28 days after transplantation (P = .042 and P = .012, respectively). Understanding the role of fibrocytes in airway fibrosis after lung transplantation could lead to a paradigm shift in treatment strategy. Anti-CXCL12 antibody afforded protection against infiltrating fibrocytes and reduced the deterioration of the tracheal allografts. Thus, the CXCR4/CXCL12 axis is a novel target for the treatment of fibro-obliteration after lung transplantation, and the quantification of fibrocyte populations could provide clinicians with a biomarker of fibrosis, allowing individualized drug therapy. Copyright © 2013 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.
Walker, K.Z.; Milner, L.J.; Boniface, G.R.
The detection of thrombi in rabbits has been investigated with 131 I-labelled DD-3B6/22, a monoclonal antibody (Mab) reactive at high affinity (Kd=2.68x10 -10 M) with human D Dimer (DD). DD-3B6/22 bound well to both 'fresh' and 'aged' human clots in an in vitro assay but showed poor binding to rabbit clots. However, reactivity was restored to rabbit blood if it was seeded, before clotting, with human DD covalently coupled to Sepharose beads. Thus, a rabbit model was developed in which blood was allowed to clot around DD-Sepharose beads introduced into the jugular vein. Gamma camera imaging showed that intact 131 I-labelled DD-3B6/22 localised to these clots within 24 h. Uptake at this time was 0.202%±0.012% injected dose per gram (%ID/g) compared with 0.086±0.018%ID/g after injection of control antibody. 131 I-labelled F(ab') 2 fragments of DD-3B6/22 allowed earlier scintigraphic detection of the clot which was evident 4 h after injection. Uptake in the clot at 24 h was 0.154±0.038% ID/g compared with 0.109±0.027% ID/g for a control F(ab') 2 . As antigen levels in the clot are estimated to be less than 300 μg DD, thus representing a very small human clot, the DD-3B6/22 Mab would appear to have a good potential for the sensitive detection of thrombi in a clinical setting. (orig.)
Sandrock, D.; Blossey, H.C.; Steinroeder, M.; Munz, D.L.
We compared three different scintigraphic techniques for the localization of neck recurrences and metastases in seven patients with medullary thyroid carcinoma one month to eight years after the first surgical intervention. Three successive scintigraphic studies were performed in five patients (6 x 3 studies) within two weeks using 201Tl chloride, 111In-labeled F(ab')2 fragments of the anti-carcinoembryonic antigen (anti-CEA) monoclonal antibody (MoAb) BW 431/31, and 131I meta-iodo-benzylguanidine (MIBG). Additionally, 11 studies were performed with the 111In-labeled MoAb fragment BW 431/31 (seven studies) or the 99mTc-labeled intact anti-CEA MoAb BW 431/26 (four studies). The gold standards for classifying scintigraphic results were biopsy, histology, surgery, and cytology. Six regions were classified as positive or negative in each study: thyroid region, four quadrants (lymph node regions) around the thyroid, and the region of the upper mediastinum. Of 36 sites, 201Tl was true positive (TP) in seven sites, false-positive (FP) in one site, true negative (TN) in 22 sites, and false-negative (FN) in six sites, resulting in a sensitivity of 54% and a specificity of 96%. 131I MIBG was TP in four sites, FP in none of the sites, TN in 23 sites, and FN in nine sites, with a sensitivity of 31% and a specificity of 100%. Immunoscintigraphy (102 sites overall) was TP in 16 sites, FP in five sites, TN in 77 sites, and FN in four sites, resulting in a sensitivity of 80% and a specificity of 94%. Immunoscintigraphy with 111In/99mTc anti-CEA F(ab')2 fragment/intact antibody is superior to scintigraphy with 201Tl and 131I MIBG
LaGraff, John R; Chu-LaGraff, Quynh
Unlabeled primary immunoglobulin G (IgG) antibodies and its F(ab')2 and Fc fragments were attached to oxygen-plasma-cleaned glass substrates using either microcontact printing (MCP) or physical adsorption during bath application from dilute solutions. Fluorescently labeled secondary IgGs were then bound to surface-immobilized IgG, and the relative surface coverage was determined by measuring the fluorescence intensity. Results indicated that the surface coverage of IgG increased with increasing protein solution concentration for both MCP and bath-applied IgG and that a greater concentration of IgG was transferred to a glass substrate using MCP than during physisorption during bath applications. Scanning force microscopy (SFM) showed that patterned MCP IgG monolayers were 5 nm in height, indicating that IgG molecules lie flat on the substrate. After incubation with a secondary IgG, the overall line thickness increased to around 15 nm, indicating that the secondary IgG was in a more vertical orientation with respect to the substrate. The surface roughness of these MCP patterned IgG bilayers as measured by SFM was observed to increase with increasing surface coverage. Physisorption of IgG to both unmodified patterned polydimethylsiloxane (PDMS) stamps and plasma-cleaned glass substrates was modeled by Langmuir adsorption kinetics yielding IgG binding constants of K(MCP) = 1.7(2) x 10(7) M(-1) and K(bath) = 7.8(7) x 10(5) M(-1), respectively. MCP experiments involving primary F(ab')2 and Fc fragments incubated in fluorescently labeled fragment-specific secondary IgGs were carried out to test for the function and orientation of IgG. Finally, possible origins of MCP stamping defects such as pits, pull outs, droplets, and reverse protein transfer are discussed.
Casey, Joanne Lois
Tumour targeting using radiolabelled antibodies for radioimmunodetection (RAID) and radioimmunotherapy (RIT) has been studied for many years. The main factors that have limited clinical success are low tumour uptake, immunogenicity and poor therapeutic ratios. This thesis has applied current technology to make advances in this area of research. The effect of physical parameters (antibody size, valency, affinity and charge) on the design of tumour targeting agents was studied by constructing divalent (DFM) and trivalent (TFM) forms of the murine anti-CEA antibody A5B7 Fab' by chemical cross-linking. This involves partial reduction of the hinge disulphides to expose thiol (-SH) groups and subsequent reaction with a maleimide cross-linker to form a thioether bond at the hinge region. Previous studies have suggested that the stability of thioether bonds is superior to naturally occurring disulphide bonds present at the hinge region of IgG and F(ab')2. The aim was to compare the functional affinities and in vivo tumour targeting in nude mice bearing human tumour xenografts of DFM and TFM to similar sized parent IgG and F(ab')2. Radiolabelling with 131I and 90Y was also compared with a view to determine which combination would be optimal for RIT. Results clearly demonstrated a significantly faster on-rate of DFM compared to all other antibody forms and estimated dosimetry analysis suggested that DFM would be the most suitable antibody form radiolabelled with 131I for RIT. Both F(ab')2 and DFM showed high kidney uptake levels on labelling with which is unacceptable for RIT. Despite the improved tumour: blood ratios for TFM, the increased estimated dose to normal tissues and lower therapeutic effect in RIT studies suggests that the most promising combination with the radionuclide appears to be IgG. A humanised version of A5B7 hFab' has been constructed previously in order to reduce its immunogenicity in man. The in vivo stability of hDFM proved to be superior to hF(ab')2 in the nude mouse xenograft model. To study the safety, stability and tumour targeting of hDFM a clinical trial using 131I was described here including details of production, characterisation, pharmacokinetics and dosimetry. ScFv's are known to have favourable tumour targeting characteristics compared to whole antibodies for RAID. To evaluate the clinical potential of a scFv, the methodology to prepare a phage derived scFv with the aid of a subcloned hexahistidine tail was described here. To enhance the clinical potential of scFv's a construct consisting of a hinge region containing a single cysteine residue was constructed. This enabled site-specific 99mTc-labelling and could facilitate multimerisation. One of the major limitations revealed by this and other studies is the problem associated with renal accretion of antibody fragments. Various modification techniques and blocking effects were used here in attempt to reduce the kidney uptake levels in mouse models. Reduction of the pi of A5B7 Fab by attachment of NHS-ester groups was effective in lowering kidney uptake levels, but losses in immunoreactivity could limit this approach. Attachment of PEG (5kD) to DFM did not adversely affect immunoreactivity and increased the circulation time of DFM in vivo. This has implications for reducing kidney uptake levels at early time points, in addition PEG is known to reduce immunogenicity of proteins.
Progress in three areas of research is summarized. These are as follows: Labeling Monoclonal antibodies (MAbs) with Tc-99m and Re-186; human melanoma tumors and specific MAbs; evaluation of biological response modifiers (BRM). The techniques of labeling MAbs (IgM, IgG, F(ab') 2 or F(ab')) with Tc-99m was developed in the author's laboratory in 1989 and that with Re-186 in 1992. The techniques are in daily use in the laboratory since then and are adapted to a convenient kit formulation. The metal ions are bound at MAb sulfhydryls generated by a controlled reduction of a pair of disulfide groups. At least two types of MAbs labeled with Tc-99m by this method have been administered into patients and excellent diagnostic results have been obtained. Over the past two and a half years the author has been successfully growing human melanoma tumors in athymic Balb/c nude mice. The cell LINE, WM-9, was obtained from Dr. D Herlyn's laboratory at Wistar Institute in Philadelphia. Sufficient quantities of antihuman melanoma specific antibodies ME 31.3 (Wistar, IgG-1) and MEM-136 (Hybritech, IgG-2A) and their F(ab') 2 fragments are also available in the laboratory. The use of BRM is a rapidly evolving field. Over the past four years, the author has evaluated a number of BRMs in a quest for agents that may augment MAb tumor uptake. These included interferon-α; a pokeweed mitogen and Ukrain, an alkaloid separated from a plant Chelideonium Majis. In these preliminary studies, normal Balb/c mice were used and the BRMs were given i.p. one hour prior to the i.v. administration of tumor necrosis factor or an MAb (TNT-F(ab') 2 ) labeled with Tc-99m which served as an imaging agent. Animals were sacrificed at 1.5 hr or 4 hrs post-injection. Highlights of the work are given here in a table
Kitamura, Naoto; Shigematsu, Naoyuki; Nakahara, Tadaki; Kanoh, Momoe; Hashimoto, Jun; Kunieda, Etsuo; Kubo, Atsushi
Immunoliposome (PEG, GAH, liposome; PGL), consisting of F(ab') 2 fragment of monoclonal antibody, GAH and polyethyleneglycol-coated (PEGylated) liposome was provided. Immunoliposome, PGL was labeled with technetium-99m (Tc-99m) by two methods: labeling F(ab') 2 fragment with Tc-99m; Tc-99m-PGL, and entrapping Tc-99m into liposome; PGL[Tc-99m]. The objective of this study was to compare the biodistribution of Tc-99m-PGL and PGL[Tc-99m] in human gastric cancer xenografted mice. Tc-99m-PGL, PGL[Tc-99m], and Tc-99m-entrapped liposome; Lipo[Tc-99m] were prepared. They were injected into human gastric cancer, MKN45, xenografted mice via the tail vein, and their biodistribution was studied. No marked accumulation of either PGL[Tc-99m] or Lipo[Tc-99m] was observed in the stomach. The uptake of Tc-99m-PGL by the liver, spleen, and lung was higher than that by the tumor. On the other hand, the uptake of PGL[Tc-99m] by the lung and spleen was markedly lower as compared with that of Tc-99m-PGL; the accumulation of PGL[Tc-99m] was lower in the lung and higher in the spleen as compared with that of the tumor. Although the liver uptake of PGL[Tc-99m] was markedly decreased as compared with that of Tc-99m-PGL, it was higher than the uptake of the tumor. The Tc-99m-PGL was strongly taken up by the tumor, with a high level of incorporation also seen in the stomach. These findings suggest the need for further study of the labeling stability. PGL[Tc-99m] appears to show promise for high tumor uptake and retention. This is an important implication for the potential application of immunoliposomes entrapped with Re-186, instead of Tc-99m, in internal radiotherapy. (author)
Ketut Karuni Nyanakumari Natih
Full Text Available The aim of this research was to prepare rabbit Immunoglobulin G as anti-idiotype antibody (Ab2 ofAvian Influenza Virus (AIV H5N1. A polyclonal antibody was collected from guinea pigs immunized withinactivated AI vaccine H5N1of Legok strain. Antibody of H5N1 AI in serum was detected by Agar gelprecipitation test (AGPT and an Inhibition Hemmaglutination test (IHT. The highest titre of antibodywas obtained one week after the third immunization. Serum of guinea pigs containing IgG was purifiedusing the Montage Antibody purification kit & spin column with Prosep A media (Millipore. The AI H5N1IgG concentration was 8 mg/ml. AI H5N1 IgG, was then digested with pepsin to obtain F(ab2 fraction andwas called Ab1. The concentration of IgG and F(ab2 and purity of IgG were determined by UVspectrophotometer which showed Ab1 concentration 1 mg/ml. Molecular weight was estimated by sodiumdodecyl sulfate- polyacrilamide gel electrophoresis (SDS-PAGE. Ab2 was produced by immunization ofrabbit with Ab1. The first immunization was carried out by subcutaneous injection with 500 ?g of Ab1emulsified in Complete Freund Adjuvant. The immunization was repeated with the same dose of Ab1emulsified in Incomplete Freund Adjuvan at 1 week intervals. One week after the second immunization,rabbit’s serum was harvested and IgG was purified using the Montage Antibody purification kit & spincolumn with Prosep A media (Millipore. The rabbit IgG, called Ab2, was an anti-idiotypic antibody againstAIV-H5N1. In AGPT, a precipitation line appeared between Ab1 and Ab2. A partial reaction appearedbetween Ab2 and the AI H5N1 antigen was also detected. The results indicated that Ab2 is a possiblecandidate of imunogen for protection against an AI virus H5N1 infection.
Full Text Available BACKGROUND: We posit that the same mononuclear phagocytes (MP that serve as target cells and vehicles for a host of microbial infections can be used to improve diagnostics and drug delivery. We also theorize that physical and biological processes such as particle shape, size, coating and opsonization that affect MP clearance of debris and microbes can be harnessed to facilitate uptake of nanoparticles (NP and tissue delivery. METHODS: Monocytes and monocyte-derived macrophages (MDM were used as vehicles of superparamagnetic iron oxide (SPIO NP and immunoglobulin (IgG or albumin coated SPIO for studies of uptake and distribution. IgG coated SPIO was synthesized by covalent linkage and uptake into monocytes and MDM investigated related to size, time, temperature, concentration, and coatings. SPIO and IgG SPIO were infused intravenously into naïve mice. T(2 measures using magnetic resonance imaging (MRI were used to monitor tissue distribution in animals. RESULTS: Oxidation of dextran on the SPIO surface generated reactive aldehyde groups and permitted covalent linkage to amino groups of murine and human IgG and F(ab'(2 fragments and for Alexa Fluor(R 488 hydroxylamine to form a Schiff base. This labile intermediate was immediately reduced with sodium cyanoborohydride in order to stabilize the NP conjugate. Optical density measurements of the oxidized IgG, F(ab'(2, and/or Alexa Fluor(R 488 SPIO demonstrated approximately 50% coupling yield. IgG-SPIO was found stable at 4 degrees C for a period of 1 month during which size and polydispersity index varied little from 175 nm and 200 nm, respectively. In vitro, NP accumulated readily within monocyte and MDM cytoplasm after IgG-SPIO exposure; whereas, the uptake of native SPIO in monocytes and MDM was 10-fold less. No changes in cell viability were noted for the SPIO-containing monocytes and MDM. Cell morphology was not changed as observed by transmission electron microscopy. Compared to unconjugated SPIO, intravenous injection of IgG-SPIO afforded enhanced and sustained lymphoid tissue distribution over 24 hours as demonstrated by MRI. CONCLUSIONS: Facilitated uptake of coated SPIO in monocytes and MDM was achieved. Uptake was linked to particle size and was time and concentration dependent. The ability of SPIO to be rapidly taken up and distributed into lymphoid tissues also demonstrates feasibility of macrophage-targeted nanoformulations for diagnostic and drug therapy.
Electrospray mass spectrometry (ESI-MS) has been used to examine a range of intact monoclonal antibodies (MAbs), antibody fragments such as F(ab') 2 , F ab and F c , chemically-modified fragments and a range of other chemically-modified peptides and proteins as part of a broader study aimed at establishing ESI-MS as a method for the characterisation of radioimmunoconjugates (radiolabelled monoclonal antibodies). For example, the addition of up to 10 biotin molecules to the 'papain-sensitive' 50 kDa F ab fragment can be easily detected in ESI mass spectra. For intact MAbs, however, it is only possible to detect average shifts in the mass of intact antibodies following modification. Successful ESI-MS analysis of complexes formed between chelators and other small molecules conjugated to synthetic peptides, hen egg-white Iysozyme (HEL) (M r 14 306) and horse heart myoglobin (M r 16 951) has been demonstrated. ESI-MS offers considerable advantages compared with existing methods for the characterisation of chemically-conjugated proteins including speed and sensitivity of analysis and the capability for obtaining specific structural information. The conditions for ESI-MS of intact MAbs and MAb fragments have been examined in detail and it was found that 150 kDa MAbs generally required lower sample concentration and higher skimmer potentials compared with the 50 kDa F ab fragment and other lower molecular weight proteins. In addition, the m/z range over which ions from MAbs were observed was higher (m/z ∼2000-4500) than for smaller proteins. ESI-MS was also found to be useful for probing the action of the protease papain, that is used to generate MAb fragments (F(ab) '2, F ab and F c ). Further, different sensitivities to papain for different MAb preparations was demonstrated. Finally, the tandem mass spectra of a range of peptides modified by iodine and biotin were examined. In the case of biotinylated peptides, a characteristic fragment ion was identified that could potentially be used to screen peptides produced from enzymatic preparations of modified proteins. 295 refs., tabs., figs
Merino, J; Qin, H Y; Schurmans, S; Gretener, D; Grau, G E; Lambert, P H
BALB/c mice rendered tolerant to alloantigens by neonatal injection of semi-allogeneic (C57BL/6 x BALB/c)F1 spleen cells develop a thrombocytopenia in association with an autoimmune lupus-like syndrome. The possible mechanisms involved in the thrombocytopenia were investigated. The development of thrombocytopenia was first detected at 3 weeks of age coinciding with the start of the other autoimmune manifestations and was always related to a state of tolerance and B cell chimerism. There was a significant increase of megakaryocytes in bone marrow and spleens from thrombocytopenic tolerant mice and radiolabeled platelets from these mice were more rapidly eliminated from the bloodstream than normal platelets when injected into normal recipients. A significant correlation between the spleen weight and the decrease of the circulating platelets was observed, although some mice with severe thrombocytopenia had only a moderate spleen enlargement. Thrombocytopenia significantly correlates with the levels of platelet-associated IgG (PAIgG) but not with anti-single-stranded DNA antibodies or circulating immune complexes. Platelets from mice with high levels of PAIgG had a shorter life-span when injected into normal mice than those from mice with low or normal PAIgG. The possibility that PAIgG are partially due to antibodies reacting specifically with platelet membrane components was analyzed. First, F(ab')2 Ig fragments from tolerant mice were shown to bind to normal platelets, in contrast to F(ab')2 Ig fragments from normal mice. Second, some monoclonal antibodies produced by hybridomas derived from tolerant mice reacted in vitro with platelets and induced a transient thrombocytopenia after i.v. injection into normal mice. These data suggest that the thrombocytopenia observed in tolerant mice is the result of a peripheral hyperdestruction of platelets associated with (a) hypersplenism, (b) nonspecific fixation of immunoglobulins, probably as immune complexes and (c) with autoantibodies reacting specifically with platelets. It may represent an interesting model for human chronic idiopathic thrombocytopenia.
Justin M Scheer
Full Text Available The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab'(2-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.
Alonso Martinez, L. M.; Xiques Castillo, A.; Leyva Montanna, R.; Perez-Malo Cruz, M.; Zamora Barrabi, M.; Manresa Sanchez, Y.
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)
Sjögren, Jonathan; Andersson, Linda; Mejàre, Malin; Olsson, Fredrik
Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.
Moldovan, Carmen; Mihailescu, Carmen; Stan, Dana; Ruta, Lavinia; Iosub, Rodica; Gavrila, Raluca; Purica, Munizer; Vasilica, Schiopu
This article presents the characterization of two substrates, silicon and polymer coated with gold, that are functionalized by mixed self-assembled monolayers (SAMs) in order to efficiently immobilize the anti-Escherichia coli O157:H7 polyclonal purified antibody. A biosurface functionalized by SAMs (self-assembled monolayers) technique has been developed. Immobilization of goat anti-E. coli O157:H7 antibody was performed by covalently bonding of thiolate mixed self-assembled monolayers (SAMs) realized on two substrates: polymer coated with gold and silicon coated with gold. The F(ab') 2 fragments of the antibodies have been used for eliminating nonspecific bindings between the Fc portions of antibodies and the Fc receptor on cells. The properties of the monolayers and the biofilm formatted with attached antibody molecules were analyzed at each step using infrared spectroscopy (FTIR-ATR), atomic force microscopy (AFM), scanning electron microscopy (SEM) and cyclic voltammetry (CV). In our study the gold-coated silicon substrates approach yielded the best results. These experimental results revealed the necessity to investigate each stage of the immobilization process taking into account in the same time the factors that influence the chemistry of the surface and the further interactions as well and also provide a solid basis for further studies aiming at elaborating sensitive and specific immunosensor or a microarray for the detection of E. coli O157:H7.
Kleinschnitz, Christoph; Braeuninger, Stefan; Pham, Mirko; Austinat, Madeleine; Nölte, Ingo; Renné, Thomas; Nieswandt, Bernhard; Bendszus, Martin; Stoll, Guido
Models of photochemically-induced thrombosis are widely used in cerebrovascular research. Photothrombotic brain infarctions can be induced by systemic application of photosensitizing dyes followed by focal illumination of the cerebral cortex. Although the ensuing activation of platelets is well established, their contribution for thrombosis and tissue damage has not formally been proved. Infarction to the cerebral cortex was induced in mice by Rose Bengal and a cold light source. To assess the functional role of platelets, animals were platelet-depleted by anti-GPIbalpha antibodies or treated with GPIIb/IIIa-blocking F(ab)(2) fragments. The significance of the plasmatic coagulation cascade was determined by using blood coagulation factor XII (FXII)-deficient mice or heparin. Infarct development and infarct volumes were determined by serial MRI and conventional and electron microscopy. There was no difference in development and final size of photothrombotic infarctions in mice with impaired platelet function. Moreover, deficiency of FXII, which initiates the intrinsic pathway of coagulation and is essential for thrombus formation, or blockade of FXa, the key protease during the waterfall cascade of plasmatic coagulation, by heparin likewise did not affect lesion development. Our data demonstrate that platelet activation, factor XII-driven thrombus formation, and plasmatic coagulation pathways downstream of FX are not a prerequisite for ensuing tissue damage in models of photothrombotic vessel injury indicating that other pathomechanisms are involved. We suggest that this widely used model does not depend on platelet- or plasmatic coagulation-derived thrombosis.
Full Text Available Crotaline snakebites (Protobothrops mucrosquamatus and Trimeresurus stejnegeri are a common medical emergency in Taiwan that can be effectively treated by a bivalent F(ab'2 antivenom. We investigated the differences in the clinical outcomes of patients who received different therapeutic regimens of antivenom in a medical center where clinical toxicologists followed the poison control center (PCC guidelines (medical group and surgeons did not (surgical group. The medical records of inpatients with crotaline snakebites between 1991 and 2005 were reviewed and information on demographics, treatments, adverse effects of antivenom, and complications was abstracted and analyzed. A total of 179 patients (90 medical, 89 surgical were eligible for study. There was no significant intergroup difference in baseline characteristics except that the dose of antivenom and the probability of antibiotic use were both higher in the surgical group (5.9 ± 4.2 vials vs. 2.7 ± 1.6 vials; 93% vs. 60%. Multiple logistic regression adjusting for age, gender, calendar year of envenomation, severity of envenomation, and antibiotic use did not disclose evidence of any difference in various clinical outcomes between medical and surgical patients. The lower dose of antivenom recommended by the PCC may be as effective and safe as the higher dose used in the surgical group for the treatment of crotaline snakebites.
Sierralta, W D; Jakob, F; Thole, H; Engel, P; Jungblut, P W
Endometrium was collected by curettage from castrated pigs, either untreated or exposed to estradiol in vivo by intrauterine injection, and processed for electron microscopy. The resin LR Gold was used for embedding, and sections were floated on droplets of 10 nm diameter gold particles, coated with the immunoglobulin-G1 (IgG1) fraction or its Fab2 fragment of a monospecific polyclonal antiserum raised in goats against the C-terminal half of the estradiol receptor. On average, only one gold particle per microns 2 became attached in the cytoplasmic area of untreated cells, whereas four were found over the nuclear area. These figures rose to 2-3/microns 2 and 15-26/microns 2, respectively, within 10 min after exposure to estradiol. The labeling intensities of nuclei in cell clusters and of coprocessed nuclei released from cells ruptured during curettage were identical in all situations. Nuclear pores were frequently tagged after estradiol treatment. The proportions of tagging densities in nuclei of untreated and estradiol-exposed cells corresponded to those of receptor contents measured in extracts of isolated nuclei by ligand binding. This correlation was not seen for the cytoplasmic compartment of untreated cells, the scarce tagging of which is interpreted by hidden antigenic determinants. Our morphological analyses support the conclusions drawn from biochemical data (Sierralta et al., 1992) of an estradiol-promoted translocation of receptor from the cytoplasm into the nucleus.
on resting B cells (even in the presence of intact F23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1+ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab')2......Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect...... fragments of F23.1 induced E9.D4 to activate B cells as efficiently as intact F23.1 and B cell populations that had been incubated with F23.1 were not activated when cultured with E9.D4, although T cells recognized cell-presented F23.1 and were weakly activated. Reduction of the density of F23.1 adsorbed...
Hnatowich, D.J.; Mardirossian, G.; Rusckowski, M.; Roy, S.; Busche, H.; Griffin, T.W.; Brill, A.B.
The FO23C5 anti-carcinoembryonic antigen (CEA) F(ab') 2 antibody was radiolabelled with sup(111)In via diethylenetriaminepentaacetic acid (DTPA) and directly with 99 Tc m by stannous ion and mercaptoethanol antibody reduction to compare the pharmacokinetics of these three agents. Four patients received 15 mCi 99 Tc m -Fab' 1 week before receiving 1 mCi 111 In-F(ab') 2 . Five additional patients received only the 99 Tc m -Fab'. The biodistribution of 99 Tc m was as expected for a labelled Fab' fragment: relative to 111 In, 99 Tc m cleared rapidly from circulation and into kidneys and urine. Liver levels of 111 In and 99 Tc m were surprisingly similar at 1 day although initial 111 In levels were lower and increased while 99 Tc m levels were higher and decreased. Spleen levels were also similar. In 4/9 patients receiving 99 Tc m , hepatobiliary clearance was observed at levels which could confuse interpretation whereas this mode of clearance was observed in only 1/4 patients receiving 111 In. Image quality was superior with 111 In versus 99 Tc m at 1 day postadministration as judged by counting rates and background activity whereas the opposite was true at 2-3 h postadministration. (author)
Overall F(ab') 2 and antitetanus-f(ab') 2 - fragments were labelled with 125 I and injected i.th. into normal juvenile cats and adult rats. One group of rats was normal; in the other, unilateral local tetanus had been induced by injection of tetanus toxin into a M. gastrocnemius. The animals were sacrificed 24 h after the i.th. injection, and tissue samples were taken for histoautoradiography. 125 I-antitetanus-F(ab') 2 permeated into the extracellular space of the spinal cord, roots, and ganglia but not into the neuronal intracellular space. 125 I-overall-F(ab') showed identical permeation behaviour. 125 I-antitetanus-F(ab') 2 reacted with tetanus toxin issuing from the motoneurons after i.th. injection, forming an immunocomplex around the motorneurons. The immunocomplex was not formed around pseudo-unipolar ganglian cells in the spinal ganglia even though some of the ganglian cells contained tetanus toxin, and 125 I-antitetanus-F(ab') 2 was present in the extracellular space. As an explanation, it was suggested that tetanus toxin does not permeate into the extracellular space through the membrane of the pseudo-unipolar ganglian cells so that immune reactions will not occur. These findings help to explain the widely divergent results of tetanus therapy by means of i.th. injection of tetanus antitoxin. Recommendations for future therapy measures are derived from the findings. (orig./MG) [de
Haisma, H.; Hilgers, J.
Many monoclonal antibodies directed against tumor-associated antigens have been identified, but so far none of these are tumor specific. Polyclonal and monoclonal antibodies have been used for imaging of a wide variety of tumors with success. Radiolabeling of antibody is usually done with iodine isotopes of which 123 I is the best candidate for radioimmunodetection purposes. The labeling of antibodies through chelates makes it possible to use metal radioisotopes like 111 In, which is the best radioisotope for imaging with monoclonal antibodies due to its favorable half-life of 2.5 days. Usually imaging cannot be performed within 24 h after injection, but clearance of antibody can be increased by using F(ab) 2 of Fab. Another approach is to clear non-bound antibody by a second antibody, directed against the first. The detection limit of immunoimaging is about 2 cm, but will be improved by tomography or SPECT. There is still a high false positive and false negative rate, which makes it impossible to use radioimmunodetection as the only technique for diagnosis of tumors. In combination with other detection techniques, tumor imaging with monoclonal antibodies can improve diagnosis. 44 refs.; 3 tabs
Rial, A; Morais, V; Rossi, S; Massaldi, H
A competitive ELISA for potency determination of bothropic equine antivenom was developed and compared to the conventional in vivo ED(50) assay, with the aim of partially substituting the in vivo assay in the monitoring of antivenom immunoglobulin levels. On this purpose, blood samples were taken at different times during and after the immunization protocol of the lot of horses used for production of snake antivenom at the Instituto de Higiene, Uruguay. Both the competitive ELISA and the ED(50) assay were performed on those samples. In addition, a group of five commercial pepsin-digested antivenoms were tested by both methods. A significant (P<0.001) correlation (Pearson's r=0.957) was found between the ELISA titres and the corresponding ED(50) values, indicating that the in vitro test can estimate the neutralizing antibody capacity of the sera as well as the in vivo assay. By means of this new ELISA, it was found that the immunized animals maintained good venom antibody titres, in the order of 20-50% of the maximum achieved, even 10 month after the end of the immunization schedule. The main advantage of our ELISA design is its ability to correctly estimate the neutralization capacity of crude hyperimmune plasma and antivenom sera independently of their antibody composition in terms of whole IgG or F(ab')(2) fragment.
Sørensen, Inge Juul; Nielsen, EH; Andersen, Ove
Serum amyloid P component (SAP), a member of the conserved pentraxin family of plasma proteins, binds calcium dependently to its ligands. The authors investigated SAPs interaction with the complement proteins C4b binding protein (C4bp) and C1q by ELISA, immunoelectrophoresis and electron microscopy....... Binding of these proteins to SAP was demonstrated when SAP was immobilized using F(ab')2 anti-SAP, but not when SAP reacted with these proteins in liquid phase; thus the binding to human SAP was markedly phase state dependent. Presaturation of solid phase SAP with heparin, which binds SAP with high...... affinity, did not interfere with the subsequent binding of C4bp or C1q to SAP. In contrast, collagen I and IV showed partial competition with the binding of C1q to SAP. Using fresh serum, immobilized native SAP bound C4bp whereas binding of C1q/C1 could not be demonstrated. Altogether the results indicate...
Bose, R; Marsh, D G; Delespesse, G
Anti-idiotypes (aId) reacting with anti-Lol I (Lolp I; Rye I) antibodies were detected by their ability to bind to radioiodinated F(ab')2 anti-Lol I. Sera were tested after removal of anti-Lol I and anti-heavy and light chain activity by adsorption on Lol I-Sepharose 4B and normal human serum Sepharose 4B. The binding of aId to Id was inhibited by affinity purified anti-Lol I but not by certain unrelated immunoglobulins; in some sera this binding was also inhibited by Lol I. The levels of aId were measured in serial bleedings collected over a 1 year period from Lol I-sensitive patients, allergic donors not sensitive to Lol I and non-allergic persons. In Lol I-allergic patients the levels of aId were significantly influenced by seasonal exposure to pollen and by immunotherapy with extracts of grass pollen. Moreover, in 12 out of 16 cases, there was also a significant inverse relationship between changes in serum levels of aId and of IgG or IgE anti-Lol I. Most interestingly, aId were also detected in non-allergic individuals; in this case, the levels of aId were not influenced by the pollen season. The data suggest that Id-aId interactions may play a role in the regulation of anti-Lol I antibody production. PMID:3492316
Hébert, J; Bernier, D; Mourad, W
Anti-idiotypic antibodies (anti-Id Abs) are involved in the regulation of a number of immune responses including the IgE antibody production. In atopic patients, the increased synthesis of IgE antibodies could be related to a defective production of regulatory anti-Id Abs. In the present study, we first developed a sensitive assay for measuring the levels of anti-Id Abs directed against antibodies specific for Lol p I, the major allergenic determinant of Lolium perenne (rye grass). In this assay, we used previously described murine monoclonal anti-Lol p I antibodies that were shown to share epitopic specificities with human anti-Lol p I IgE and IgG antibodies, thus short-cutting the need for purification of F(ab')2 fragments of human IgG Abs and insuring optimal specificity and sensitivity. Levels of anti-Id Abs against two anti-Lol p I monoclonal antibodies (290A-167, 348A-6) were higher in normal volunteers than in untreated atopic patients. Specific immunotherapy increased the levels of anti-Id Abs to those of normal volunteers. These observations suggest a role for the Id-anti-Id network in the regulation of IgE antibody production.
Wahl, R.L.; Geatti, O.; Liebert, M.; Beers, B.; Jackson, G.; Laino, L.; Kronberg, S.; Wilson, B.S.; Beierwaltes, W.H.
Radiolabeled monoclonal antibody (MoAb) administration subcutaneously (sq), so that preferential uptake is to the lymphatics, holds significant promise for the detection of lymph node metastases. Only limited information is available about clearance rates of intralymphatically administered MoAbs. I-131 labeled intact IgG (225.28S), F(ab's)2 (225.28S) or IgM (FT162) were administered sq to anesthetized Balb/C mice. Eight mice were studied with each MoAb, 4 with a foot-pad injection, 4 with an anterior abdominal injection. Gamma camera images were collected into a computer, over the first 6 hrs after injection with the animals anesthetized and immobile. Animals were then allowed to move about freely. Additional images were then acquired out to 48 hrs. Regions of interest wre selected over the injection site and the kinetics of antibody egress determined. Clearance rates from local sq injection sites are influenced by motion and somewhat by location. The class and fragment status of the MoAb appear relatively less important in determining clearance rates from sq injections than they are in determining whole-body clearance after iv injections. Additional studies using Fab fragments and additional monoclonals will be useful in extending these observations
Chen, D.C.P.; Siegel, M.E.; Chen, F.; Taylor, O.R.; Epstein, A.L.
The TNT-1 antibody was developed to bind intracellular nuclear antigens that are accessible only in degenerative or necrotic cells. Since about 50% of tumor cells are in various stages of cell degeneration or death, this antibody could serve as a pantumor antibody for tumor detection. After intravenous injection of 10 μg of TNT-1F(ab')2 fragments labeled with 20 μCi of I-131, serial images were obtained at 1 and 4 hours and daily for 6 days in mice bearing various human tumors. Accumulation of TNT-1 was imaged in a necrotic tumor as early as 4 hours after injection and because more intense at 48 hours. The tumor-muscle ratio was as high as 29:1. Intense accumulation was noted in the necrotic tumor, about nine times that of healthy tumor. In conclusion, TNT-1, a pantumor antibody, can detect necrotic tumors in animal models. It may be an ideal imaging agent for cancer detection
As the lung cancer is the leading cause of death from cancer at males, the exact staging is essential. Monoclonal antibodies marked with radionuclides like 131 I, 111 In, 99m Tc, etc., allow detecting and staging the small cell lung cancer with sensibility 90%, specificity 45% and accuracy 85%. It is suggested this method to be applied simultaneously with computerized tomography. The diagnostic possibility of radioimmunoscintigraphy (RIS) in earlier detection, recurrence or metastasis as well as follow up the effect of therapy performed at patients with lung cancer are reviewed. RIS is performed with IODOMAB-R-2 (Sorin Biomedica) 131 I antiCEA Mob F(ab') 2 , dose 92.5-185 MBq. Planar images were performed 72 hours after i.v. injection. Four patients with epidermoid squamous cell cancer were examined. Positive results were obtained at 3 patients and one false negative. In general sensitivity of radioimmunoscintigraphy of lung cancer is 75-90%. However there are difficulties at its application linked with necessity of permanent availability of radiolabelled antibodies with high specific activity at the moment of their injection. Despite all radioimmunoscintigraphy is developing as an useful diagnostic method for evaluation and follow up of lung cancer patients
Chatal, J.F.; Douillard, J.Y.; Kremer, M.; Curtet, C.; Le Mevel, B.; Fumoleau, P.; Bourdoiseau, M.
Two monoclonal antibodies, 17-1A and 19-9, with recognized human gastrointestinal cancers in cell cultures, were labeled with iodine 131 for immunoscintigraphic application. With the intact 131 I-17-1A antibody, 21 out of 35 (60%) primary or secondary colorectal cancer sites were visualized, whereas all 21 nonepitheliomatous colic cancer sites or noncolic cancer sites were negative. With F(ab') 2 fragments of the 19-9 antibody, 18 out of 27 (67%) colorectal cancer sites were positive. With both radioantibodies, the bestly contrasted tumor images were late, 4 to 5 days after injection. A study with paired-label technique, associating a specific iodine-131-labeled antibody with a non-specific iodine-125-labeled immunoglobulin, demonstrated, that tumor uptake was indeed specific for the 17-1A or 19-9 antibody in tumor and normal colon fragments obtained during operations on 4 patients. A preliminary prospective study showed that only immunoscintigraphy was able to confirm and localize a recurrence of rectal cancer in one patient. A larger series will be necessary to validate the clinical benefit of the technique, as compared with the results of other diagnostic techniques, before immunoscintigraphy can be proposed for routine clinical use [fr
Zhang, Wei; Wei, Menglian; Carvalho, Wildemar S P; Serpe, Michael J
A biosensor for mouse Immunoglobulin G (IgG) was generated from responsive polymer-based interference filters (etalons). To accomplish this, an excess amount of alkaline phosphatase-modified goat anti-mouse IgG (AP-GAM, F(ab') 2 fragment specific to mouse IgG) was added to mouse IgG, and allowed to react for some time. After a given reaction time, the bound AP-GAM could be isolated from the unbound, excess AP-GAM by addition of goat anti-mouse IgG (Fc fragment specific)-modified magnetic microspheres (GAM-M) that bind the mouse IgG bound to AP-GAM. After application of a magnetic field, the free, unbound AP-GAM was isolated from the mixture and exposed to an etalon that has its upper Au surface modified with phosphate-containing polymer that can be degraded by AP-GAM. By the phosphate-containing polymer being degraded by the excess AP-GAM, the cleaved phosphate groups can diffuse into the interference filter's active polymer layer that yields a change in the optical properties that can be related to the amount of IgG in the sample. This concept is extremely straightforward to implement, and can be modified to detect a variety of other analytes of interest. Copyright © 2017 Elsevier B.V. All rights reserved.
McCabe, D.P.; Flynn, E.J.
Antibodies specific for barbiturates were generated in rabbits by administration of a barbiturate-bovine gamma globulin conjugate. Antibodies with a high degree of specificity for barbiturates were obtained after 2-3 months of immunization. Anti-barbiturate antisera were used to passively immunize pregnant animals on day 15 of gestation. The disposition of 3 H-Phenobarbital (PhB) in fetal tissue in these mice were compared to normal rabbit serum(NRS)-treated pregnant mice. Following intravenous injection of 3 H-PhB (25 pmoles), there were significantly higher levels of radioactivity in the serum of the immunized mice when compared to controls. Both the distribution half-life and the elimination half-life were reduced in the immunized animals (17.2 vs 88.3 min. and 123.7 vs 308.8 min., respectively) when compared to the NRS-treated mice. In contrast, levels of total radioactivity were significantly reduced in whole fetal homogenates in animals pretreated with whole antisera, Fab, or F(ab') 2 fragments as compared to control animals. The data indicate that there was a decrease in the ability of 3 H-PhB or its metabolites to diffuse into fetal tissue at various time points and that antisera or antisera fragments were instrumental in preventing this diffusion
Full Text Available Imaging tumors and their response to treatment could be a valuable biomarker toward early assessment of therapy in patients with cancer. Phosphatidylserine (PS is confined to the inner leaflet of the plasma membrane in normal cells but is externalized on tumor vascular endothelial cells (ECs and tumor cells, and PS exposure is further enhanced in response to radiation and chemotherapy. In the present study, we evaluated the potential of a PS-targeting human F(ab'2 antibody fragment, PGN650, to detect exposure of PS in tumor-bearing mice. Tumor uptake of PGN650 was measured by near-infrared optical imaging in human tumor xenografts in immunodeficient mice. PGN650 specifically targeted tumors and was shown to target CD31-positive ECs and tumor cells. Tumor uptake of PGN650 was significantly higher in animals pretreated with docetaxel. The peak tumor to normal tissue (T/N ratio of probe was observed at 24 hours postinjection of probe, and tumor binding was detected for at least 120 hours. In repeat dose studies, PGN650 uptake in tumors was significantly higher following pretreatment with docetaxel compared to baseline uptake prior to treatment. PGN650 may be a useful probe to detect PS exposed in tumors and to monitor enhanced PS exposure to optimize therapeutic agents to treat tumors.
Monoclonal antibodies (MAbs) provide an attractive method of selectively localizing sufficient boron atoms around tumour cells to capture neutrons. Assuming that 10(8)-10(10) 10B atoms are needed for one capture event and that 10(3)-10(4) atoms can be coupled to each antibody molecule, then 10(5)-10(6) antibody molecules gathered on an individual cell will destroy that cell. Binding to normal tissues, on the other hand, would need to be at least 20-fold less than that to tumour tissues to avoid toxic effects of neutrons on surrounding tissues. Preclinical studies in animals show that several MAbs may bind to melanoma cells in sufficient quantities in vitro to localize the required amount of boron per cell. Whether this will occur in vivo, however, may depend not only on antigen density but a variety of other properties of the tumour cells and MAbs. These include the Ig class and affinity of the antibody and whether the antibody is internalized into the tumour cell. The ratio of uptake between tumour and normal tissue is governed by such factors as the percentage of tumour cells within a tumour expressing the antigen and whether the MAb react with normal tissues. Use of Fab or F(ab)2 preparations of the MAb may increase the uptake ratio by preventing uptake of MAb by cells with Fc receptors. In contrast to preclinical animal studies, tumour/normal tissue uptake ratios in phase I studies in humans have been disappointingly low.80 references
Haisma, H.J.; Moseley, K.R.; Battaile, A.; Griffiths, T.C.; Knapp, R.C.
Radiolabeled monoclonal antibodies may be useful for radioimmunotherapy of gynecologic tumors. Iodine 131-labeled F(ab')2 fragments of a monoclonal antibody, OC 125, with specificity for ovarian carcinoma, were used to study the distribution and pharmacokinetics of this antibody in patients with gynecologic tumors. The radiolabeled antibody was injected intravenously or intraperitoneally into 10 patients suspected of having ovarian cancer. Blood and urine samples were used for pharmacokinetic studies, and biopsy specimens were examined for the uptake of antibody. The serum half-life of the labeled antibody was 30 hours after intravenous administration, with 20% of the injected dose per liter detected at 24 hours. After intraperitoneal injection, the appearance of antibody in serum was slow, with a maximum level of 1.4% of the injected dose per liter at 24 hours. Urinary excretion of the radiolabeled antibody was similar for intravenous and intraperitoneal administration, with approximately 50% of the injected dose excreted after 48 hours. Intraperitoneal administration of the radiolabeled antibody resulted in a higher uptake of antibody in the tumor and a lower uptake of antibody in normal tissues. On the basis of this limited study, intraperitoneal administration of radiolabeled antibody is preferred over intravenous administration for radioimmunotherapy of ovarian cancer
Lynch, D M; Lynch, J M; Howe, S E
A quantitative ELISA assay for the measurement of in vivo bound platelet-associated IgG (PAIgG) using intact patient platelets is presented. The assay requires quantitation and standardization of the number of platelets bound to microtiter plate wells and an absorbance curve using quantitated IgG standards. Platelet-bound IgG was measured using an F(ab')2 peroxidase labeled anti-human IgG and o-phenylenediamine dihydrochloride (OPD) as the substrate. Using this assay, PAIgG for normal individuals was 2.8 +/- 1.6 fg/platelet (mean +/- 1 SD; n = 30). Increased levels were found in 28 of 30 patients with clinical autoimmune thrombocytopenia (ATP) with a range of 7.0-80 fg/platelet. Normal PAIgG levels were found in 26 of 30 patients with nonimmune thrombocytopenia. In the sample population studied, the PAIgG assay showed a sensitivity of 93%, specificity of 90%, a positive predictive value of 0.90, and a negative predictive value of 0.93. The procedure is highly reproducible (CV = 6.8%) and useful in evaluating patients with suspected immune mediated thrombocytopenia.
Howe, S E; Lynch, D M; Lynch, J M
An enzyme-linked immunosorbent assay (ELISA) using F(ab')2 peroxidase-labeled antihuman immunoglobulin and o-phenylenediamine dihydrochloride (OPD) as a substrate was developed to measure serum platelet bindable IgG (S-PBIgG). The assay was made quantitative by standardizing the number of normal "target" platelets bound to microtiter plate wells, and by incorporating quantitated IgG standards with each microtiter plate tested to prepare a standard calibration curve. By this method, S-PBIgG for normal individuals was 3.4 +/- 1.6 fg per platelet (mean +/- 1 SD; n = 40). Increased S-PBIgG levels were detected in 36 of 40 patients with clinical autoimmune thrombocytopenia (ATP), ranging from 7.0 to 85 fg per platelet. Normal S-PBIgG levels were found in 34 of 40 patients with nonimmune thrombocytopenia. This method showed a sensitivity of 90 percent, specificity of 85 percent, and in the sample population studied, a positive predictive value of 0.86 and a negative predictive value of 0.90. This assay is highly reproducible (coefficient of variation was 6.8%) and appears useful in the evaluation of patients with suspected immune-mediated thrombocytopenia.
Richter, C; Schnabel, A; Csernok, E; De Groot, K; Reinhold-Keller, E; Gross, W L
In this uncontrolled study 15 patients with ANCA-associated systemic vasculitis, who were poor responders to conventional therapy, were treated with single or multiple courses of intravenous immunoglobulin (IVIG), 30 g/day over 5 days. Clinical and serological evaluation was performed before and 4 weeks after IVIG. Six of the 15 patients experienced clinically significant benefit from IVIG. Improvement was confined to single organ manifestations (skin, ENT findings), no improvement was seen with conjunctivitis and scleritis, pericarditis or nephritis. No patient experienced complete remission after IVIG. Repeated courses of IVIG at 4-week intervals were no more effective than single courses. In six anti-proteinase 3 (PR3)-positive patients pretreatment sera were incubated with F(ab')2 fragments of the IVIG preparation in vitro to measure the inhibitory effect of IVIG on anti-PR3 activity. An inhibition of anti-PR3 activity by 25-70% was observed; this did not correlate with clinical effects. Approximately 40% of patients benefited from IVIG treatment, though complete remission of disease activity did not occur. Neither clinical characteristics nor the inhibitory effect of the IVIG preparation on serum anti-PR3 activity in vitro predicted clinical response to this treatment modality.
Tibben, J.G.; Massuger, L.F.A.G.; Claessens, R.A.M.J.; Schijf, C.P.T.; Strijk, S.P.; Kenemans, P.; Corstens, F.H.M.; Pak, K.Y.
Fab' fragments of the monoclonal antibody OV-TL 3, that recognizes an ovarian carcinoma-associated antigen (OA3), were labelled with 99 Tc m using D-glucarate as a ligand. Twenty patients suspected of having primary or recurrent ovarian cancer received intravenously 1 mg of the Fab' labelled with 740 MBq 99 Tc m . Both planar and single photon emission computed tomographic (SPECT) scintigraphy were performed up to 30 h after intravenous infusion. Imaging results were compared with X-ray computed tomography (CT), ultrasonography (US) and CA 125 serum level. Blood clearance was fast (t 1/2 of 9.5 h). Thirty-seven per cent of the injected dose (% ID) was excreted in the urine within the first 24 h, whereas 7% ID was excreted in the 24 h faeces. In one patient with an OA3 negative ovarian carcinoma, radioimmunoscintigraphy (RIS) did not visualize the tumour. In two other patients a benign ovarian cyst was found, also showing no elevated uptake. In 13 out of 17 patients ovarian cancer lesions were detected with RIS, whereas CT and US detected lesions in, respectively, 15 and 12 patients. Of 36 surgically defined tumour deposits larger than 1 cm in diameter, 53% were detected and localized with RIS, whereas CT and US detected 61 and 40%, respectively. Radioimmunoscintigraphy with 99 Tc m -OV-TL 3 Fab' is less distressing for the patients but the overall imaging performance is not improved when compared with 111 In-OV-TL 3 F(ab') 2 . (Author)
Chen Zhinan; Mi Li; Xu Jing
Purpose: HAb18G/CD147 is a hepatocellular carcinoma (HCC)-associated antigen. We developed iodine ( 131 I) metuximab injection (Licartin), a novel 131 I-labeled HAb18G/CD147-specific monoclonal antibody F(ab') 2 fragment, and evaluated its safety, pharmacokinetics, and clinical efficacy on HCC in Phase I/II trials. Methods and Materials: In a Phase I trial, 28 patients were randomly assigned to receive the injection in 9.25-, 18.5-, 27.75-, or 37-MBq/kg doses by hepatic artery infusion. In a multicenter Phase II trial, 106 patients received the injection (27.75 MBq/kg) on Day 1 of a 28-day cycle. Response rate and survival rate were the endpoints. Results: No life-threatening toxic effects were found. The safe dosage was 27.75 MBq/kg. The blood clearance fitted a biphasic model, and its half-life was 90.56-63.93 h. In the Phase II trial, the injection was found to be targeted and concentrated to tumor tissues. Of the 73 patients completing two cycles, 6 (8.22%) had a partial response, 14 (19.18%) minor response, and 43 (58.90%) stable disease. The 21-month survival rate was 44.54%. The survival rate of progression-free patients was significantly higher than that of patients with progressive disease after either one or two cycles (p 131 I) metuximab injection is safe and active for HCC patients
Gadkar, Kapil; Pastuskovas, Cinthia V; Le Couter, Jennifer E; Elliott, J Michael; Zhang, Jianhuan; Lee, Chingwei V; Sanowar, Sarah; Fuh, Germaine; Kim, Hok Seon; Lombana, T Noelle; Spiess, Christoph; Nakamura, Makia; Hass, Phil; Shatz, Whitney; Meng, Y Gloria; Scheer, Justin M
To design and select the next generation of ocular therapeutics, we performed a comprehensive ocular and systemic pharmacokinetic (PK) analysis of a variety of antibodies and antibody fragments, including a novel-designed bispecific antibody. Molecules were administrated via intravitreal (IVT) or intravenous (IV) injections in rabbits, and antibody concentrations in each tissue were determined by ELISA. A novel mathematical model was developed to quantitate the structure-PK relationship. After IVT injection, differences in vitreal half-life observed across all molecules ranged between 3.2 and 5.2 days. Modification or elimination of the fragment crystallizable (Fc) region reduced serum half-life from 9 days for the IgG to 5 days for the neonatal Fc receptor (FcRn) null mAb, to 3.1 to 3.4 days for the other formats. The F(ab')2 was the optimal format for ocular therapeutics with comparable vitreal half-life to full-length antibodies, but with minimized systemic exposure. Concomitantly, the consistency among mathematical model predictions and observed data validated the model for future PK predictions. In addition, we showed a novel design to develop bispecific antibodies, here with activity targeting multiple angiogenesis pathways. We demonstrated that protein molecular weight and Fc region do not play a critical role in ocular PK, as they do systemically. Moreover, the mathematical model supports the selection of the "ideal therapeutic" by predicting ocular and systemic PK of any antibody format for any dose regimen. These findings have important implications for the design and selection of ocular therapeutics according to treatment needs, such as maximizing ocular half-life and minimizing systemic exposure.
Jarzabek, Monika A; Proctor, William R; Vogt, Jennifer; Desai, Rupal; Dicker, Patrick; Cain, Gary; Raja, Rajiv; Brodbeck, Jens; Stevens, Dale; van der Stok, Eric P; Martens, John W M; Verhoef, Cornelis; Hegde, Priti S; Byrne, Annette T; Tarrant, Jacqueline M
Drug-related sinusoidal dilatation (SD) is a common form of hepatotoxicity associated with oxaliplatin-based chemotherapy used prior to resection of colorectal liver metastases (CRLM). Recently, hepatic SD has also been associated with anti-delta like 4 (DLL4) cancer therapies targeting the NOTCH pathway. To investigate the hypothesis that NOTCH signaling plays an important role in drug-induced SD, gene expression changes were examined in livers from anti-DLL4 and oxaliplatin-induced SD in non-human primate (NHP) and patients, respectively. Putative mechanistic biomarkers of bevacizumab (bev)-mediated protection against oxaliplatin-induced SD were also investigated. RNA was extracted from whole liver sections or centrilobular regions by laser-capture microdissection (LCM) obtained from NHP administered anti-DLL4 fragment antigen-binding (F(ab')2 or patients with CRLM receiving oxaliplatin-based chemotherapy with or without bev. mRNA expression was quantified using high-throughput real-time quantitative PCR. Significance analysis was used to identify genes with differential expression patterns (false discovery rate (FDR) < 0.05). Eleven (CCL2, CCND1, EFNB2, ERG, ICAM1, IL16, LFNG, NOTCH1, NOTCH4, PRDX1, and TGFB1) and six (CDH5, EFNB2, HES1, IL16, MIK67, HES1 and VWF) candidate genes were differentially expressed in the liver of anti-DLL4- and oxaliplatin-induced SD, respectively. Addition of bev to oxaliplatin-based chemotherapy resulted in differential changes in hepatic CDH5, HEY1, IL16, JAG1, MMP9, NOTCH4 and TIMP1 expression. This work implicates NOTCH and IL16 pathways in the pathogenesis of drug-induced SD and further explains the hepato-protective effect of bev in oxaliplatin-induced SD observed in CRLM patients.
Full Text Available Nimotuzumab is an anticancer agent which belongs to the inhibitor group of Epidermal Growth Factor Receptor (EGFR. This monoclonal antibody has a relatively high molecular weight which makes slow penetration on tumor cell, as concequence, it is less attractive in imaging kinetics, and potentially elicits antibodies respons. Therefore in this study nimotuzumab was fragmented to form bivalent antibody [F(ab’2] and then labeled with 125I to form 125I-F(ab’2-nimotuzumab which can be used further as a precursor for preparing 125I-F(ab’2-nimotuzumab-NLS (NLS = nuclear localizing sequences radiopharmaceutical for radioimmunotherapy. The aims of this study were to obtain characteristics of 125I-F(ab’2-nimotuzumab by comparing with the 125I labeled-intact nimotuzumab (125I-nimotuzumab. This study was initiated by purifying nimotuzumab by mean of dialysis. The purified nimotuzumab was then fragmented by using pepsin. The F(ab'2-nimotuzumab formed was then purified from its by-products which formed in fragmentation process by using a PD-10 column (consisted Sephadex G25. The intact nimotuzumab and its F(ab’2 fragment were then labeled with the 125I to form 125I-nimotuzumab and 125I-F(ab’2-nimotuzumab. The radiochemical purity are 98.27 % and 93.24 % ,respectively. Stability test results show that, both of 125I-nimotuzumab and 125I-F(ab’2-nimotuzumab more stable at 4 °C than at room temperature storage and 37 °C
Haryuni, R.D.; Bahtiar, A.; Soenarjo, S.; Harahap, Y.; Mutalib, A.; Ramli, M.; Hermanto, S.; Ardiyatno, C.N.; Susilo, V.Y.; Haffid, D.
Nimotuzumab is an anticancer agent which belongs to the inhibitor group of Epidermal Growth Factor Receptor (EGFR). This monoclonal antibody has a relatively high molecular weight which slows penetration on tumor cells, making it less attractive in imaging kinetics and potentially elicits antibodies responses. Therefore, in this study nimotuzumab was fragmented to form a bivalent antibody [F(ab’) 2 ] and then labeled with 125 I to form 125 I-F(ab’) 2 -nimotuzumab which can be used further as a precursor for preparing 125 I-F(ab’) 2 -nimotuzumab-NLS (NLS = nuclear localization sequence) radiopharmaceutical for radioimmunotherapy. The aims of this study was to obtain characteristics of 125 I-F(ab’) 2 -nimotuzumab by comparing with the 125 I labeled-intact nimotuzumab ( 125 I-nimotuzumab). This study was initiated by purifying nimotuzumab by mean of dialysis. The purified nimotuzumab was then fragmented by using pepsin. The F(ab') 2 -nimotuzumab formed was then purified from its by-products which formed in fragmentation process by using a PD-10 column (consisted Sephadex G25). The intact nimotuzumab and its F(ab’)2 fragment were then labeled with the 125 I to form 125 I-nimotuzumab and 125 I-F(ab’) 2 -nimotuzumab. The radiochemical purity are 98.27 % and 93.24 %, respectively. Stability test results show that, both 125 I-nimotuzumab and 125 I-F(ab’) 2 -nimotuzumab are more stable at 4 °C than at room temperature storage and 37 °C. (author)
Inami, Koichi; Abe, Masaaki; Takeda, Kazuyoshi; Hagiwara, Yoshiaki; Maeda, Masahiro; Segawa, Tatsuya; Suyama, Masafumi; Watanabe, Sumio; Hino, Okio
Mesothelioma is an aggressive cancer often caused by chronic asbestos exposure, and its prognosis is very poor despite the therapies currently used. Due to the long latency period between asbestos exposure and tumor development, the worldwide incidence will increase substantially in the next decades. Thus, novel effective therapies are warranted to improve the prognosis. The ERC/mesothelin gene (MSLN) is expressed in wide variety of human cancers, including mesotheliomas, and encodes a precursor protein cleaved by proteases to generate C-ERC/mesothelin and N-ERC/mesothelin. In this study, we investigated the antitumor activity of C-ERC/mesothelin-specific mouse monoclonal antibody, 22A31, against tumors derived from a human mesothelioma cell line, ACC-MESO-4, in a xenograft experimental model using female BALB/c athymic nude mice. Treatment with 22A31 did not inhibit cell proliferation of ACC-MESO-4 in vitro; however, therapeutic treatment with 22A31 drastically inhibited tumor growth in vivo. 22A31 induced antibody-dependent cell-mediated cytotoxicity by natural killer (NK) cells, but not macrophages, in vitro. Consistently, the F(ab')(2) fragment of 22A31 did not inhibit tumor growth in vivo, nor did it induce antibody-dependent cell mediated cytotoxicity (ADCC) in vitro. Moreover, NK cell depletion diminished the antitumor effect of 22A31. Thus, 22A31 induced NK cell-mediated ADCC and exerted antitumor activity in vivo. 22A31 could have potential as a therapeutic tool to treat C-ERC/mesothelin-expressing cancers including mesothelioma.
Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon
The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230
Zhao, Dawen; Stafford, Jason H.; Zhou, Heling; Thorpe, Philip E.
Phosphatidylserine (PS), normally restricted to the inner leaflet of the plasma membrane, becomes exposed on the outer surface of viable (non-apoptotic) endothelial cells in tumor blood vessels, probably in response to oxidative stresses present in the tumor microenvironment. In the present study, we optically imaged exposed PS on tumor vasculature in vivo using PGN635, a novel human monoclonal antibody that targets PS. PGN635 F(ab')2 was labeled with the near infrared (NIR) dye, IRDye 800CW. Human glioma U87 cells or breast cancer MDA-MB-231 cells were implanted subcutaneously or orthotopically into nude mice. When the tumors reached ~5 mm in diameter, 800CW- PGN635 was injected via a tail vein and in vivo dynamic NIR imaging was performed. For U87 gliomas, NIR imaging allowed clear detection of tumors as early as 4 h later, which improved over time to give a maximal tumor/normal ratio (TNR = 2.9 +/- 0.5) 24 h later. Similar results were observed for orthotopic MDA-MB-231 breast tumors. Localization of 800CW-PGN635 to tumors was antigen specific since 800CW-Aurexis, a control probe of irrelevant specificity, did not localize to the tumors, and pre-administration of unlabeled PGN635 blocked the uptake of 800CW-PGN635. Fluorescence microscopy confirmed that 800CW-PGN635 was binding to PS-positive tumor vascular endothelium. Our studies suggest that tumor vasculature can be successfully imaged in vivo to provide sensitive tumor detection.
A phospholipase C (PLC) which can hydrolyze 14 C-phosphatidylcholine was purified from bull seminal plasma. This PLC has an optimum at pH 7.2 and its PI was about 5.0. The enzyme was inhibited by EDTA, Cd 2+ , Pb 2+ , Ni 2+ , Fe 2+ , and Zn 2+ . PLC consists of two subunits one 69,000 and the other 55,000 daltons. The purified PLC was examined for induction of capacitation and acrosome reaction of guinea pig spermatozoa. Sperm were examined for the acrosome reaction 10 min after addition of 3.4 mM Ca 2+ . Fifty percent of the sperm underwent the acrosome reaction while the control had less than 5% acrosome reacted sperm. The antiserum to the inneracrosomal membrane isolated from sperm was labeled with FITC conjugated goat anti-guinea pig IgG. The conjugated antibody was used to localize sperm antigens. The antigens located on the IAM were only fluoresced when rabbit sperm were treated with methanol and/or MgCl 2 . Therefore anti-IAM antibody did not bind to the sperm plasma membrane. In vivo capacitated rabbit sperm were incubated with anti-IAM antibody (intact IgG and F(ab') 2 fragments) for 30 min prior to addition of rabbit eggs. After 24 h the eggs were examined for cleavage. The control eggs were fertilized (90%) while the antibody completely inhibited the fertilization of ova in vitro. The eggs incubated with antibody prior to the addition of sperm were still fertilizable. Thus, anti-IAM did not have any noticeable effect on the eggs. It was also shown that antibody inhibited fertilization of zona-free rabbit eggs in vitro as well
Ushijima, Miho; Uruno, Takehito; Nishikimi, Akihiko; Sanematsu, Fumiyuki; Kamikaseda, Yasuhisa; Kunimura, Kazufumi; Sakata, Daiji; Okada, Takaharu; Fukui, Yoshinori
A hallmark of humoral immune responses is the production of antibodies. This process involves a complex cascade of molecular and cellular interactions, including recognition of specific antigen by the B cell receptor (BCR), which triggers activation of B cells and differentiation into plasma cells (PCs). Although activation of the small GTPase Rac has been implicated in BCR-mediated antigen recognition, its precise role in humoral immunity and the upstream regulator remain elusive. DOCK2 is a Rac-specific guanine nucleotide exchange factor predominantly expressed in hematopoietic cells. We found that BCR-mediated Rac activation was almost completely lost in DOCK2-deficient B cells, resulting in defects in B cell spreading over the target cell-membrane and sustained growth of BCR microclusters at the interface. When wild-type B cells were stimulated in vitro with anti-IgM F(ab') 2 antibody in the presence of IL-4 and IL-5, they differentiated efficiently into PCs. However, BCR-mediated PC differentiation was severely impaired in the case of DOCK2-deficient B cells. Similar results were obtained in vivo when DOCK2-deficient B cells expressing a defined BCR specificity were adoptively transferred into mice and challenged with the cognate antigen. In addition, by generating the conditional knockout mice, we found that DOCK2 expression in B-cell lineage is required to mount antigen-specific IgG antibody. These results highlight important role of the DOCK2-Rac axis in PC differentiation and IgG antibody responses.
Käsermann, Fabian; Boerema, David J; Rüegsegger, Monika; Hofmann, Andreas; Wymann, Sandra; Zuercher, Adrian W; Miescher, Sylvia
It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG) might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA) lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1) or acidified lactose (E2) were analyzed for total IgG, F(ab')(2) and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2); again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10%) of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.
Full Text Available It has been proposed that the anti-inflammatory effects of intravenous immunoglobulin (IVIG might be due to the small fraction of Fc-sialylated IgG. In this study we biochemically and functionally characterized sialic acid-enriched IgG obtained by Sambucus nigra agglutinin (SNA lectin fractionation. Two main IgG fractions isolated by elution with lactose (E1 or acidified lactose (E2 were analyzed for total IgG, F(ab'(2 and Fc-specific sialic acid content, their pattern of specific antibodies and anti-inflammatory potential in a human in vitro inflammation system based on LPS- or PHA-stimulated whole blood. HPLC and LC-MS testing revealed an increase of sialylated IgG in E1 and more substantially in the E2 fraction. Significantly, the increased amount of sialic acid residues was primarily found in the Fab region whereas only a minor increase was observed in the Fc region. This indicates preferential binding of the Fab sialic acid to SNA. ELISA analyses of a representative range of pathogen and auto-antigens indicated a skewed antibody pattern of the sialylated IVIG fractions. Finally, the E2 fraction exerted a more profound anti-inflammatory effect compared to E1 or IVIG, evidenced by reduced CD54 expression on monocytes and reduced secretion of MCP-1 (CCL2; again these effects were Fab- but not Fc-dependent. Our results show that SNA fractionation of IVIG yields a minor fraction (approx. 10% of highly sialylated IgG, wherein the sialic acid is mainly found in the Fab region. The tested anti-inflammatory activity was associated with Fab not Fc sialylation.
Juçara C. Parra
Full Text Available Infection with Schistosoma mansoni induces humoral and T cell mediated responses and leads to delayed hipersensitivity that results in granulomatous inflamatory disease around the parasite eggs. Regulation of these responses resulting in a reduction in this anti-egg inflamatory disease is appsrently determined by idiotypic repertoires of the patient, associated with genetic background and multiple external factors. We have previously reported on idiotype/anti-idiotype-receptor transactions in clinical human schistosomiasis. These findings support a hypothesis that anti-SEA cross-reactive idiotypes develop in some patients during the course of a chronic infection and participate in regulation of anti-SEA cellular immune responses. We repport here on experiments wich extend those observations to the regulation of granulomatous hypersensitivity measured by an in vitro granuloma model. T cells from chronic intestinal schistosomiasis patients were stimulated in vitro with anti-SEA idiotypes and assayed in an autologous in vitro granuloma assay for modulation of granuloma formation. These anti-SEA idiotype reactive T cells were capable of regulating autologous in vitro granuloma formation. This regulatory activity, initiated with stimulatory anti-SEA idiotypic antibodies, was antigenically specific and was dependent on the present of intact (F(ab'2 immunoglobulin molecules. The ability to elicit this regulatory activity appears to be dose dependent and is more easily demonstrated in chronically infected intestinal patients or SEA sensitized individuals. These data support the hypothesis that anti-SEA cross reactive idiotypes are important in regulating granulomatous hypersensitivy in chronic intestinal schistosomiasis patients and these cross-reactive idiotypes appear to play a major role in cell-cell interactions which result in the regulation of anti-SEA cellular immune responses.
Schiff, D E; Kline, L; Soldau, K; Lee, J D; Pugin, J; Tobias, P S; Ulevitch, R J
THP-1-derived cell lines were stably transfected with constructs encoding glycophosphatidylinositol (GPI)-anchored or transmembrane forms of human CD14. CD14 expression was associated with enhanced phagocytosis of serum (heat-inactivated)-opsonized Escherichia coli (opEc). Both the GPI-anchored and transmembrane forms of CD14 supported phagocytosis of opEc equally well. Lipopolysaccharide-binding protein (LBP) played a role in CD14-dependent phagocytosis as evidenced by inhibition of CD14-dependent phagocytosis of opEc with anti-LBP monoclonal antibody (mAb) and by enhanced phagocytosis of E. coli opsonized with purified LBP. CD14-dependent phagocytosis was inhibited by a phosphatidylinositol (PI) 3-kinase inhibitor (wortmannin) and a protein tyrosine kinase inhibitor (tyrphostin 23) but not a protein kinase C inhibitor (bisindolyl-maleimide) or a divalent cation chelator (ethylenediaminetetraacetate). Anti-LBP mAb 18G4 and anti-CD14 mAb 18E12 were used to differentiate between the pathways involved in CD14-dependent phagocytosis and CD14-dependent cell activation. F(ab')2 fragments of 18G4, a mAb to LBP that does not block cell activation, inhibited ingestion of opEc by THP1-wtCD14 cells. 18E12 (an anti-CD14 mAb that does not block LPS binding to CD14 but does inhibit CD14-dependent cell activation) did not inhibit phagocytosis of LBP-opEc by THP1-wtCD14 cells. Furthermore, CD14-dependent phagocytosis was not inhibited by anti-CD18 (CR3 and CR4 beta-chain) or anti-Fcgamma receptor mAb.
Kayili, H Mehmet; Salih, Bekir
Hydrophobic silicon-based material having magnetic properties was fairly synthesized by a classical sol-gel approach. Pepsin enzyme was encapsulated in the sol-gel material and the enzyme activity was evaluated in consequence of the digestion of some common proteins such as α- and β-casein, cytochrome c, myoglobin, and bovine serum albumin (BSA) both in a single protein batch and in the protein mixture. The optimum digestion time of the studied proteins using pepsin-encapsulated magnetic sol-gel material was found to be 20min. To produce the magnetic sol-gel material for convenient and easy proteomics applications, Fe3O4 was doped inside sol-gel material during the gelation step. It was observed that the activity of encapsulated pepsin was not affected by the amount of Fe3O4. Poly(ethylene glycol) was also inserted in sol-gel bulk to obtain suitable roughness and increase the hydrophilicity of the material surface to let protein molecules reach to the sol-gel material easily. The digestion of the protein mixture and non-fat bovine milk was performed with the pepsin-encapsulated magnetic sol-gel material and the digested solutions were analyzed using SDS-PAGE, MALDI-TOF-MS and LC-MS/MS for the protein identification. Reusability of the pepsin-encapsulated sol-gel material was examined and it was determined that they could be used at least 20 times. Finally, IgG digestions with a fast incubation time period were carried out using pepsin-encapsulated sol-gel material for generation of (Fab)2 product to evaluate the kinetic performance of the material. Copyright © 2016 Elsevier B.V. All rights reserved.
Boutonnat, J; Bonnefoix, T; Mousseau, M; Seigneurin, D; Ronot, X
Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.
De Schryver, Marjorie; Leemans, Annelies; Pintelon, Isabel; Cappoen, Davie; Maes, Louis; Caljon, Guy; Cos, Paul; Delputte, Peter L
Sialoadhesin (Sn) is a surface receptor expressed on resident macrophages with the ability to bind with sialic acids. During inflammation, an upregulation of Sn is observed. Upon binding of monoclonal antibodies to Sn, the receptor becomes internalized and this has been observed in multiple species. The latter characteristic, combined with the strong upregulation of Sn on inflammatory macrophages and the fact that Sn-positive macrophages contribute to certain inflammatory diseases, makes Sn an interesting entry portal for phenotype-modulating or cytotoxic drugs. Such drugs or toxins can be linked to Sn-specific antibodies which should enable their targeted uptake by macrophages. However, the activity of such drugs depends not only on their internalization but also on the intracellular trafficking and final fate in the endolysosomal system. Although information is available for porcine Sn, the detailed mechanisms of human and mouse Sn internalization and subsequent intracellular trafficking are currently unknown. To allow development of Sn-targeted therapies, differences across species and cellular background need to be characterized in more detail. In the current report, we show that internalization of human and mouse Sn is dynamin-dependent and clathrin-mediated, both in primary macrophages and CHO cell lines expressing a recombinant Sn. In primary macrophages, internalized Sn-specific F(ab') 2 fragments are located mostly in the early endosomes. With Fc containing Sn-specific antibodies, there is a slight shift towards lysosomal localization in mouse macrophages, possibly because of an interaction with Fc receptors. Surprisingly, in CHO cell lines expressing Sn, there is a predominant lysosomal localization. Our results show that the mechanism of Sn internalization and intracellular trafficking is concurrent in the tested species. The cellular background in which Sn is expressed and the type of antibody used can affect the intracellular fate, which in turn can impact the activity of antibody-based therapeutic interventions via Sn. Copyright © 2016 Elsevier GmbH. All rights reserved.
Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F
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.
Ferro-Flores, G.; Garcia-Salinas, L.; Paredes-Gutierrez, L.; Hashimoto, K.; Melendez-Alafort, L.; Murphy, C.A.
A direct labelling technique via ethane-1-hydroxy-1,1-diphosphonic acid (EHDP) as a weak competing ligand was developed for the preparation of several biomolecules: 188 Re-monoclonal antibody ior cea1 against carcinoembryonic antigen ( 188 Re-MoAb), biotinylated 188 Re-MoAb ( 188 Re-MoAb-biotin), 188 Re-polyclonal IgG ( 188 Re-IgG), 188 Re-peptide (somatostatine analogue peptide b-(2-naphtyl)-D-Ala-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-amide), 188 Re-MoAb fragments ( 188 Re-F(ab') 2 ) and biotinylated 188 Re-F(ab') 2 ( 188 Re-F(ab') 2 -biotin). The reaction conditions such as pH, temperature, weak ligand concentration and stannous chloride concentration were optimized during the radiolabelling of each biomolecule. Before the labelling procedure, disulphide bridge groups of the biomolecules were reduced with 2-mercaptoethanol (2-ME). To obtain 188 Re labelled antibodies and peptides in high radiochemical yields (>90%) via EHDP, it was necessary to use acidic conditions and a high concentration of stannous chloride to allow the redox reaction Re +7 →Re +5 :Re +4 . The labelling of MoAb and F(ab') 2 with 188 Re via EHDP was also evaluated employing a pretargeted technique by avidin-biotin strategy in normal mice, demonstrating that the 188 Re-labelled biotinylated antibodies are stable complexes in vivo. The 188 Re-peptide complex prepared by this method, was stable for 24 h and no radiolytic degradation was observed. (author)
Ermakov, Evgeny A; Smirnova, Ludmila P; Bokhan, Nikolay A; Semke, Arkadiy V; Ivanova, Svetlana A; Buneva, Valentina N; Nevinsky, Georgy A
We present first evidence showing that some electrophoretically homogeneous IgGs from the sera of patients with schizophrenia (36.4%) and their Fab and F(ab)2 fragments as well as from healthy donors (33.3%) possess catalase activity. The relative catalase activity of IgGs from the sera of individual schizophrenia patients (and healthy donors) significantly varied from patient to patient, but the activity of IgGs from healthy donors is on average 15.8-fold lower than that for schizophrenia patients. After extensive dialysis of purified IgGs against EDTA chelating metal ions, the relative catalase activity of IgGs decreases on average approximately 2.5-3.7-fold; all IgGs possess metal-dependent and independent catalase activity. The addition of external Me2+ ions to dialyzed and non-dialyzed IgGs leads to a significant increase in their activity. The best activator of dialyzed and non-dialyzed IgGs is Co2+, the activation by Cu2+, Mn2+, and Ni2+ ions were rare and always lower than by Co2+. Every IgG preparation demonstrates several individual sets of very well expressed pH optima in the pH range from 4.0 to 9.5. These data speak for the individual repertoire of catalase IgGs in every person and an extreme diversity of abzymes in their pH optima and activation by different metal ions. It is known that antioxidant enzymes such as superoxide dismutases, catalases, and glutathione peroxidases represent critical defense mechanisms preventing oxidative modifications of DNA, proteins, and lipids. Catalase activity of human IgGs could probably also play a major role in the protection of organisms from oxidative stress and toxic compounds.
Happ, J.; Baum, R.P.; Frohn, J.; Weimer, B.; Hoer, G.; Halbsguth, A.; Lochner, B.; Brandhorst, I.
The present study was done in order to examine if the use of 111 In-DTPA-labeled MAb fragments in place of 131 I-labeled MAb fragments increases the sensitivity of tomographic immunoscintigraphy to reach the level of that of planar imaging techniques. In 11 patients with various primary tumors, local recurrences or metastases [colorectal carcinoma (n=7), ovarian carcinoma (n=2), papillary thyroid carcinoma (n=1), undifferentiated carcinoma of the lung (n=1)], immuniscintigraphy (IS) was carried out using 111 In-DTPA-labeled F(ab') 2 fragments of various MAbs (anti-CEA, OC 125, anti-hTG) and planar and tomographic imaging were compared intraindividually. By conventional diagnostic procedures, the presence of a tumor mass was confirmed (transmission computer tomography, ultrasound) or verified ( 131 I whole-body scintigraphy, histology) in all cases. Immunoscintigraphy was positive in 9 out of 11 cases by ECT and in 10 out of 11 cases by planar imaging. When using 111 In-labeled MAb fragments, intraindividual comparison of ECT and planar imaging resulted in a similar sensitivity. The increased sensitivity of ECT using this tracer in contrast to 131 I-labeled MAb fragments may be attributed to the fact that the physical properties of 111 In are much more suitable for the gamma cameras most commonly used (single detector, 3/8'' crystal); using 111 In-labelled MAb fragments, count rates sufficient for ECT can be obtained within a reasonable acquisition time. This allows to combine IS with the advantages of ECT regarding tumour localization and prevention of artefacts due to superposition of background. (orig.) [de
Takahashi, S; Maecker, H T; Levy, R
An anti-T cell receptor (TcR) monoclonal antibody (mAb), LC4, directed against a human leukemic T cell line, SUP-T13, caused DNA fragmentation ("apoptosis") and cell death upon binding to this cell line. Cross-linking of receptor molecules was necessary for this effect since F(ab')2, but not Fab', fragments of LC4 could induce cell death. Five anti-CD3 mAb tested also caused apoptosis, but only when they were presented on a solid phase. Interestingly, soluble anti-CD3 mAb induced calcium flux and had an additive effect on the calcium flux and interleukin 2 receptor expression induced by LC4, but these anti-CD3 mAb reversed the growth inhibition and apoptosis caused by LC4. The calcium ionophore A23187, but not the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), also induced apoptosis, suggesting that protein kinase C activation alone does not cause apoptosis, although PMA is growth inhibitory. These results suggest that two distinct biological phenomena can accompany stimulation of the TcR/CD3 complex. In both cases, calcium flux and interleukin 2 receptor expression is induced, but only in one case is apoptosis and cell death seen. The signal initiating apoptosis can be selectively prevented by binding CD3 portion of the receptor in this cell line. This difference in signals mediated by the TcR/CD3 complex may be important in explaining the process of thymic selection, as well as in choosing anti-TcR mAb for therapeutic use.
Bapat, K.; Venkatesh, M.; Pillai, M.R.A.; Sarma, H.D.; Sainis, K.B.
Monoclonal antibodies were raised against myosin, a specific indicator of myocardial infarction and labelled with 125 I and 99m Tc. Human cardiac myosin was isolated from normal human heart and was used for raising the monoclonal antibodies by the hybridoma technique. Antibody producing clones were identified by ELISA and cloning was done by the limiting dilution technique. Of the 13 clones obtained, 4 were deemed suitable for further studies. The antibodies were grown in ascites, purified, isotyped and their cross reactions with other forms of myosin were estimated. All the clones showed negligible cross reaction with rabbit myosin, but reacted to different extents with bovine skeletal myosin. The most avid antibody Mab-4G4 was chosen for further labelling studies. Mab-4G4 was labelled with 125 I using different oxidising agents such as iodogen, chloramine-T and lactoperoxidase. Purified radioiodinated antibody with radiochemical purity >95% could be obtained by gel filtration. Immunoreactivity was retained as tested by binding to myosin immobilised on a solid support. Mab-4G4 was also labelled with 99m Tc using stannous tartrate as the reducing agent. Radiolabelling yield was ∼60%, the purity was >95% and the immunoreactivity was retained. Both the labelled preparations were tested for bio-distribution in normal and infarcted rats. The activity accumulation in the infarcted region was ∼ 1.5 and 3.5 times as that in normal heart muscle for 125 I and 99m Tc labelled Mab-4G4 respectively. The major problem with the iodinated antibody was the in vivo deiodination resulting in very high percentage of activity in the thyroid. Although the fraction of the total activity associated with the infarcted heart is not very impressive, the fact that the activities with the infarcted and normal hearths are significantly different is heartening. With further optimisation of labelling and use of F(ab)'2 fragments, better delineation of the infarct sites is aspired. (author)
Schuhmacher, Jochen; Klivenyi, Gabor; Kaul, Sepp; Henze, Marcus; Matys, Ronald; Hauser, Harald; Clorius, John
We recently demonstrated the feasibility of combining enhanced tumor-to-tissue contrast and PET imaging for immunoscintigraphic tumor localization in pancreas and colon carcinoma bearing nude mice. Contrast enhancement was obtained with a multistep targeting technique that consists of the sequential administration of an antitumor/antihapten bispecific antibody (BS-MAb), a blocker to saturate the antihapten binding sites of the BS-MAb that remains in circulation, and a low molecular weight Ga chelate, labeled with the positron emitter 68 Ga, which serves as the hapten. To evaluate the efficacy of this pretargeting technique for breast cancer localization, we synthesized a BS-MAb from the F(ab') 2 fragments of the anti-MUC1 MAb 12H12 which reacts with the vast majority of human breast carcinomas, and the F(ab') fragment of an anti-Ga chelate MAb using a bifunctional chemical linker. The BS-MAb was tested for its affinity and its biokinetics in nude mice bearing a human mammary carcinoma. Equilibrium binding of the BS-MAb for mammary carcinoma cells was low (1.2 x 10 7 M -1 ) while the binding capacity of cells was high (8.4 x 10 6 BS-MAbs per cell). Tumor uptake of the 67 Ga labeled chelate in pretargeted animals was to 5.8 ± 0.8% iD/g resulting in a tumor-to-blood ratio of 2.6 at 1h postinjection. This compares with a ratio of 0.65 and 0.85 obtained with 125 I-labeled native 12H12 at 24h and 48h postinjection. No difference in the tumor uptake of both the 68 Ga and 67 Ga labeled chelate was observed. PET imaging of mice, started 1h postinjection of the 68 Ga chelate, clearly visualized all tumors
Croteau Nancy L
Full Text Available Abstract Background Obligate intracellular pathogens belonging to the Chlamydiaceae family possess a number of mechanisms by which to manipulate the host cell and surrounding environment. Such capabilities include the inhibition of apoptosis, down-regulation of major histocompatability complex (MHC and CD1/d gene expression, and the acquisition of host-synthesized nutrients. It is also documented that a limited number of host-derived macromolecules such as β-catenin and sphingomyelin accumulate within the inclusion. Results This report provides evidence that immunoglobulin, inherently present in the extracellular environment in vivo and in vitro, enters infected cells and accumulates within the chlamydial inclusion. Using epi-fluorescent and confocal microscopy, this selective uptake of Ig is shown to occur among human leukocytes in vivo as well as cells cultured in vitro. These findings were confirmed by detection of IgG in the lysate of infected cells by western blot hybridization. Sequestered antibodies appear to be present during the entire course of the chlamydial developmental cycle and are distributed throughout this compartment. IgG pre-labeled with fluorescein, when added to the supernatant of infected cell cultures, was also imported and readily visualized. Accumulation of these molecules within the inclusion and the failure of bovine serum albumin or F(ab'2 fragments to accumulate in a similar manner suggests the process of entry is specific for intact IgG molecules and not a result of pinocytosis, diffusion, or any other mass endocytic event. Conclusion Sequestration of a host cell-derived protein within the chlamydial inclusion, although unexpected, is not an unprecedented occurrence. However, selective accumulation of an exogenous host protein, such as extracellular IgG, has not been previously reported in connection with chlamydial infections. The selectivity of this process may indicate that this uptake plays an important role in pathogen physiology or virulence during infection and the phenomenon itself may give rise to novel diagnostic and therapeutic approaches.
de Graauw, E; Sitaru, C; Horn, M; Borradori, L; Yousefi, S; Simon, H-U; Simon, D
Bullous pemphigoid (BP) is an autoimmune bullous disease of the skin characterized by subepidermal blister formation due to tissue-bound and circulating autoantibodies to the hemidesmosomal antigens BP180 and BP230. Although eosinophils and their toxic mediators are found abundantly in BP lesions, their role in blister formation has remained unclear. To investigate the role of eosinophils in the pathogenesis of BP with a specific focus on blister formation and to define conditions inducing dermal-epidermal separation (DES). In an ex vivo human model of BP, normal human skin cryosections were incubated with purified human peripheral blood eosinophils with or without activation in the presence or absence of BP autoantibodies, brefeldin A, diphenyleneiodonium, DNase or blocking F(ab') 2 fragments to CD16, CD18, CD32 and CD64. Dermal-epidermal separation was assessed by light microscopy studies and quantified using Fiji software. Following activation with IL-5 and in the presence of BP autoantibodies, eosinophils induced separation along the dermal-epidermal junction of ex vivo skin. Dermal-epidermal separation was significantly reduced by blocking any of the following: Fcγ receptor binding (P = 0.048), eosinophil adhesion (P = 0.046), reactive oxygen species (ROS) production (P = 0.002), degranulation (P eosinophil extracellular trap (EET) formation (P = 0.048). Our results provide evidence that IL-5-activated eosinophils directly contribute to BP blister formation in the presence of BP autoantibodies. Dermal-epidermal separation by IL-5-activated eosinophils depends on adhesion and Fcγ receptor activation, requires elevated ROS production and degranulation and involves EET formation. Thus, targeting eosinophils may be a promising therapeutic approach for BP. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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.
Goettel, Wolfgang; Xia, Eric; Upchurch, Robert; Wang, Ming-Li; Chen, Pengyin; An, Yong-Qiang Charles
Variation in seed oil composition and content among soybean varieties is largely attributed to differences in transcript sequences and/or transcript accumulation of oil production related genes in seeds. Discovery and analysis of sequence and expression variations in these genes will accelerate soybean oil quality improvement. In an effort to identify these variations, we sequenced the transcriptomes of soybean seeds from nine lines varying in oil composition and/or total oil content. Our results showed that 69,338 distinct transcripts from 32,885 annotated genes were expressed in seeds. A total of 8,037 transcript expression polymorphisms and 50,485 transcript sequence polymorphisms (48,792 SNPs and 1,693 small Indels) were identified among the lines. Effects of the transcript polymorphisms on their encoded protein sequences and functions were predicted. The studies also provided independent evidence that the lack of FAD2-1A gene activity and a non-synonymous SNP in the coding sequence of FAB2C caused elevated oleic acid and stearic acid levels in soybean lines M23 and FAM94-41, respectively. As a proof-of-concept, we developed an integrated RNA-seq and bioinformatics approach to identify and functionally annotate transcript polymorphisms, and demonstrated its high effectiveness for discovery of genetic and transcript variations that result in altered oil quality traits. The collection of transcript polymorphisms coupled with their predicted functional effects will be a valuable asset for further discovery of genes, gene variants, and functional markers to improve soybean oil quality.
Full Text Available CD6 is associated with T-cell modulation and is implicated in several autoimmune diseases. We previously demonstrated that Itolizumab, a CD6 domain 1 (CD6D1 specific humanized monoclonal antibody, inhibited the proliferation and cytokine production by T lymphocytes stimulated with anti-CD3 antibody or when co-stimulated with ALCAM. Aberrant IL-17 producing CD4+ helper T-cells (Th17 have been identified as pivotal for the pathogenesis of certain inflammatory autoimmune disorders, including psoriasis. Itolizumab has demonstrated efficacy in human diseases known to have an IL-17 driven pathogenesis. Here, in in vitro experiments we show that by day 3 of human PBMC activation using anti-CD3 and anti-CD28 co-stimulation in a Th17 polarizing milieu, 15-35% of CD4+ T-cells overexpress CD6 and there is an establishment of differentiated Th17 cells. Addition of Itolizumab reduces the activation and differentiation of T cells to Th17 cells and decreases production of IL-17. These effects are associated with the reduction of key transcription factors pSTAT3 and RORγT. Further, transcription analysis studies in these conditions indicate that Itolizumab suppressed T cell activation by primarily reducing cell cycle, DNA transcription and translation associated genes. To understand the mechanism of this inhibition, we evaluated the effect of this anti-human CD6D1 mAb on ALCAM-CD6 as well as TCR-mediated T cell activation. We show that Itolizumab but not its F(ab'2 fragment directly inhibits CD6 receptor hyper-phosphorylation and leads to subsequent decrease in associated ZAP70 kinase and docking protein SLP76. Since Itolizumab binds to CD6 expressed only on human and chimpanzee, we developed an antibody binding specifically to mouse CD6D1. This antibody successfully ameliorated the incidence of experimental autoimmune encephalitis in the mice model. These results position CD6 as a key molecule in sustaining the activation and differentiation of T cells and an important target for modulating autoimmune diseases.
Full Text Available Abstract Background In recent few years is underlined that altered balance of pro- and anti-inflammatory cytokines play an important role in the pathogenesis of AITD. The aim of this study was to estimate intracellular INF-γ and IL-4 levels in thyroid-infiltrating lymphocytes and thyrocytes isolated from thyroid tissues in 54 adolescent patients aged 8-21 years, with Graves' disease (GD; n = 18, Hashimoto's thyroiditis (HT; n = 18 and non-toxic multinodular goiter (NTMG; n = 18. Methods Fresh thyroid tissues were taken on culture medium RPMI -1640, it was mechanically prepared. In next step were added cell activators -12- myristate 13- the acetate (PMA and Ionomycin as well as the inhibitor of transportation of proteins - Breferdin A. They were cultured 24 hours in 50 ml flasks at 37°C in a 5-95% CO2-air water-saturated atmosphere. After that, thyrocytes were identified by mouse mAb directed against human TPO epitope 64 conjugated with rabbit anti-mouse antibodies IgG (Fab'2 labeled by FITC. After incubation at room temperature to each of samples added reagent A fixative the cellular membrane. In next step into the cell suspensions were added reagent B to permeabilization of cellular membrane and specific anti-IL-4-PE or anti-IFN-γ-PE mAbs. Identification of intracellular cytokines in T lymphocytes was performed in the same procedure with application of anti-CD4-PerCP and anti-CD8-PerCP mAbs specific for T lymphocytes. The cells were analyzed in a flow cytometry (Coulter EPICS XL. Results In examined group of patients with GD we observed statistically significant higher mean percentage of cells with phenotype CD4+IL-4 (p Conclusions We conclude that human thyrocytes in autoimmune thyroid disorders could be a source of cytokine production and that their activation influences local interaction with T lymphocytes inflowing to the thyroid gland.
Wilbur, D.S.; Hamlin, D.K.; Quinn, J.; Vessella, R.L.
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
Park, Kyung Bae; Kim, Jae Rok; Awh, Ok Doo; Sin, Byung Cheul; Park, Woong Woo; Han, Kwang Hee
1. Development of 165 Dy-HMA for the treatment of rheumatoid arthritis 1) Irradiation of twelve mg of 164 Dy 2 O 3 showing specific activity of 2x10 13 n/cm 2 sec for four hours gave 165 Dy 2 O 3 showing specific activity(∼480 mCi/mg Dy 2 O 3 ) and radionuclidic purity(>99.9%). 2) 165 Dy-HMA was prepared in yield of 80 - 85% from the 165 DyCl 3 solution which was made by dissolving 165 Dy 2 O 3 with hydrochloric acid and adjusting pH to 3.0, and then followed by the treatment with aqueous sodium hydroxide solution. 3) Serial filtration using polycarbonate filter (1 - 10μm) of 165 Dy-HMA suspension in saline after treatment with sonificator exhibited that the majority of particles are in the 3 - 8μm range. 4) Even though the 165 Dy-HMA suspension in saline was left to stand for 24 hours at room temperature, there was no significant change in particle size resulting in high stability of 165 Dy-HMA. 2. Study on the 99mTc labelling of bioactive material 1) The labelling of antibody [F(ab') 2 ] coupled to DTPA with Na99mTcO 4 in the presence of sodium dithionite(μg) gave labelling yield of 40% determined by ITLC-SG. 2) The labelling of 500μl of antibody solution in phosphate buffer(0.2M, pH 7.4, 1.5mg antibody/ml) with Na 131 I(3 - 5mCi) in the presence of chloramine-T(0.14mg) for 30minutes at room temperature exibited labelling yield of 60 - 70%, radiochemical purity of 97%, specific activity of 1.2 - 3.5 mCi/mg, respectively, determined by ITLC-SG. 3) The results obtained from the animal experiment in rabbit to study the specificity and sensitivity of 131 I labelled antibody exhibited hot uptake until 72 hours in the case of tuberculous infection, with the highest target/background ratio(2.52) at 24 hours after injection of 131 I-F(ab') 2 4) In the case of rabbit infected with syphilitic orchitis, it exhibited the highest target/background ratio(3.51) at 2 hours after injection and showed fast decrease after 24 hours. (Author)
Saurat, J.H.; Schifferli, J.; Steiger, G.; Dayer, J.M.; Didierjean, L.
Since autoantibodies (Abs) to cytokines may modify their biologic activities, high-affinity binding factors for interleukin-1 alpha (IL-1 alpha BF) were characterized in human sera. IL-1 alpha BF was identified as IgG (1) by sucrose density-gradient centrifugation followed by immunodiffusion autoradiography, (2) by ligand-blotting method, (3) by ligand binding to affinity-immobilized serum IgG, and (4) by IgG affinity purification followed by sucrose density-gradient centrifugation. IL-1 alpha binding activity resided in the F(ab)2 fragment. The apparent equilibrium constant was in the range of IgG found after immunization with conventional antigens (i.e., 10(-9) to 10(-10) mol/L). Anti-IL-1 alpha IgG auto-Abs represented only an extremely small fraction of total IgG (less than 1/10(-5)). Some sera with IL-1 alpha BF and purified IgG thereof were able to inhibit by 96% to 98% the binding of human recombinant IL-1 alpha to its receptor on murine thymoma EL4-6.1 cells, whereas other sera did not. When 125I-labeled anti-IL-1 alpha IgG complexes were injected into rats, they prolonged the plasma half-life of 125I-labeled IL-1 alpha several fold and altered its tissue distribution. The predominant class was IgG (12/19), mainly IgG4 (9/19), but in five of the sera, anti-IL-1 alpha IgA was also detected. In a screening of 271 sera, IL-1 alpha BF was detected in 17/98 normal subjects and was not more frequent in several control groups of patients, except in patients with Schnitzler's syndrome (fever, chronic urticaria, bone pain, and monoclonal IgM paraprotein) (6/9; p less than 0.005). The pathologic significance of these auto-Abs remains to be determined
ZALUTSKY, MICHAEL R.
The general objective of this research is to combine genetically derived molecular constructs reactive with tenascin, with appropriate radionuclides and labeling methods in order to generate more effective diagnostic and therapeutic reagents for oncologic nuclear medicine. Tenascin, a polymorphic extracellular matrix glycoprotein, is of interest because of its high expression on glioma, melanoma, as well as prostate and breast carcinoma. Recently, we have also documented high levels of tenascin in lymphomas, particularly those of higher grade, making the potential clinical impact of tenascin-specific radiodiagnostics and therapeutics even greater. An essential feature of our work plan is the ability to exploit our extensive clinical experience in order to design second-generation constructs with properties which could improve clinical efficacy. To date, we have treated over 150 brain tumor patients with 131I-labeled murine 81C6, an antibody which binds specifically to the alternatively spliced fibronectin type III repeats CD of the tenascin molecule. During the current grant period, we have made several observations which form the basis for our proposed specific aims. First, tissue distribution and catabolism experiments in animal models have demonstrated enhanced stability for a chimeric construct composed of murine variable regions and human IgG2 constant domains. Furthermore, pharmacokinetic studies in patients with 131I-labeled chimeric 81C6 have shown significantly longer retention in glioma tumor resection cavities compared with its murine parent. Second, we have initiated the first clinical trial of an endoradiotherapeutic labeled with the 7.2-hr ?-particle emitter 211At. Twelve glioma patients have received 211At-labeled chimeric 81C6 directly into their brain tumor resection cavity, and very encouraging results have been obtained. Now that the feasibility of human studies with 211At, has been demonstrated, the development and evaluation of anti-tenascin constructs with optimized properties for use in tandem with short half life radionuclides such as 211At (as well as 1.8-hr 18F for PET imaging) is warranted. Our specific aims are: (1) to construct a bivalent, anti-tenascin molecule containing murine 81C6 variable regions and the human IgG2 hinge region. Both the CH2 domain deletion construct (?CH2) and F(ab?)2 will be investigated; (2) to construct a single-chain Fv dimer or multimer with adequate stability, affinity and immunoreactivity for use in tandem with 211At for therapy and 18F for imaging; (3) to generate higher affinity scFv constructs reactive with the alternatively spliced fibronectin type III repeats CD of the tenascin molecule via phage display technology and site-directed mutagenesis; (4) to label promising anti-tenascin constructs with radioiodine, 211At, and 18F and evaluate their potential as radiodiagnostic and radiotherapeutic agents. The proposed studies include: characterization of affinity and immunoreactivity after labeling; evaluation of tissue distribution and projected dosimetry in normal mice, and athymic rodents with subcutaneous, intracranial and neoplastic meningitis xenografts; investigation of the nature of low and high molecular weight labeled catabolites generated in mice; and assessment of cytotoxicity in vitro and in vivo models of human glioma, and possibly, other tenascin expressing tumors; and (5) to investigate strategies for labeling scFv monomers and dimers which will minimize retention of the radiohalogen in the kidneys through the use of negatively charged templates
Full Text Available Abstract Background Human monoclonal antibodies (MAbs are needed for colon cancer radioimmunotherapy (RIT to allow for repeated injections. Carcinoembryonic antigen (CEA being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs. Methods XenoMouse®-G2 animals were immunized with CEA. Among all the antibodies produced, two of them, VG-IgG2κ and VG-IgM, were selected for characterization in vitro in comparison with the human-mouse chimeric anti-CEA MAb X4 using flow cytometry, surface plasmon resonance, and binding to radiolabeled soluble CEA and in vivo in human colon carcinoma LS174T bearing nude mice. Results Flow cytometry analysis demonstrated binding of MAbs on CEA-expressing cells without any binding on NCA-expressing human granulocytes. In a competitive binding assay using five reference MAbs, directed against the five Gold CEA epitopes, VG-IgG2κ and VG-IgM were shown to be directed against the Gold 4 epitope. The affinities of purified VG-IgG2κ and VG-IgM were determined to be 0.19 ± 0.06 × 108 M-1 and 1.30 ± 0.06 × 108 M-1, respectively, as compared with 0.61 ± 0.05 × 108 M-1 for the reference MAb X4. In a soluble phase assay, the binding capacities of VG-IgG2κ and VG-IgM to soluble CEA were clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10-7 M was needed to precipitate approximatively 1 ng 125I-rhCEA as compared with 10-9 M for MAb X4, suggesting a preferential binding of the human MAbs to solid phase CEA. In vivo, 24 h post-injection, 125I-VG-IgG2κ demonstrated a high tumor uptake (25.4 ± 7.3%ID/g, close to that of 131I-X4 (21.7 ± 7.2%ID/g. At 72 h post-injection, 125I-VG-IgG2κ was still concentrated in the tumor (28.4 ± 11.0%ID/g whereas the tumor concentration of 131I-X4 was significantly reduced (12.5 ± 4.8%ID/g. At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. A pertinent analysis of VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver. Conclusion Our human anti-CEA IgG2κ is a promising candidate for radioimmunotherapy in intact form, as F(ab'2 fragments, or as a bispecific antibody.
R. A. Vinogradov
differences (p>0.05. MMSE3 showed a significant improvement in the test results in each group compared to MMSE1 and MMSE2. MMSE3 results were significantly better (p<0.01 in group 1 than in group 2. MoCA revealed most significant differences in group 1 results with a significant decrease in cognitive dysfunction both in MoCA1, MoCA2, MoCA3 either within the group (p><0.05 or compared to MoCA3 data between CE group and TBA group (p><0.01. FAB showed that the FAB2 test amounted to 16.2 points in the group with CE and 14.6 points in the group with TBA of ICAs (p><0.05. A significant improvement in the performance of mnestic functions was noted when examining patients in the dynamics of FAB3: 17.3 and 15.6 points (p><0.05, respectively. According to Hamilton Depresion Rating Scale and Beck Depression Inventory, both groups of patients showed a moderate level of anxiety and depression (12.4 and 15.8 according to Hamilton Depression Rating Scale (p><0.05, 12.3 and 14.4 according to Beck Depression Inventory (p>0.05. During the second test, the depressive mood of patients was reduced (on the Hamilton scale 8.4 and 13.8 points.CONCLUSION When comparing HMFs in patients who underwent a different surgical approach (CE vs TBA of ICAs in the treatment of obliterating atherosclerotic lesions of ICAs, we found that: 1 the maximum improvement in HMFs appears by the 30th day of the postoperative period in comparison with preoperative parameters; 2 the most significant improvement HMFs test parameters by the 30th day of the postoperative period is noted in the group where CE was used as the method of surgical revascularization of the brain.
This review will cover the efforts in Gothenburg to evaluate the potential of 211 At radioimmunotherapy (RIT) in the treatment of small tumor deposits of ovarian cancer in the abdominal cavity. The lifetime risk of ovarian cancer is 1% - 2% in European and American women. Despite seemingly successful surgery followed by chemotherapy, most patients will relapse, most frequently in the abdominal cavity, and succumb to the disease. Despite newer systemic chemotherapy regimens, the outcome has not improved over the past decade. RIT with various β-emitters has displayed promising results, though an international Phase III study of 90Y-labeled antibody showed no improvement in time to relapse or survival. This disappointing result might be explained by the long range of β-emitters, which results in poor irradiation of tumors less than a few millimeters in size. In treating small tumors, the short range and high LET of α-emitters such as 211 At offer a significant advantage by more effectively irradiating targeted small cell clusters. The PET and Cyclotron Unit at Rigshospitalet in Copenhagen has regularly since ∼10 years delivered 211 At to the research group in Gothenburg led by Prof. Ragnar Hultborn and Prof. Lars Jacobsson. Astatine-211 is isolated from the irradiated target by dry distillation. The 211 At-labelling method gives stable radiochemical yields of 70% - 80% with the antibody conjugate's tumor-cell binding ability essentially preserved. The activity of an antibody batch of 0.1 - 0.5 mg is approximately 300 - 500 MBq, sufficient for extensive animal experiments or for treatment of one patient. The therapeutic effect has been studied in a series of experiments in vitro and in nude mice with intraperitoneal (i.p.) growth of microscopic ovarian cancer tumors. A number of parameters related to the injected antibody conjugate and stage of tumor growth have been investigated. Studies of toxic effects for bone-marrow, kidneys, and the peritoneal membrane indicate that microscopic tumors smaller than approximately 0.1 mm are likely sterilized without any serious organ toxicity. Tumor cure probability decreases with increasing tumor size. Dosimetry, based on biokinetic modeling and a Monte Carlo program, indicates that an absorbed dose of approximately 20 Gy is needed for tumor eradication in nude mice. The tolerance level (mean absorbed dose) is estimated to be ∼0.5 Gy for bone-marrow and ∼10 Gy for kidneys. For the peritoneal membrane preliminary results indicate a tolerance level of more than ∼25 Gy. Comparisons with low-LET 60Co irradiation for tumor-growth inhibition and bone-marrow toxicity both resulted in an RBE of ∼5. Based on the promising results of the animal studies, a clinical Phase I study of 9 patients was started in 2005 (and published in 2009). Thirty to 120 MBq of 211 At-MX35 F(ab')2 was administered i.p. in 1.1 - 2.2 L of fluid (Extraneal). Dosimetric calculations were mainly based on the 211 At activity in samples of peritoneal fluid, blood, and urine 0 - 48 h post injection. Gamma camera imaging did not reveal uptake in any major organs except the thyroid. The thyroid uptake was reduced by potassium perchlorate or potassium iodide in the last four patients. No adverse effects of the treatment were observed subjectively or in the laboratory parameters. In conclusion, therapeutic absorbed doses of 211 At in microscopic tumors in the abdominal cavity of humans are achievable without significant toxicity. (author)