Bio markers and Anti-EGFR therapies for Krads wild-type tumors in metastatic colorectal cancer patients; Biomarcadores y terapeutica ANTI-EGFR en el cancer colorrectal metastasico en pacientes con K-Ras no mutado

Energy Technology Data Exchange (ETDEWEB)

Diaz Rubio Garcia, E

2009-07-01

The natural history of metastasis colorectal cancer has being clearly modified in terms of response rate, time to progression and overall survival, once the anti-EGFR monoclonal antibodies (cetuximab and panitumumab) have emerged in combination with the standard cytotoxic chemotherapy (FOLFOX and FOLFIRI). However, the benefit from cetuximab and panitumumab is only confined to KRAS-wild type (KRAS-wt) colorectal tumors, while KRAS mutated tumors do not respond to these drugs. The 65 % of colorectal tumors are KRAS-wt tumors, but efficacy of antiEGFR therapies is detected only in 60-70 % of these KRAS-wt tumors. Other biomarkers and molecular pathways must be involved in the response of the antiEGFR therapies for the KRAS-wt colorectal tumors, such as the EGFR ligands, the EGFR-phosphorilated levels, the number of EGFR copies, the status of the KRAS effected B-RAF and the alternative intracellular signaling pathways PIK3CA/PTEN/AKT and JAK/STAT. A battery of these biomarkers is needed to select the most sensitive patients to the antiEGFR therapies. This pattern may represent a novel favorable cost-effectiveness tool to develop tailored treatments. A review of these biomarkers and molecular pathways, involved in the antiEGFR therapies response, is performed. (Author) 68 refs.

BDNF/TrkB signaling protects HT-29 human colon cancer cells from EGFR inhibition

Energy Technology Data Exchange (ETDEWEB)

Brunetto de Farias, Caroline [Cancer Research Laboratory, University Hospital Research Center (CPE-HCPA), Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, RS (Brazil); Children' s Cancer Institute, 90420-140 Porto Alegre, RS (Brazil); Laboratory of Neuropharmacology and Neural Tumor Biology, Department of Pharmacology, Institute for Basic Health Sciences, Federal University of Rio Grande do Sul, 90050-170 Porto Alegre, RS (Brazil); National Institute for Translational Medicine (INCT-TM), 90035-003 Porto Alegre, RS (Brazil); Heinen, Tiago Elias; Pereira dos Santos, Rafael [Cancer Research Laboratory, University Hospital Research Center (CPE-HCPA), Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, RS (Brazil); Laboratory of Neuropharmacology and Neural Tumor Biology, Department of Pharmacology, Institute for Basic Health Sciences, Federal University of Rio Grande do Sul, 90050-170 Porto Alegre, RS (Brazil); National Institute for Translational Medicine (INCT-TM), 90035-003 Porto Alegre, RS (Brazil); Abujamra, Ana Lucia [Cancer Research Laboratory, University Hospital Research Center (CPE-HCPA), Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, RS (Brazil); Children' s Cancer Institute, 90420-140 Porto Alegre, RS (Brazil); National Institute for Translational Medicine (INCT-TM), 90035-003 Porto Alegre, RS (Brazil); Schwartsmann, Gilberto [Cancer Research Laboratory, University Hospital Research Center (CPE-HCPA), Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, RS (Brazil); National Institute for Translational Medicine (INCT-TM), 90035-003 Porto Alegre, RS (Brazil); Department of Internal Medicine, School of Medicine, Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, RS (Brazil); and others

2012-08-24

Highlights: Black-Right-Pointing-Pointer BDNF protected HT-29 colorectal cancer cells from the antitumor effect of cetuximab. Black-Right-Pointing-Pointer TrkB inhibition potentiated the antitumor effect of cetuximab. Black-Right-Pointing-Pointer BDNF/TrkB signaling might be involved in resistance to anti-EGFR therapy. -- Abstract: The clinical success of targeted treatment of colorectal cancer (CRC) is often limited by resistance to anti-epidermal growth factor receptor (EGFR) therapy. The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB have recently emerged as anticancer targets, and we have previously shown increased BDNF levels in CRC tumor samples. Here we report the findings from in vitro experiments suggesting that BDNF/TrkB signaling can protect CRC cells from the antitumor effects of EGFR blockade. The anti-EGFR monoclonal antibody cetuximab reduced both cell proliferation and the mRNA expression of BDNF and TrkB in human HT-29 CRC cells. The inhibitory effect of cetuximab on cell proliferation and survival was counteracted by the addition of human recombinant BDNF. Finally, the Trk inhibitor K252a synergistically enhanced the effect of cetuximab on cell proliferation, and this effect was blocked by BDNF. These results provide the first evidence that increased BDNF/TrkB signaling might play a role in resistance to EGFR blockade. Moreover, it is possible that targeting TrkB could potentiate the anticancer effects of anti-EGFR therapy.

Clearance of 131I-labeled murine monoclonal antibody from patients' blood by intravenous human anti-murine immunoglobulin antibody

International Nuclear Information System (INIS)

Stewart, J.S.; Sivolapenko, G.B.; Hird, V.; Davies, K.A.; Walport, M.; Ritter, M.A.; Epenetos, A.A.

1990-01-01

Five patients treated with intraperitoneal 131I-labeled mouse monoclonal antibody for ovarian cancer also received i.v. exogenous polyclonal human anti-murine immunoglobulin antibody. The pharmacokinetics of 131I-labeled monoclonal antibody in these patients were compared with those of 28 other patients receiving i.p.-radiolabeled monoclonal antibody for the first time without exogenous human anti-murine immunoglobulin, and who had no preexisting endogenous human anti-murine immunoglobulin antibody. Patients receiving i.v. human anti-murine immunoglobulin antibody demonstrated a rapid clearance of 131I-labeled monoclonal antibody from their circulation. The (mean) maximum 131I blood content was 11.4% of the injected activity in patients receiving human anti-murine immunoglobulin antibody compared to 23.3% in patients not given human anti-murine immunoglobulin antibody. Intravenous human anti-murine immunoglobulin antibody decreased the radiation dose to bone marrow (from 131I-labeled monoclonal antibody in the vascular compartment) 4-fold. Following the injection of human anti-murine immunoglobulin antibody, 131I-monoclonal/human anti-murine immunoglobulin antibody immune complexes were rapidly transported to the liver. Antibody dehalogenation in the liver was rapid, with 87% of the injected 131I excreted in 5 days. Despite the efficient hepatic uptake of immune complexes, dehalogenation of monoclonal antibody was so rapid that the radiation dose to liver parenchyma from circulating 131I was decreased 4-fold rather than increased. All patients developed endogenous human anti-murine immunoglobulin antibody 2 to 3 weeks after treatment

Dexamethasone-(C21-phosphoramide-[anti-EGFR]: molecular design, synthetic organic chemistry reactions, and antineoplastic cytotoxic potency against pulmonary adenocarcinoma (A549

Directory of Open Access Journals (Sweden)

Coyne CP

2016-08-01

Full Text Available Cody P Coyne,1 Lakshmi Narayanan2 1Department of Basic Sciences, 2Department of Clinical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS, USA Purpose: Corticosteroids are effective in the management of a variety of disease states, such as several forms of neoplasia (leukemia and lymphoma, autoimmune conditions, and severe inflammatory responses. Molecular strategies that selectively “target” delivery of corticosteroids minimize or prevents large amounts of the pharmaceutical moiety from passively diffusing into normal healthy cell populations residing within tissues and organ systems. Materials and methods: The covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide-[anti-EGFR] was synthesized by reacting dexamethasone-21-monophosphate with a carbodiimide reagent to form a dexamethasone phosphate carbodiimide ester that was subsequently reacted with imidazole to create an amine-reactive dexamethasone-(C21-phosphorylimidazolide intermediate. Monoclonal anti-EGFR immunoglobulin was combined with the amine-reactive dexamethasone-(C21-phosphorylimidazolide intermediate, resulting in the synthesis of the covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide-[anti-EGFR]. Following spectrophotometric analysis and validation of retained epidermal growth factor receptor type 1 (EGFR-binding avidity by cell-ELISA, the selective anti-neoplasic cytotoxic potency of dexamethasone-(C21-phosphoramide-[anti-EGFR] was established by MTT-based vitality stain methodology using adherent monolayer populations of human pulmonary adenocarcinoma (A549 known to overexpress the tropic membrane receptors EGFR and insulin-like growth factor receptor type 1. Results: The dexamethasone:IgG molar-incorporation-index for dexamethasone-(C21-phosphoramide-[anti-EGFR] was 6.95:1 following exhaustive serial microfiltration. Cytotoxicity analysis: covalent bonding of dexamethasone to monoclonal anti-EGFR immunoglobulin

Use of Re-188 labelled anti-EGFr humanized monoclonal antibody h-R3 for radioimmunotherapy of gliomas

International Nuclear Information System (INIS)

Perera, A.; Torres, L.A.; Lopez, G.; Casaco, A.; Batista, J.F.; Pena, Y.; Coca, M.A.; Leyva, R.; Garcia, I.

2007-01-01

MBq of 188Re. In patients included in the group I transitory worsening of p re-existing neurological symptoms were observed. In this group: 1 patient with glioblastoma multiforme (GBM) died in progression after 6 months of treatment, 2 patients (one with GBM and one with anaplastic astrocitoma) developed stable disease during 3 months. One GBM patient had partial response for more than 1 year and 2 patients (one with GBM and one with anaplastic astrocitoma) were asymptomatic and in complete response after 3 years of treatment. No patient developed a HAMA response. Conclusions: Proposed procedure allowed the stable efficient labelling of h-R3 with 188Re. Preliminary results of this study strongly suggest that loco-regional radioimmunotherapy of high grade glioma using the anti-EGFr humanized monoclonal antibody h-R3 labelled with 188Re may be safe and constitute a promising therapeutic approach for these patients. (author)

Bio markers and Anti-EGFR therapies for Krads wild-type tumors in metastatic colorectal cancer patients

International Nuclear Information System (INIS)

Diaz Rubio Garcia, E.

2009-01-01

The natural history of metastasis colorectal cancer has being clearly modified in terms of response rate, time to progression and overall survival, once the antiEGFR monoclonal antibodies (cetuximab and panitumumab) have emerged in combination with the standard cytotoxic chemotherapy (FOLFOX and FOLFIRI). However, the benefit from cetuximab and panitumumab is only confined to KRAS-wild type (KRAS-wt) colorectal tumors, while KRAS mutated tumors do not respond to these drugs. The 65 % of colorectal tumors are KRAS-wt tumors, but efficacy of antiEGFR therapies is detected only in 60-70 % of these KRAS-wt tumors. Other biomarkers and molecular pathways must be involved in the response of the antiEGFR therapies for the KRAS-wt colorectal tumors, such as the EGFR ligands, the EGFR-phosphorilated levels, the number of EGFR copies, the status of the KRAS effected B-RAF and the alternative intracellular signaling pathways PIK3CA/PTEN/AKT and JAK/STAT. A battery of these biomarkers is needed to select the most sensitive patients to the antiEGFR therapies. This pattern may represent a novel favorable cost-effectiveness tool to develop tailored treatments. A review of these biomarkers and molecular pathways, involved in the antiEGFR therapies response, is performed. (Author) 68 refs.

Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

International Nuclear Information System (INIS)

Berger, Christian; Madshus, Inger Helene; Stang, Espen

2012-01-01

The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: ► Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. ► Antibody combination causes internalization of EGFR by macropinocytosis. ► Antibody-induced internalization of EGFR is independent of EGFR kinase activity. ► Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

Dual Inhibition of EGFR with Afatinib and Cetuximab in Kinase Inhibitor-Resistant EGFR-Mutant Lung Cancer with and without T790M Mutations

NARCIS (Netherlands)

Janjigian, Yelena Y.; Smit, Egbert F.; Groen, Harry J. M.; Horn, Leora; Gettinger, Scott; Camidge, D. Ross; Riely, Gregory J.; Wang, Bushi; Fu, Yali; Chand, Vikram K.; Miller, Vincent A.; Pao, William

EGFR-mutant lung cancers responsive to reversible EGFR inhibitors (gefitinib/erlotinib) develop acquired resistance, mediated by second-site EGFR T790M mutation in >50% of cases. Preclinically, afatinib (irreversible ErbB family blocker) plus cetuximab (anti-EGFR monoclonal antibody) overcomes

Development of freeze-dried kit for direct 99mTc -labeling of nimotuzumab to diagnose human EGFR positive tumors

International Nuclear Information System (INIS)

Toledo, Darien; Figueiras, Jenneby; Rojas, Gertrudis; León, Kalet; Gongora Bravo, Magdiel; Miguel Martínez, Antonio; Michel Alonso, Luis; Hernández, Ignacio; León, Mariela; Leyva, René; Hernández, Gerardo Ramses

2016-01-01

Epidermal growth factor receptor (EGFR), a 170 kDa transmembrane tyrosine kinase receptor which specifically binds epidermal growth factor (EGF) and transforming growth factor-a (TGFa) that are crucial in signaling cell proliferation, differentiation, and survival1. Overexpression of EGFR has been observed in breast cancer2, colorectal cancer, ovarian cancer, squamous cell lung carcinoma3 head and neck cancer4 and bladder cancer. Molecular imaging using radiopharmaceuticals directed towards EGFR could characterize the receptor status of tumors and thereby predict response to anti-EGFR agents for the treatment of cancer. Nimotuzumab is a humanized anti-EGFR monoclonal antibody5 designed to reduce immunogenicity and rate of clearance from the body6. Radiolabelled formulations of nimotuzumab would have applications for non-invasive imaging in order to characterize EGFR-positive tumors and thus to select patient populations that could benefit from therapy7, 8. The studies described in this work were designed to develop and evaluate the in vitro and in vivo the properties of a radiolabeled freeze dried kit of nimotuzumab and to determine its potential for radio immunodiagnostic applications. (author)

A bi-paratopic anti-EGFR nanobody efficiently inhibits solid tumour growth

Science.gov (United States)

Roovers, Rob C.; Vosjan, Maria J.W.D.; Laeremans, Toon; el Khoulati, Rachid; de Bruin, Renée C.G.; Ferguson, Kathryn M.; Verkleij, Arie J.; van Dongen, Guus A.M.S.; van Bergen en Henegouwen, Paul M. P.

2014-01-01

The epidermal growth factor receptor (EGFR) has been shown to be a valid cancer target for antibody-based therapy. At present, several anti-EGFR monoclonal antibodies (mAbs) have been successfully used, among which cetuximab and matuzumab. X-ray crystallography data show that these antibodies bind to different epitopes on the ecto-domain of EGFR, providing a rationale for the combined use of these two antibody specificities. We have previously reported on the successful isolation of antagonistic anti-EGFR nanobodies. In the present study, we aimed to improve on these molecules by combining nanobodies with specificities similar to both cetuximab and matuzumab into a single bi-paratopic molecule. Carefully designed phage nanobody selections resulted in two sets of nanobodies that specifically blocked the binding of either matuzumab or of cetuximab to EGFR and that did not compete for each others binding. A combination of nanobodies from both epitope groups into the bi-paratopic nanobody CONAN-1 was shown to block EGFR activation more efficiently than monovalent or bivalent (monospecific) nanobodies. In addition, this bi-paratopic nanobody potently inhibited EGF-dependent cell proliferation. Importantly, in an in vivo model of athymic mice bearing A431 xenografts, CONAN-1 inhibited tumour outgrowth with an almost similar potency as the whole mAb cetuximab, despite the fact that CONAN-1 is devoid of an Fc portion that could mediate immune effector functions. Compared to therapy using bivalent, mono-specific nanobodies, CONAN-1 was clearly more potent in tumour growth inhibition. These results show that the rational design of bi-paratopic nanobody-based anti-cancer therapeutics may yield potent lead molecules for further development. PMID:21520037

Production of human anti-HLA monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Walker, M.C.; Mercier, F.; Roger, J.; Varin, M.

1986-03-01

Only 40% of the several hundred anti-HLA murine monoclonal antibodies (MAbs) that have been made detect HLA-A,B,C or DR specificities previously defined by human alloantisera, the range of recognized specificities is very narrow, and few of the MAbs have proven useful as tissue typing reagents. In hopes of obtaining HLA typing reagents, the authors are developing a protocol for the production of human anti-HLA MAbs from HLA-antigen (Ag) immunized peripheral blood B cells of volunteering renal patients, immunized to one or more HLA Ags through therapeutic blood transfusions. A simple enrichment of the donor B cells has not been sufficient for anti-HLA MAb production, the authors are currently delineating the conditions necessary for increasing the number of HLA-specific donor B cells by in vitro stimulation with cells expressing the HLA Ag to which the B cell donor is immunized. For the production of MAbs, the stimulated B cells are transformed with Epstein-Barr virus and subsequently fused with KR-4 lymphoblastoid cells. Hybridomas are selected by HAT and Ouabain. Supernatants are screened for anti-HLA activity against lymphocyte targets expressing the original immunizing HLA Ag by complement mediated /sup 51/Cr release assay. Antibody specificity is determined by the complement-dependent microcytotoxicity test used for HLA typing.

Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

Energy Technology Data Exchange (ETDEWEB)

Berger, Christian [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Madshus, Inger Helene [Institute of Pathology, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway); Stang, Espen, E-mail: espsta@rr-research.no [Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo (Norway)

2012-12-10

The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.

[Study of anti-idiotype antibodies to human monoclonal antibody].

Science.gov (United States)

Harada, R; Takahashi, N; Owaki, I; Kannagi, R; Endo, N; Morita, N; Inoue, M

1992-02-01

A human monoclonal antibody, ll-50 (IgM, lambda), was generated, which reacted specifically with a major of glycolipid present in LS174T colon cancer cells. The glycolipid antigen which reacted with the ll-50 antibody was expected to four sugar residues from its TLC mobility, and it was ascertained that the glycolipid antigen which reacted with ll-50 antibody might be Lc4 antigen [Gal beta 1----3 GLcNAc beta 1----3 Gal beta 1----4 Glc beta 1----1 Cer] judging from TLC immunostaining and ELISA when the reactivity of ll-50 antibody was tested using various pure glycolipids in 3-5 sugar residues as an antigen. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated ll-50 antibody. The serum of the Lc4 antigen recognized by ll-50 antibody was significantly higher in patients with malignant disorders than that in healthy individuals (p less than 0.05). Three mouse monoclonal anti-idiotype antibodies, G3, B3 and C5 (all IgG1), were generated by the immunization of BALB/c mice with ll-50 antibody. These anti-idiotype antibodies specifically bound to to human monoclonal antibody, ll-50 and had a significant inhibitory activity towards the binding of ll-50 antibody to the Lc4 antigen. This indicated that these anti-idiotype antibodies, G3, B3, and C5, were paratope-related anti-idiotype antibodies. G3, B3, and C5 were expected to define the nearest idiotope because they could mutually inhibit ll-50 antibody. Sera in patients with malignant disorders and healthy individuals were analyzed by Sandwich assay of immobilized and biotinylated anti-idiotype antibodies, G3, B3, and C5. As to the ll-50 like antibodies defined by C5 (Id-C5+), the mean serum level in patients with malignant disorders was significantly higher than that in healthy individuals (p less than 0.05). As to the ll-50 like antibodies defined by B3 (Id-B3+), the mean serum level in patients with malignant disorders was significantly higher

EGFR targeting monoclonal antibody combines with an mTOR inhibitor and potentiates tumor inhibition by acting on complementary signaling hubs

International Nuclear Information System (INIS)

James, Roshan; Vishwakarma, Siddharth; Chivukula, Indira V; Basavaraj, Chetana; Melarkode, Ramakrishnan; Montero, Enrique; Nair, Pradip

2012-01-01

Nimotuzumab, an anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody, has been used extensively in many solid tumors and confers significant survival advantage. The antibody has limited skin toxicity and is generally well tolerated. Similar to other anti-EGFR therapies, patients may relapse a few months after treatment. In this study we show for the first time, the use of Nimotuzumab along with Sirolimus has synergistic effect on tumor inhibition as compared with the drugs used individually, in Nimotuzumab responsive and nonresponsive cell lines. In vitro studies prove that while Sirolimus (25 nmol/L) affects the signal downstream to mammalian target of rapamycin (mTOR), Nimotuzumab (83 nmol/L) downregulates pTYR, pMAPK and pSTAT3 by 40%, 20% and 30%, respectively. The combination, targeting these two different signaling hubs, may be associated with the synergistic inhibition observed. In vivo, the use of half human therapeutic equivalent doses for both the drugs substantially reduces tumors established in nude as well as severe combined immunodeficiency (SCID) mice by EGFR overexpressing A-431 cells. The drug combination reduces cell proliferation and the expression of signal transduction molecules. Treated tumors are better differentiated as compared with those established in the control mice. Tumor microarray demonstrates that Nimotuzumab and the combination groups segregate independently to the Sirolimus and the control treatment. The combination uniquely downregulated 55% of the altered tumor genes, extending beyond the typical pathways associated with Nimotuzumab and Sirolimus downstream pathways inhibition. These results would suggest that this nontoxic drug combination improves therapeutic benefit even in patients with low-EGFR expression and severely immunocompromised because of their current medication

Differences in human skin between the epidermal growth factor receptor distribution detected by EGF binding and monoclonal antibody recognition

DEFF Research Database (Denmark)

Green, M R; Couchman, J R

1985-01-01

, the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing......Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map...... whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar...

Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

Science.gov (United States)

Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

Tc-99m direct radiolabeling of monoclonal antibody ior egf/r3: quality control and image studies in mice

International Nuclear Information System (INIS)

Dias, Carla Roberta; Marczewski, Barbara; Moraes, Vanessa; Barboza, Marycel Figols de; Osso Junior, Joao Alberto

2005-01-01

Monoclonal antibodies (Mabs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in the practice of nuclear medicine. The ior egf/r3 (Centis, Cuba) is a murine monoclonal antibody against epidermal growth factor receptor (EGF-R) and has been widely used in the radioimmunodiagnosis of tumors of epithelial origin. Labeled with 99m Tc, its main application in Nuclear Medicine is the follow up, detection and evaluation of tumor recurrences. The objective of this work is to describe the preparation of a lyophilized formulation (kit) for radiolabeling the Mab ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, image studies and stability of the formulation are reported. The study demonstrated that the kit formulation can be labeled with 99m Tc at high yields and can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies.(author)

Experimental Treatment of Bladder Cancer with Bi-213-anti-EGFR MAb

International Nuclear Information System (INIS)

Seidl, Christof; Pfost, Birgit; Müller, Felix

2013-01-01

Therapy of non-muscle-invasive bladder cancer (carcinoma in situ) comprises transurethral resection of the tumour and subsequent instillation of the chemotherapeutic drug mitomycin C in order to eradicate remaining tumour cells. Yet 15 – 40% of treated patients relapse within 5 years. Therefore, new therapeutic strategies to combat tumour recurrence are needed. Alpha-particle emitting radionuclides efficiently kill single tumour cells or small tumour cell clusters. Because the epidermal growth factor receptor (EGFR) is overexpressed on bladder cancer cells, conjugates composed of the alpha-emitter Bi-213 and the anti-EGFR antibody matuzumab should provide a powerful drug to eliminate disseminated bladder cancer cells. Therefore, the aims of our study were (i) to analyse the cytotoxic effects of Bi-213-anti-EGFR radioimmunoconjugates at the cellular level, (ii) to evaluate therapeutic efficacy of intravesically applied Bi-213- anti-EGFR-Mab in a nude mouse model with intravesical human bladder cancer xenografts, (iii) to compare Bi- 213-anti-EGFR-Mab efficacy with chemotherapy using mitomycin C and (iv) to demonstrate that radioimmunotherapy is not toxic to cells of the bladder wall and of the kidneys

Tc-{sup 99m} direct radiolabeling of monoclonal antibody ior egf/r3: quality control and image studies in mice

Energy Technology Data Exchange (ETDEWEB)

Dias, Carla Roberta; Marczewski, Barbara; Moraes, Vanessa; Barboza, Marycel Figols de; Osso Junior, Joao Alberto [Instituto de Pesquisas Energeticas e Nucleares (IPEN-CNEN/SP), SP (Brazil). Centro de Radiofarmacia]. E-mail: crdias@ipen.br

2005-10-15

Monoclonal antibodies (Mabs) have been useful for immunoscintigraphic applications in clinical diagnosis since they were introduced in the practice of nuclear medicine. The ior egf/r3 (Centis, Cuba) is a murine monoclonal antibody against epidermal growth factor receptor (EGF-R) and has been widely used in the radioimmunodiagnosis of tumors of epithelial origin. Labeled with 99m Tc, its main application in Nuclear Medicine is the follow up, detection and evaluation of tumor recurrences. The objective of this work is to describe the preparation of a lyophilized formulation (kit) for radiolabeling the Mab ior egf/r3 with 99m Tc for immunoscintigraphic applications. Radiolabeling efficiency, effects on immunoreactivity, image studies and stability of the formulation are reported. The study demonstrated that the kit formulation can be labeled with 99m Tc at high yields and can be used to visualize in vivo human tumors of epithelial origin by immunoscintigraphy studies.(author)

Detection of Signal Regulatory Protein α in Saimiri sciureus (Squirrel Monkey) by Anti-Human Monoclonal Antibody

Science.gov (United States)

de Souza, Hugo Amorim dos Santos; Costa-Correa, Edmar Henrique; Bianco-Junior, Cesare; Andrade, Márcia Cristina Ribeiro; Lima-Junior, Josué da Costa; Pratt-Riccio, Lilian Rose; Daniel-Ribeiro, Cláudio Tadeu; Totino, Paulo Renato Rivas

2017-01-01

Non-human primates (NHP) are suitable models for studying different aspects of the human system, including pathogenesis and protective immunity to many diseases. However, the lack of specific immunological reagents for neo-tropical monkeys, such as Saimiri sciureus, is still a major factor limiting studies in these models. An alternative strategy to circumvent this obstacle has been the selection of immunological reagents directed to humans, which present cross-reactivity with NHP molecules. In this context and considering the key role of inhibitory immunoreceptors—such as the signal regulatory protein α (SIRPα)—in the regulation of immune responses, in the present study, we attempted to evaluate the ability of anti-human SIRPα monoclonal antibodies to recognize SIRPα in antigen-presenting S. sciureus peripheral blood mononuclear cells (PBMC). As shown by flow cytometry analysis, the profile of anti-SIRPα staining as well as the levels of SIRPα-positive cells in PBMC from S. sciureus were similar to those observed in human PBMC. Furthermore, using anti-SIRPα monoclonal antibody, it was possible to detect a decrease of the SIRPα levels on surface of S. sciureus cells after in vitro stimulation with lipopolysaccharides. Finally, using computed-based analysis, we observed a high degree of conservation of SIRPα across six species of primates and the presence of shared epitopes in the extracellular domain between humans and Saimiri genus that could be targeted by antibodies. In conclusion, we have identified a commercially available anti-human monoclonal antibody that is able to detect SIRPα of S. sciureus monkeys and that, therefore, can facilitate the study of the immunomodulatory role of SIRPα when S. sciureus is used as a model. PMID:29312325

Detection of Signal Regulatory Protein α in Saimiri sciureus (Squirrel Monkey by Anti-Human Monoclonal Antibody

Directory of Open Access Journals (Sweden)

Hugo Amorim dos Santos de Souza

2017-12-01

Full Text Available Non-human primates (NHP are suitable models for studying different aspects of the human system, including pathogenesis and protective immunity to many diseases. However, the lack of specific immunological reagents for neo-tropical monkeys, such as Saimiri sciureus, is still a major factor limiting studies in these models. An alternative strategy to circumvent this obstacle has been the selection of immunological reagents directed to humans, which present cross-reactivity with NHP molecules. In this context and considering the key role of inhibitory immunoreceptors—such as the signal regulatory protein α (SIRPα—in the regulation of immune responses, in the present study, we attempted to evaluate the ability of anti-human SIRPα monoclonal antibodies to recognize SIRPα in antigen-presenting S. sciureus peripheral blood mononuclear cells (PBMC. As shown by flow cytometry analysis, the profile of anti-SIRPα staining as well as the levels of SIRPα-positive cells in PBMC from S. sciureus were similar to those observed in human PBMC. Furthermore, using anti-SIRPα monoclonal antibody, it was possible to detect a decrease of the SIRPα levels on surface of S. sciureus cells after in vitro stimulation with lipopolysaccharides. Finally, using computed-based analysis, we observed a high degree of conservation of SIRPα across six species of primates and the presence of shared epitopes in the extracellular domain between humans and Saimiri genus that could be targeted by antibodies. In conclusion, we have identified a commercially available anti-human monoclonal antibody that is able to detect SIRPα of S. sciureus monkeys and that, therefore, can facilitate the study of the immunomodulatory role of SIRPα when S. sciureus is used as a model.

Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies

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Pietsch, E C; Dong, J; Cardoso, R; Zhang, X; Chin, D; Hawkins, R; Dinh, T; Zhou, M; Strake, B; Feng, P-H; Rocca, M; Santos, C Dos; Shan, X; Danet-Desnoyers, G; Shi, F; Kaiser, E; Millar, H J; Fenton, S; Swanson, R; Nemeth, J A; Attar, R M

2017-01-01

CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRPα. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRPα blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1 mg/kg, whereas the effector function silent mAb IgG2σ C47B222-(CHO) had minimal impact on RBC indices at 1 and 10 mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices. PMID:28234345

Immunotherapy of Alzheimer's disease (AD): from murine models to anti-amyloid beta (Abeta) human monoclonal antibodies.

Science.gov (United States)

Geylis, Valeria; Steinitz, Michael

2006-01-01

The deposition of amyloid beta (Abeta) protein is a key pathological feature in Alzheimer's disease (AD). In murine models of AD, both active and passive immunization against Abeta induce a marked reduction in amyloid brain burden and an improvement in cognitive functions. Preliminary results of a prematurely terminated clinical trial where AD patients were actively vaccinated with aggregated Abeta bear resemblance to those documented in murine models. Passive immunization of AD patients with anti-Abeta antibodies, in particular human antibodies, is a strategy that provides a more cautious management and control of any undesired side effects. Sera of all healthy adults contain anti-Abeta IgG autoimmune antibodies. Hence antigen-committed human B-cells are easily immortalized by Epstein-Barr virus (EBV) into anti-Abeta secreting cell lines. Two anti-Abeta human monoclonal antibodies which we recently prepared bind to the N-terminus of Abeta peptide and were shown to stain amyloid plaques in non-fixed brain sections from an AD patient. It is anticipated that specifically selected anti-Abeta human monoclonal antibodies could reduce and inhibit deposits of amyloid in brain while avoiding the cognitive decline that characterizes AD. In the future, this type of antibody may prove to be a promising immune therapy for the disease.

Systems approach for the selection of micro-RNAs as therapeutic biomarkers of anti-EGFR monoclonal antibody treatment in colorectal cancer

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Deyati, Avisek; Bagewadi, Shweta; Senger, Philipp; Hofmann-Apitius, Martin; Novac, Natalia

2015-01-01

miRNA plays an important role in tumourgenesis by regulating expression of oncogenes and tumour suppressors. Thus affects cell proliferation and differentiation, apoptosis, invasion and angiogenesis. miRNAs are potential biomarkers for diagnosis, prognosis and therapies of different forms of cancer. However, relationship between response of cancer patients towards targeted therapy and the resulting modifications of the miRNA transcriptome in the context of pathway regulation is poorly understood. With ever-increasing pathways and miRNA-mRNA interaction databases, freely available mRNA and miRNA expression data in multiple cancer therapy have produced an unprecedented opportunity to decipher the role of miRNAs in early prediction of therapeutic efficacy in diseases. Efficient translation of -omics data and accumulated knowledge to clinical decision-making are of paramount scientific and public health interest. Well-structured translational algorithms are needed to bridge the gap from databases to decisions. Herein, we present a novel SMARTmiR algorithm to prospectively predict the role of miRNA as therapeutic biomarker for an anti-EGFR monoclonal antibody i.e. cetuximab treatment in colorectal cancer.

An in vivo C. elegans model system for screening EGFR-inhibiting anti-cancer drugs.

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Young-Ki Bae

Full Text Available The epidermal growth factor receptor (EGFR is a well-established target for cancer treatment. EGFR tyrosine kinase (TK inhibitors, such as gefinitib and erlotinib, have been developed as anti-cancer drugs. Although non-small cell lung carcinoma with an activating EGFR mutation, L858R, responds well to gefinitib and erlotinib, tumors with a doubly mutated EGFR, T790M-L858R, acquire resistance to these drugs. The C. elegans EGFR homolog LET-23 and its downstream signaling pathway have been studied extensively to provide insight into regulatory mechanisms conserved from C. elegans to humans. To develop an in vivo screening system for potential cancer drugs targeting specific EGFR mutants, we expressed three LET-23 chimeras in which the TK domain was replaced with either the human wild-type TK domain (LET-23::hEGFR-TK, a TK domain with the L858R mutation (LET-23::hEGFR-TK[L858R], or a TK domain with the T790M-L858R mutations (LET-23::hEGFR-TK[T790M-L858R] in C. elegans vulval cells using the let-23 promoter. The wild-type hEGFR-TK chimeric protein rescued the let-23 mutant phenotype, and the activating mutant hEGFR-TK chimeras induced a multivulva (Muv phenotype in a wild-type C. elegans background. The anti-cancer drugs gefitinib and erlotinib suppressed the Muv phenotype in LET-23::hEGFR-TK[L858R]-expressing transgenic animals, but not in LET-23::hEGFR-TK[T790M-L858R] transgenic animals. As a pilot screen, 8,960 small chemicals were tested for Muv suppression, and AG1478 (an EGFR-TK inhibitor and U0126 (a MEK inhibitor were identified as potential inhibitors of EGFR-mediated biological function. In conclusion, transgenic C. elegans expressing chimeric LET-23::hEGFR-TK proteins are a model system that can be used in mutation-specific screens for new anti-cancer drugs.

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines

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Cavazzoni Andrea

2012-12-01

Full Text Available Abstract Background The epidermal growth factor receptor (EGFR is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. Results In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. Conclusion Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

77 FR 9678 - Prospective Grant of Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies...

Science.gov (United States)

2012-02-17

.../patent applications for the technology family, to Sanomab, Ltd. The patent rights in these inventions... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Prospective Grant of Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies for the Treatment of Human...

77 FR 41792 - Prospective Grant of Co-Exclusive License: The Development of Human Anti-CD22 Monoclonal...

Science.gov (United States)

2012-07-16

... applications for the technology family, to Customized Therapeutics. The patent rights in these inventions have... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Prospective Grant of Co-Exclusive License: The Development of Human Anti-CD22 Monoclonal Antibodies for the Treatment of Human...

EGFR Amplification as a Target in Gastroesophageal Adenocarcinoma: Do Anti-EGFR Therapies Deserve a Second Chance?

Science.gov (United States)

Strickler, John H

2018-06-01

Anti-EGFR therapies have failed to improve survival for unselected patients with metastatic gastroesophageal cancer, but in a subset of patients, EGFR amplification may predict treatment benefit. Maron and colleagues report the clinical activity of anti-EGFR therapies in a cohort of patients with EGFR -amplified metastatic gastroesophageal cancer and utilize serial blood and tumor tissue collection to identify molecular drivers of treatment sensitivity and resistance. Their insights offer a path to overcome technical limitations associated with EGFR amplification and facilitate molecularly targeted therapeutic strategies. Cancer Discov; 8(6); 679-81. ©2018 AACR See related article by Maron et al., p. 696 . ©2018 American Association for Cancer Research.

Reactivity of eleven anti-human leucocyte monoclonal antibodies with lymphocytes from several domestic animals

DEFF Research Database (Denmark)

Aasted, Bent; Blixenkrone-Møller, Merete; Larsen, Else Bang

1988-01-01

Nine commercially available monoclonal antibodies and two monoclonal antibodies from The American Type Culture Collection, raised against various human leucocyte surface antigens, were tested on lymphocytes from cow, sheep, goat, swine, horse, cat, dog, mink, and rabbit as well as man. Four...... antibodies bound to lymphocytes from some of the animals. These were the antibodies against CD8 and CD4 antigen, the antibody to C3b-receptor, and the antibody to the HLA-DR antigen. The CD8 antigen-reactive antibody reacted with lymphocytes from mink, cat, dog, and sheep, while the CD4 antigen......-reactive antibody reacted with lymphocytes from mink. The anti-C3b-R antibody reacted with lymphocytes from horse, swine, dog, and cat, and the anti-HLA-DR reacted with lymphocytes from cow, goat, sheep, horse, dog, cat, and mink....

Immunoscintigraphy of human tumors transplanted in nude mice with radiolabeled anti-ras p21 monoclonal antibodies

International Nuclear Information System (INIS)

Katoh, Y.; Nakata, K.; Kohno, K.; Shima, M.; Satoh, A.; Kusumoto, Y.; Ishii, N.; Kohji, T.; Shiku, H.; Nagataki, S.

1990-01-01

Anti-ras p21 monoclonal antibody (RASK-3) was used for immunoscintigraphy of human cancer cell lines in nude mice. Iodine-125-labeled RASK-3 was injected into nude mice with either human colon cancers (FCC-1 or BM-314) or lung cancer (KNS-62). Clear images were obtained in all three cancers 7 days after the injection of antibody. No localization of 125 I-labeled control monoclonal antibody was observed. The ratio of tissue/blood radioactivity and % ID/g in the tumor were significantly higher than other organs by Day 8. The specific localization index examined by 131 I-RASK-3 and 125 I-control monoclonal antibody was also higher in the tumor than in other tissues. In the in vitro study, binding of RASK-3 to tumor cells increased significantly by treatment of cells with either lysolecithin or periodate-lysine-paraformaldehyde, which confirmed the intracellular localization of ras p21. The mechanism by which anti-ras p21 antibodies accumulate in tumor sites could be the necrotic changes in tumor cells or changes in membrane permeability of non-necrotic cells. These results provide a strong rationale for the utilization of ras p21 as a target antigen in the imaging of a variety of human cancers

Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

International Nuclear Information System (INIS)

Qian Weizhu; Wang Ling; Li Bohua; Wang Hao; Hou Sheng; Hong Xueyu; Zhang Dapeng; Guo Yajun

2008-01-01

Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application

Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII.

OpenAIRE

Broze, G J; Hickman, S; Miletich, J P

1985-01-01

Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on We...

Tumor-targeted Nanobullets: Anti-EGFR nanobody-liposomes loaded with anti-IGF-1R kinase inhibitor for cancer treatment.

Science.gov (United States)

van der Meel, Roy; Oliveira, Sabrina; Altintas, Isil; Haselberg, Rob; van der Veeken, Joris; Roovers, Rob C; van Bergen en Henegouwen, Paul M P; Storm, Gert; Hennink, Wim E; Schiffelers, Raymond M; Kok, Robbert J

2012-04-30

The epidermal growth factor receptor (EGFR) is a validated target for anti-cancer therapy and several EGFR inhibitors are used in the clinic. Over the years, an increasing number of studies have reported on the crosstalk between EGFR and other receptors that can contribute to accelerated cancer development or even acquisition of resistance to anti-EGFR therapies. Combined targeting of EGFR and insulin-like growth factor 1 receptor (IGF-1R) is a rational strategy to potentiate anti-cancer treatment and possibly retard resistance development. In the present study, we have pursued this by encapsulating the kinase inhibitor AG538 in anti-EGFR nanobody-liposomes. The thus developed dual-active nanobody-liposomes associated with EGFR-(over)expressing cells in an EGFR-specific manner and blocked both EGFR and IGF-1R activation, due to the presence of the EGFR-blocking nanobody EGa1 and the anti-IGF-1R kinase inhibitor AG538 respectively. AG538-loaded nanobody-liposomes induced a strong inhibition of tumor cell proliferation even upon short-term exposure followed by a drug-free wash-out period. Therefore, AG538-loaded nanobody-liposomes are a promising anti-cancer formulation due to efficient intracellular delivery of AG538 in combination with antagonistic and downregulating properties of the EGa1 nanobody-liposomes. Copyright © 2011 Elsevier B.V. All rights reserved.

A murine monoclonal anti-idiotypic antibody detects a common idiotope on human, mouse and rabbit antibodies to allergen Lol p IV.

Science.gov (United States)

Zhou, E M; Dzuba-Fischer, J M; Rector, E S; Sehon, A H; Kisil, F T

1991-09-01

A syngeneic mouse monoclonal anti-idiotypic antibody (anti-Id), designated as B1/1, was generated against a monoclonal antibody (MoAb 91) specific for Ryegrass pollen allergen Lol p IV. This anti-Id recognized an idiotope (Id) that was also present on other monoclonal antibodies with the same specificity as MoAb 91. Observations that (i) the anti-Id inhibited the binding of MoAb 91 to Lol p IV and (ii) the Id-anti-Id interaction could be inhibited by Lol p IV indicated that the Id was located within or near the antigen combining site. These properties served to characterize B1/1 as an internal image anti-Id. Evidence that an immune response in different species to Lol p IV elicits the formation of antibodies which express a common Id was provided by the observations that (i) the Id-anti-Id interactions could be inhibited by mouse, human and rabbit antisera to Lol p IV and (ii) the binding of these antisera to Lol p IV could be inhibited by the anti-Id. Interestingly, the internal image anti-Id B1/1 also recognized an Id on a monoclonal antibody which was directed to an epitope of Lol p IV, different from that recognized by MoAb 91.

High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals.

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Stefan Ryser

Full Text Available We report here the cloning of native high affinity anti-TIM-3 and anti-KIR IgG monoclonal antibodies (mAbs from peripheral blood mononuclear cells (PBMC of healthy human donors. The cells that express these mAbs are rare, present at a frequency of less than one per 105 memory B-cells. Using our proprietary multiplexed screening and cloning technology CellSpot™ we assessed the presence of memory B-cells reactive to foreign and endogenous disease-associated antigens within the same individual. When comparing the frequencies of antigen-specific memory B-cells analyzed in over 20 screening campaigns, we found a strong correlation of the presence of anti-TIM-3 memory B-cells with memory B-cells expressing mAbs against three disease-associated antigens: (i bacterial DNABII proteins that are a marker for Gram negative and Gram positive bacterial infections, (ii hemagglutinin (HA of influenza virus and (iii the extracellular domain of anaplastic lymphoma kinase (ALK. One of the native anti-KIR mAbs has similar characteristics as lirilumab, an anti-KIR mAb derived from immunization of humanized transgenic mice that is in ongoing clinical trials. It is interesting to speculate that these native anti-TIM-3 and anti-KIR antibodies may function as natural regulatory antibodies, analogous to the pharmacological use in cancer treatment of engineered antibodies against the same targets. Further characterization studies are needed to define the mechanisms through which these native antibodies may function in healthy and disease conditions.

Anti-EGFR immunonanoparticles containing IL12 and salmosin genes for targeted cancer gene therapy.

Science.gov (United States)

Kim, Jung Seok; Kang, Seong Jae; Jeong, Hwa Yeon; Kim, Min Woo; Park, Sang Il; Lee, Yeon Kyung; Kim, Hong Sung; Kim, Keun Sik; Park, Yong Serk

2016-09-01

Tumor-directed gene delivery is of major interest in the field of cancer gene therapy. Varied functionalizations of non-viral vectors have been suggested to enhance tumor targetability. In the present study, we prepared two different types of anti-EGF receptor (EGFR) immunonanoparticles containing pDNA, neutrally charged liposomes and cationic lipoplexes, for tumor-directed transfection of cancer therapeutic genes. Even though both anti-EGFR immunonanoparticles had a high binding affinity to the EGFR-positive cancer cells, the anti-EGFR immunolipoplex formulation exhibited approximately 100-fold higher transfection to the target cells than anti-EGFR immunoliposomes. The lipoplex formulation also showed a higher transfection to SK-OV-3 tumor xenografts in mice. Thus, IL12 and/or salmosin genes were loaded in the anti-EGFR immunolipoplexes and intravenously administered to mice carrying SK-OV-3 tumors. Co-transfection of IL12 and salmosin genes using anti-EGFR immunolipoplexes significantly reduced tumor growth and pulmonary metastasis. Furthermore, combinatorial treatment with doxorubicin synergistically inhibited tumor growth. These results suggest that anti-EGFR immunolipoplexes containing pDNA encoding therapeutic genes could be utilized as a gene-transfer modality for cancer gene therapy.

Anti-EGFR Therapy: Mechanism and Advances in Clinical Efficacy in Breast Cancer

Directory of Open Access Journals (Sweden)

John F. Flynn

2009-01-01

Full Text Available This review will focus on recent advances in the application of antiepidermal growth factor receptor (anti-EGFR for the treatment of breast cancer. The choice of EGFR, a member of the ErbB tyrosine kinase receptor family, stems from evidence pinpointing its role in various anti-EGFR therapies. Therefore, an increase in our understanding of EGFR mechanism and signaling might reveal novel targets amenable to intervention in the clinic. This knowledge base might also improve existing medical treatment options and identify research gaps in the design of new therapeutic agents. While the approved use of drugs like the dual kinase inhibitor Lapatinib represents significant advances in the clinical management of breast cancer, confirmatory studies must be considered to foster the use of anti-EGFR therapies including safety, pharmacokinetics, and clinical efficacy.

Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model.

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Xiaodong Xiao

2010-10-01

Full Text Available Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252 and two anti-V-specific human mAb (m253, m254 by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.

Clinical observation in nasopharyngeal carcinoma (NPC) treated with the anti-EGFR monoclonal antibody followed by helical tomotherapy

International Nuclear Information System (INIS)

Hou Jun; Feng Linchun; Cai Boning; Lu Na; Du Lei; Ma Lin; Xu Shouping; Xie Chuanbin

2011-01-01

Objective: To evaluate the clinical outcome and the acute toxicity in nasopharyngeal carcinoma (NPC) treated with tomotherapy followed by the anti-EGFR monoclonal antibody. Methods: Between March 2008 and November 2009, 34 newly diagnosed NPC patients were treated with helical tomotherapy combined with nimotuzumab or cetuximab. All the patients underwent tomotherapy at the dose of 70 Gy/33F for the gross tumor volume (pGTV ns ) and positive lymphnodes (GTV nd ), and 60 Gy/33F for the high risk clinical target volume (PTV 1 ), and 56 Gy/33 F for the low risk clinical target volume (PTV 2 ), respectively. 17 patients in group N were given weekly injection of 200 mg for 6-7 times and 17 patients in group C were given initial dosage 400 mg/m 2 followed by subsequent weekly dosage of 250 mg/m 2 for 6-7 times. Acute lesions were evaluated with the RTOG/EORTC criteria. Result: The median follow-up time was 22 months. The effective rates (CR + PR) in 3, 6 and 12 months were 14/17, 12/17, 12/17 in group N and 15/17, 14/17, 14/17 in group C. The 1 year survival rate was 15/17 in group Nand 17/17 in group C. Nimotuzumab had less acute mucositis reaction (u=2.25, P< 0.05), weight loss (t=2.56, P=0.02) and rash (u=4.36, P<0.01) compared with cetuximab. Conclusions: Helical tomotherapy combined with nimotuzumab or cetuximab was effective and made no difference in the short-term efficacy and 1 year survival rate for the patients with NPC. Nimotuzumab has less acute reaction than cetuximab. More studies should be done to prove long-term effects. (authors)

Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

Science.gov (United States)

Iwamoto, Ryo; Takagi, Mika; Akatsuka, Jun-Ichi; Ono, Ken-Ichiro; Kishi, Yoshiro; Mekada, Eisuke

2016-04-01

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

Immunotherapy for the treatment of colorectal tumors: focus on approved and in-clinical-trial monoclonal antibodies

Directory of Open Access Journals (Sweden)

Françoso A

2017-01-01

Full Text Available Alex Françoso,1 Patricia Ucelli Simioni1–3 1Department of Biomedical Science, Faculty of Americana, Americana, 2Department of Genetics, Evolution and Bioagents, Institute of Biology, University of Campinas, Campinas, 3Department of Biochemistry and Microbiology, Institute of Biosciences, Universidade Estadual Paulista, Rio Claro, São Paulo, Brazil Abstract: Colorectal cancer is considered a disease of the elderly population. Since the number of geriatric patients continues to rise, monoclonal antibody therapy is the most promising therapy in the recent research. Presently, the monoclonal antibodies most frequently used in the treatment of colorectal tumors are bevacizumab, cetuximab, panitumumab, and ramucirumab. Bevacizumab is a monoclonal antibody that acts on VEGF. Cetuximab and panitumumab act on EGFR. Ramucirumab binds directly to the ligand-binding pocket of VEGFR-2 to block the binding of VEGF-A, VEGF-C, and VEGF-D. These monoclonal antibodies, alone or in association with radiotherapy or chemotherapy, are presenting good results and are increasing patient survival, despite the side effects. Due to the limited number of molecules available, several studies are trying to develop new monoclonal antibodies for the treatment of colorectal tumors. Among those being studied, some recent molecules are in phase I and/or II trials and are yielding advantageous results, such as anti-DR5, anti-Fn14, anti-IGF-1R, anti-EGFR, anti-NRP1, and anti-A33 antibodies. This has been successful in reducing side effects and in treating nonresponsive patients. Keywords: monoclonal antibodies, colorectal tumor, bevacizumab, cetuximab, panitumumab, ramucirumab

Monoclonal anti-melanoma antibodies and their possible clinical use

International Nuclear Information System (INIS)

Hellstroem, K.E.; Hellstroem, Ingegerd; Washington Univ., Seattle; Washington Univ., Seattle

1985-01-01

Cell surface antigens of human melanoma, as defined by monoclonal antibodies, are discussed and in particular the three antigens p97, a GD3 ganglioside and a proteoglycan. The potential diagnostic uses of antibodies to melanoma antigens are reviewed including in vitro diagnosis by immuno-histology, in vitro diagnosis by serum assays and in vivo diagnosis by tumour imaging using radioactively labelled antibodies. The potential therapeutic uses of monoclonal antibodies to melanoma antigens are also reviewed including targets for antibody therapy, the use of antibodies alone, radiolabelled antibodies, antibody-toxin conjugates, antibody-drug conjugates, anti-idiotypic antibodies and vaccines. (UK)

First clinical evaluation of radioimmunoimaging using anti-human lung cancer monoclonal antibodies

International Nuclear Information System (INIS)

Zhou Qian

1991-01-01

Anti-human large cell lung cancer monoclonal antibodies (McAb) 2E3 and 6D1 were produced in the laboratory. Immunohistochemical studies and radiobinding assay showed these antibodies possessed high specificity against lung cancer cells. 28 patients with lung masses were investigated with 131 I-labeled McAb 6D1 and/or 2E3 scintigraphy. 19 of them were histologically proven and 13 were diagnosed primary lung carcinoma. Radioimmunoimaging visualized 10/13 of the primary lung cancers with a detection rate of 77%. Only 1 case of the non-cancer patients and a false localization, giving a true negative rate of 83%. Pathologically the squamous cell lung carcinoma had the highest localization and the small cell lung carcinoma next, but the detection rate was 100% for both. The adenocarcinoma of lung was less sensitive to these McAbs, with a detection rate of only 33% (1 of 3 cases). We conclude that radioimmunoimaging with anti-human large cell lung cancer McAbs is more specific and effective in detecting primary lung cancers and differentiating lung masses than with antibodies against other tumor associated antigens

Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies.

NARCIS (Netherlands)

G.F. Rimmelzwaan (Guus); J. van Es (Johan); G.A. Drost; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

1991-01-01

textabstractMonoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these

Anti-EGFR therapy radiosensitizes human lung adenocarcinoma xenograft in nude mouse

International Nuclear Information System (INIS)

Wang Hui; Li Tianran; Tian Jiahe; Qu Baolin; Zhu Hui

2008-01-01

Objective: To investigate the effect of Gefitinib on radiosensitivity of human lung adenocarcinoma xenograft in nude mouse. Methods: Human lung adenocarcinoma cell line A549 was used to establish nude mouse xenograft tumor model. The mice were derided into 4 groups: control, irradiation alone, Gefinitib alone and radiation combined with Genifitib. Radiation schedule was 3 fractions of 5 Gy, once daily. Gefitinib was daily administered by gavage at 100 mg/(kg·day -1 ) for 14 days. In the combination group, radiotherapy was performed 2 hours after Gefitinib administration. Tumor diameter was measured every other day. Percentage of tumor growth inhibition, growth delay time and regrowth delay time were evaluated. Results: For A549 xenografts in radiation alone, gefitinib alone and combination therapy groups, the percentage of tumor growth inhibition was 22.7%, 12.4% and 38.2%, respectively (F=25.75, P=0.000). Tumor growth delay time was 6.0, 7.8 and 21.6 days, respectively (F=70.49, P=0.000). Tumor regrowth delay time in combination therapy and irradiation alone groups was 23.4 and 10.2 days. (F=174.24, P= 0.000). Sensitizing enhancement ratio of combination group was 1.5 in growth and 1.7 in regrowth. Conclusions: Anti-EGFR therapy enhances the radiosensitivity of human lung adenocarcinoma xenograft in nude mouse. (authors)

Erkitinib, a novel EGFR tyrosine kinase inhibitor screened using a ProteoChip system from a phytochemical library

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Kim, Eung-Yoon; Choi, Young-Jin [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of); Park, Chan-Won [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Kang, In-Cheol, E-mail: ickang@hoseo.edu [Biochip Research Center, Hoseo University, Asan 336-795 (Korea, Republic of); Dept. of Biological Science, Hoseo University, Asan 336-795 (Korea, Republic of); Innopharmascreen, Inc., Asan 336-795 (Korea, Republic of)

2009-11-20

Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer. Therefore PTK inhibitors are currently under intensive investigation as potential drug candidates. Herein, we report on a ProteoChip-based screening of an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor, Erkitinibs, from phytochemical libraries. PLC-{gamma}-1 was used as a substrate immobilized on a ProteoChip and incubated with an EGFR kinase to phosphorylate tyrosine residues of the substrate, followed by a fluorescence detection of the substrate recognized by a phospho-specific monoclonal antibody. Erkitinibs inhibited HeLa cell proliferation in a dose-dependent manner. In conclusion, these data suggest that Erkitinibs can be a specific inhibitor of an EGFR kinase and can be further developed as a potent anti-tumor agent.

Erkitinib, a novel EGFR tyrosine kinase inhibitor screened using a ProteoChip system from a phytochemical library

International Nuclear Information System (INIS)

Kim, Eung-Yoon; Choi, Young-Jin; Park, Chan-Won; Kang, In-Cheol

2009-01-01

Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer. Therefore PTK inhibitors are currently under intensive investigation as potential drug candidates. Herein, we report on a ProteoChip-based screening of an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor, Erkitinibs, from phytochemical libraries. PLC-γ-1 was used as a substrate immobilized on a ProteoChip and incubated with an EGFR kinase to phosphorylate tyrosine residues of the substrate, followed by a fluorescence detection of the substrate recognized by a phospho-specific monoclonal antibody. Erkitinibs inhibited HeLa cell proliferation in a dose-dependent manner. In conclusion, these data suggest that Erkitinibs can be a specific inhibitor of an EGFR kinase and can be further developed as a potent anti-tumor agent.

Radioimmunodetection of human melanoma tumor xenografts with human monoclonal antibodies

International Nuclear Information System (INIS)

Gomibuchi, Makoto; Saxton, R.E.; Lake, R.R.; Katano, Mitsuo; Irie, R.F.

1986-01-01

A human IgM monoclonal antibody has been established that defines a tumor-associated membrane antigen expressed on human melanoma cells. The antigen has been identified as the ganglioside GD2. In this paper, the authors describe the potential usefulness of the human monoclonal antibody for radioimaging. Nude mice bearing tumors derived from a human melanoma cell line were used as a model. Antibody activity was degradated significantly after labeling with 131 I by the use of a modified chloramine-T method. After testing various concentrations, labeled antibody of a specific activity of 2.8μCi/μg produced the best results. Balb/c nude mice bearing a GD2-positive M14 melanoma cell line were injected with 10-30μg of labeled antibody, and its radiolocalization in different organs and in the whole body were evaluated. The best tumor image was obtained on Day 6. The labeled antibody uptake ratio between tumor and muscle was 9.2:1; the ratio between tumor and liver was 1.4:1. These studies represent the first report of experimental tumor imaging with human monoclonal antibody. Human monoclonals will probably prove to be superior reagents for tumor imaging in melanoma patients if the problem of anti-body radiolysis is resolved. (author)

A sensitive radioimmunoassay for the detection of monoclonal anti-idiotype antibodies

International Nuclear Information System (INIS)

Morahan, G.

1983-01-01

A radioimmunoassay was developed in order to detect anti-idiotypic antibodies in the supernatants of hybrid cells. This assay is both sensitive and specific for anti-idiotypic (but not anti-allotypic) antibodies. Monoclonal antibodies present in test supernatants are bound by an anti-immunoglobulin coated solid phase. Subsequent incubation with a source of mouse immunoglobulin 'blocks' unreacted anti-immunoglobulin antibodies on the solid phase. Anti-idiotypic antibodies are then detected by their ability to bind 125 I-labelled idiotype-bearing antibody. This paper describes the use of this assay to detect monoclonal anti-idiotypic antibodies in 2 systems; the cross-reactive idiotype of A/J anti-ABA antibodies, and the idiotype expressed by the myeloma protein HOPC 8. Similarly, 125 I-labelled anti-idiotype antibodies may be used in this assay to detect monoclonal idiotype-bearing antibodies. Further modifications are described which would allow the detection of monoclonal anti-allotype antibodies. (Auth.)

Establishment of EMab-134, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody for Detecting Squamous Cell Carcinoma Cells of the Oral Cavity.

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Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Chang, Yao-Wen; Harada, Hiroyuki; Kato, Yukinari

2017-12-01

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, activates downstream signaling cascades in many tumors. In this study, we established novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. We immunized mice with a combination of the extracellular domain of EGFR and EGFR-overexpressing LN229 glioblastoma cells (LN229/EGFR) and performed the first screening using enzyme-linked immunosorbent assay. Next, we selected mAbs using flow cytometry. Among 156 established clones, two mAbs, EMab-51 (IgG 1 , kappa) and EMab-134 (IgG 1 , kappa), reacted with EGFR in Western blot analysis; EMab-134 showed a much higher sensitivity compared with EMab-51. We compared the binding affinities of EMab-51 and EMab-134 using flow cytometry; the calculated K D values for EMab-51 and EMab-134 against SAS cells/HSC-2 cells were 9.2 × 10 -9 M/9.9 × 10 -9 M and 2.6 × 10 -9 M/8.3 × 10 -9 M, respectively, indicating that EMab-134 has a higher affinity to EGFR-expressing cells. Immunohistochemical analysis of EMab-51 and EMab-134 showed sensitive and specific reactions against oral cancer cells; EMab-134 demonstrated a much higher sensitivity (36/38 cases; 94.7%) to oral squamous cell carcinomas compared with EMab-51 (6/38 cases; 15.8%). This novel anti-EGFR mAb, EMab-134, could be advantageous for detecting EGFR in the pathological analysis of EGFR-expressing cancers.

Anti-interleukin-17 monoclonal antibody ixekizumab in chronic plaque psoriasis

DEFF Research Database (Denmark)

Leonardi, Craig; Matheson, Robert; Zachariae, Claus

2012-01-01

Type 17 helper T cells have been suggested to play a pathological role in psoriasis. They secrete several proinflammatory cytokines, including interleukin-17A (also known as interleukin-17). We evaluated the safety and efficacy of ixekizumab (LY2439821), a humanized anti-interleukin-17 monoclonal...... antibody, for psoriasis treatment....

99mTc labeled anti EGFR Nanobody pentamer for tumor radioimmunoimaging

International Nuclear Information System (INIS)

Ding Zhiling; Lan Xiaoli; Li Chongjiao; Pei Zhijun; Zhang Yongxue; Wang Lifei; Gao Bin

2014-01-01

Novel Nanobody has small molecular weight and lower affinity. Appropriate polymer would be more suitable for radioimmunoimaging. In this study, we labeled anti EGFR Nanobody pentamer with 99m Tc to prepare tumor targeting imaging agent and to investigate its binding characteristics of tumor cells and tissues in vitro and in vivo, and to explore the feasibility of 99m Tc-EGFR Nanobody pentamer for tumor radioimmunoimaging compared with anti EGFR Nanobody monomer. EGFR Nanobody labeled with 99m Tc through tricarbonyl intermediate. The labeled compounds were purified by an ultra centrifugal filter; The labeling efficiency was determined by thin layer chromatography (TLC), and the radiochemical purity more than 95%. In vitro, 99m Tc-EGFR Nanobody monomer and pentamer have the specific binding capability with EGFR overexpression A431 tumor cell. the binding rate of 99m Tc-EGFR Nanobody monomer higher than that of pentamer (11.32% ± 2.73% vs 5.80% ± 0.92%, P < O.05). In A431 xenografted tumor was clearly displayed after intravenous injection of 99m Tc-EGFR Nanobody pentamer at l.5 h, T/NT maximum was 2.9 (1.5 h), whereas, the tumor tissues was not obviously found using 99m Tc-EGFR Nanobody monomer. The negative EGFR expression OCM-I xenografted tumor was not showed in both monomer and pentamer tracer. The experiment indicated that 99m Tc-EGFR Nanobody pentamer are appropriate for tumor radioimmunoimaging and has the potential value for the further study. (authors)

Transient human anti-mouse antibodies (HAMA) interference in CA 125 measurements during monitoring of ovarian cancer patients treated with murine monoclonal antibody.

NARCIS (Netherlands)

Oei, A.L.M.; Sweep, F.C.; Massuger, L.F.A.G.; Olthaar, A.J.; Thomas, C.M.G.

2008-01-01

OBJECTIVE: To investigate the influence of human anti-mouse antibodies (HAMA) on serial CA 125 measurements in serum of patients with epithelial ovarian cancer following single intraperitoneal (IP) therapy with Yttrium-90-labeled human milk fat globule 1 murine monoclonal antibody ((90)Y-muHMFG1) as

Technetium-99m direct radiolabeling of monoclonal antibody ior egf/r3

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Morales, Alejo A. Morales; Crespo, Francisco Zayas; Gandolff, Gilda Nunez; Escobar, Normando Iznaga; Perez, Niuvis Perez; Hernandez, Juan C. Izquierdo

1998-01-01

Monoclonal antibodies (MAbs) are being widely used for imaging studies, coupled mainly with {sup 99m}Tc. The antibody ior egf/r3 is a MAb against human epidermal growth factor receptor (hEGF-r), and we have developed a method for optimum labeling of this MAb with {sup 99m}Tc. The reduction was performed with 2-mercaptoethanol (2-ME) at a molar ratio of 2000:1 (2-ME:MAb) and methylene diphosphonate as transchelant. The integrity of reduced MAb was checked by mean of native polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography on Superose 12 (purity >99%). Radio colloids remained lower than 2%, and the labeling efficiency was 98.5%. The number of sulfhydryl groups generated was quantified using Ellman's reagent and was found to be 6.65 {+-} 0.69 per antibody molecule. In vitro stability studies in several challenging conditions (DTPA, human serum albumin and human serum) were performed, and no significant loss in binding percentage was seen. Radio receptor assay was used to test immunoreactivity of the reduced MAb. Both labeled and unlabeled MAbs were able to compete for binding to the hEGF-r with radioiodinated EGF. Biodistribution studies in BALB/c mice are reported.

Technetium-99m direct radiolabeling of monoclonal antibody ior egf/r3

International Nuclear Information System (INIS)

Morales, Alejo A. Morales; Crespo, Francisco Zayas; Gandolff, Gilda Nunez; Escobar, Normando Iznaga; Perez, Niuvis Perez; Hernandez, Juan C. Izquierdo

1998-01-01

Monoclonal antibodies (MAbs) are being widely used for imaging studies, coupled mainly with 99m Tc. The antibody ior egf/r3 is a MAb against human epidermal growth factor receptor (hEGF-r), and we have developed a method for optimum labeling of this MAb with 99m Tc. The reduction was performed with 2-mercaptoethanol (2-ME) at a molar ratio of 2000:1 (2-ME:MAb) and methylene diphosphonate as transchelant. The integrity of reduced MAb was checked by mean of native polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography on Superose 12 (purity >99%). Radio colloids remained lower than 2%, and the labeling efficiency was 98.5%. The number of sulfhydryl groups generated was quantified using Ellman's reagent and was found to be 6.65 ± 0.69 per antibody molecule. In vitro stability studies in several challenging conditions (DTPA, human serum albumin and human serum) were performed, and no significant loss in binding percentage was seen. Radio receptor assay was used to test immunoreactivity of the reduced MAb. Both labeled and unlabeled MAbs were able to compete for binding to the hEGF-r with radioiodinated EGF. Biodistribution studies in BALB/c mice are reported

EGFR signaling in colorectal cancer: a clinical perspective

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Saletti P

2015-01-01

Full Text Available Piercarlo Saletti,1 Francesca Molinari,2 Sara De Dosso,1 Milo Frattini2 1Oncology Institute of Southern Switzerland, Bellinzona, 2Laboratory of Molecular Pathology, Institute of Pathology, Locarno, Switzerland Abstract: Colorectal cancer (CRC remains a formidable health burden worldwide, with up to 50% of patients developing metastases during the course of their disease. This group of CRC patients, characterized by the worst prognosis, has been extensively investigated to improve their life expectancy. Main efforts, focused on the epidermal growth-factor receptor (EGFR, which plays a pivotal role in CRC pathogenesis, have led to the development and introduction in clinical practice of specific targeted therapies (ie, monoclonal antibodies. Subsequently, the scientific community has tried to identify molecular predictors of the efficacy of such therapies. However, it has become clear that EGFR alterations occurring in CRC are difficult to investigate, and therefore their predictive role is unclear. In contrast, the clinical role of two downstream members (KRAS and NRAS has been clearly demonstrated. Currently, EGFR-targeted therapies can be administered only to patients with wild-type KRAS and NRAS genes. Our review addresses the medical management of metastatic CRC. Specifically, we describe in detail the molecular biology of metastatic CRC, focusing on the EGFR signaling pathway, and we discuss the role of current and emerging related biomarkers and therapies in this field. We also summarize the clinical evidence regarding anti-EGFR monoclonal antibodies and examine potential future perspectives. Keywords: colorectal cancer, EGFR, gene mutations, cetuximab, panitumumab

Structural considerations for functional anti-EGFR × anti-CD3 bispecific diabodies in light of domain order and binding affinity.

Science.gov (United States)

Asano, Ryutaro; Nagai, Keisuke; Makabe, Koki; Takahashi, Kento; Kumagai, Takashi; Kawaguchi, Hiroko; Ogata, Hiromi; Arai, Kyoko; Umetsu, Mitsuo; Kumagai, Izumi

2018-03-02

We previously reported a functional humanized bispecific diabody (bsDb) that targeted EGFR and CD3 (hEx3-Db) and enhancement of its cytotoxicity by rearranging the domain order in the V domain. Here, we further dissected the effect of domain order in bsDbs on their cross-linking ability and binding kinetics to elucidate general rules regarding the design of functional bsDbs. Using Ex3-Db as a model system, we first classified the four possible domain orders as anti-parallel (where both chimeric single-chain components are variable heavy domain (VH)-variable light domain (VL) or VL-VH order) and parallel types (both chimeric single-chain components are mixed with VH-VL and VL-VH order). Although anti-parallel Ex3-Dbs could cross-link the soluble target antigens, their cross-linking ability between soluble targets had no correlation with their growth inhibitory effects. In contrast, the binding affinity of one of the two constructs with a parallel-arrangement V domain was particularly low, and structural modeling supported this phenomenon. Similar results were observed with E2x3-Dbs, in which the V region of the anti-EGFR antibody clone in hEx3 was replaced with that of another anti-EGFR clone. Only anti-parallel types showed affinity-dependent cancer inhibitory effects in each molecule, and E2x3-LH (both components in VL-VH order) showed the most intense anti-tumor activity in vitro and in vivo . Our results showed that, in addition to rearranging the domain order of bsDbs, increasing their binding affinity may be an ideal strategy for enhancing the cytotoxicity of anti-parallel constructs and that E2x3-LH is particularly attractive as a candidate next-generation anti-cancer drug.

The evolving role of monoclonal antibodies in colorectal cancer: early presumptions and impact on clinical trial development.

Science.gov (United States)

Eng, Cathy

2010-01-01

Targeted biologic agents have an established role in treating metastatic colorectal cancer (mCRC). Bevacizumab, a recombinant monoclonal antibody against the vascular endothelial growth factor ligand is approved by the U.S. Food and Drug Administration (FDA) for bevacizumab-naïve patients. Cetuximab, a chimeric monoclonal antibody (mAb) against the epidermal growth factor receptor (EGFR) is FDA approved as a single agent, or in combination with irinotecan, in both irinotecan-naïve and refractory patients, and has additional efficacy in combination with oxaliplatin. Panitumumab, a fully human EGFR mAb, is FDA approved as a single agent in refractory patients but has additional efficacy in combination with chemotherapy. After reaching a temporary therapeutic plateau of FDA-approved agents for the treatment of mCRC, pivotal results have developed that critically affect the care for these patients. Correlative data from randomized trials of EGFR inhibitors across disease settings have demonstrated higher response rates, specifically for patients with wild-type K-RAS tumors. The interpretation of the B-RAF mutation and other molecular markers may further define the appropriateness of anti-EGFR therapy. Recent literature revealed that the first-line use of combined anti-EGFR therapy plus bevacizumab resulted in inferior outcomes and additional toxicities. Furthermore, the role of biologic agents for locally advanced colon cancer cannot be advocated at this time. With impending changes in the health care system, the economic impact of mAbs will continue to be scrutinized. Hence, as the significance of molecular markers continues to develop, their role as it pertains to the appropriate use of biologic agents in the treatment of mCRC will continue to evolve.

Location of Primary Tumor and Benefit From Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies in Patients With RAS and BRAF Wild-Type Metastatic Colorectal Cancer

Science.gov (United States)

Moretto, Roberto; Cremolini, Chiara; Rossini, Daniele; Pietrantonio, Filippo; Battaglin, Francesca; Mennitto, Alessia; Bergamo, Francesca; Loupakis, Fotios; Marmorino, Federica; Berenato, Rosa; Marsico, Valentina Angela; Caporale, Marta; Antoniotti, Carlotta; Masi, Gianluca; Salvatore, Lisa; Borelli, Beatrice; Fontanini, Gabriella; Lonardi, Sara; De Braud, Filippo

2016-01-01

Introduction. Right- and left-sided colorectal cancers (CRCs) differ in clinical and molecular characteristics. Some retrospective analyses suggested that patients with right-sided tumors derive less benefit from anti-epidermal growth factor receptor (EGFR) antibodies; however, molecular selection in those studies was not extensive. Patients and Methods. Patients with RAS and BRAF wild-type metastatic CRC (mCRC) who were treated with single-agent anti-EGFRs or with cetuximab-irinotecan (if refractory to previous irinotecan) were included in the study. Differences in outcome between patients with right- and left-sided tumors were investigated. Results. Of 75 patients, 14 and 61 had right- and left-sided tumors, respectively. None of the right-sided tumors responded according to RECIST, compared with 24 left-sided tumors (overall response rate: 0% vs. 41%; p = .0032), and only 2 patients with right-sided tumors (15%) versus 47 patients with left-sided tumors (80%) achieved disease control (p < .0001). The median duration of progression-free survival was 2.3 and 6.6 months in patients with right-sided and left-sided tumors, respectively (hazard ratio: 3.97; 95% confidence interval: 2.09–7.53; p < .0001). Conclusion. Patients with right-sided RAS and BRAF wild-type mCRC seemed to derive no benefit from single-agent anti-EGFRs. Implications for Practice: Right- and left-sided colorectal tumors have peculiar epidemiological and clinicopathological characteristics, distinct gene expression profiles and genetic alterations, and different prognoses. This study assessed the potential predictive impact of primary tumor site with regard to anti-epidermal growth factor receptor (EGFR) monoclonal antibody treatment in patients with RAS and BRAF wild-type metastatic colorectal cancer. The results demonstrated the lack of activity of anti-EGFRs in RAS and BRAF wild-type, right-sided tumors, thus suggesting a potential role for primary tumor location in driving treatment choices

Nimotuzumab promotes radiosensitivity of EGFR-overexpression esophageal squamous cell carcinoma cells by upregulating IGFBP-3

Directory of Open Access Journals (Sweden)

Zhao Lei

2012-12-01

Full Text Available Abstract Background Epidermal growth factor receptor (EGFR is suggested to predict the radiosensitivity and/or prognosis of human esophageal squamous cell carcinoma (ESCC. The objective of this study was to investigate the efficacy of Nimotuzumab (an anti-EGFR monoclonal antibody on ESCC radiotherapy (RT and underlying mechanisms. Methods Nimotuzumab was administrated to 2 ESCC cell lines KYSE30 and TE-1 treated with RT. Cell growth, colony formation and apoptosis were used to measure anti-proliferation effects. The method of RNA interference was used to investigate the role of insulin-like growth factor binding protein-3 (IGFBP-3 in ESCC cells radiosensitivity treated with Nimotuzumab. In vivo effect of Nimotuzumab on ESCC radiotherapy was done using a mouse xenograft model. Results Nimotuzumab enhanced radiation response of KYSE30 cells (with high EGFR expression in vitro, as evidenced by increased radiation-inhibited cell growth and colony formation and radiation-mediated apoptosis. Mechanism study revealed that Nimotuzumab inhibited phosphorylated EGFR (p-EGFR induced by EGF in KYSE30 cells. In addition, knockdown of IGFBP-3 by short hairpin RNA significantly reduced KYSE30 cells radiosensitivity (PP>0.05. In KYSE30 cell xenografts, Nimotuzumab combined with radiation led to significant tumor growth delay, compared with that of radiation alone (P=0.029, and also with IGFBP-3 up-regulation in tumor tissue. Conclusions Nimotuzumab could enhance the RT effect of ESCC cells with a functional active EGFR pathway. In particular, the increased ESCC radiosensitivity by Nimotuzumab might be dependent on the up-regulation of IGFBP-3 through EGFR-dependent pathway.

Predominant antitumor effects by fully human anti-TRAIL-receptor2 (DR5) monoclonal antibodies in human glioma cells in vitro and in vivo

Science.gov (United States)

Nagane, Motoo; Shimizu, Saki; Mori, Eiji; Kataoka, Shiro; Shiokawa, Yoshiaki

2010-01-01

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2 L) preferentially induces apoptosis in human tumor cells through its cognate death receptors DR4 or DR5, thereby being investigated as a potential agent for cancer therapy. Here, we applied fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) to specifically target one of death receptors for TRAIL in human glioma cells, which could also reduce potential TRAIL-induced toxicity in humans. Twelve human glioma cell lines treated with several fully human anti-human TRAIL receptor mAbs were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. The cellular sensitivity was closely associated with cell-surface expression of DR5. Expression of c-FLIPL, Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAbs. Primary cultures of glioma cells were also relatively resistant to anti-DR5 mAbs, exhibiting both lower DR5 and higher c-FLIPL expression. Downregulation of c-FLIPL expression resulted in the sensitization of human glioma cells to anti-DR5 mAbs, whereas overexpression of c-FLIPL conferred resistance to anti-DR5 mAb. Treatment of tumor-burden nude mice with the direct agonist anti-DR5 mAb KMTR2 significantly suppressed growth of subcutaneous glioma xenografts leading to complete regression. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with KMTR2 significantly elongated lifespan without tumor recurrence. These results suggest that DR5 is the predominant TRAIL receptor mediating apoptotic signals in human glioma cells, and sensitivity to anti-DR5 mAbs was determined at least in part by the expression level of c-FLIPL and Akt. Specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas. PMID:20511188

EGFR targeted therapy in non-small cell lung cancer: potential role of cetuximab

Directory of Open Access Journals (Sweden)

Chad A Reade

2009-05-01

Full Text Available Chad A Reade1, Apar Kishor Ganti1,21Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA; 2Section of Oncology-Hematology, Department of internal Medicine, VA Medical Center, Omaha, NE, USAAbstract: Chemotherapy alone has limited ability to significantly improve survival in non-small lung cancer (NSCLC beyond what has already been achieved. The epidermal growth factor (EGF pathway plays a vital role in the pathogenesis and progression of NSCLC. Two classes of drugs inhibit the EGF receptor (EGFR pathway: small molecules that inhibit the intracellular tyrosine kinase activity of the receptor, and monoclonal antibodies that target the extracellular domain in the ligand-binding region. Cetuximab is a human – mouse chimeric immunoglobulin G1 class monoclonal antibody directed against EGFR. Preclinical studies with cetuximab suggested that there was inhibition of growth of human NSCLC cell lines. Cetuximab is currently the focus of intense investigation in various patient populations with NSCLC. This review focuses on clinical trials of cetuximab in NSCLC and identifies future directions with this agent.Keywords: non-small cell lung cancer, EGFR, cetuximab, monoclonal antibodies

Anti-EGFR-Targeted Therapy for Esophageal and Gastric Cancers: An Evolving Concept

Directory of Open Access Journals (Sweden)

Tomislav Dragovich

2009-01-01

Full Text Available Cancers of the esophagus and stomach present a major health burden worldwide. In the past 30 years we have witnessed some interesting shifts in terms of epidemiology of esophago gastric cancers. Regardless of a world region, the majority of patients diagnosed with esophageal or gastric cancers die from progression or recurrence of their disease. While there are many active cytotoxic agents for esophageal and stomach cancers, their impact on the disease course has been modest at best. Median survival for patients with advanced gastroesophageal cancer is still less than a year. Therefore, novel strategies, based on our understanding of biology and genetics, are desperately needed. Epidermal growth factor receptor (EGFR pathway has been implicated in pathophysiology of many epithelial malignancies, including esophageal and stomach cancers. EGFR inhibitors, small molecule tyrosine kinase inhibitors and monoclonal antibodies, have been explored in patients with esophageal and gastric cancers. It appears that tumors of the distal esophagus and gastroesophageal junction (GEJ may be more sensitive to EGFR blockade than distal gastric adenocarcinomas. Investigations looking into potential molecular predictors of sensitivity to EGFR inhibitors for patients with esophageal and GEJ cancers are ongoing. While we are still searching for those predictors, it is clear that they will be different from ones identified in lung and colorectal cancers. Further development of EGFR inhibitors for esophageal and GEJ cancers should be driven by better understanding of EGFR pathway disregulation that drives cancer progression in a sensitive patient population.

Interdisciplinary management of EGFR-inhibitor-induced skin reactions: a German expert opinion.

Science.gov (United States)

Potthoff, K; Hofheinz, R; Hassel, J C; Volkenandt, M; Lordick, F; Hartmann, J T; Karthaus, M; Riess, H; Lipp, H P; Hauschild, A; Trarbach, T; Wollenberg, A

2011-03-01

Anti-epidermal growth factor receptor treatment strategies, i.e. monoclonal antibodies such as cetuximab and panitumumab, or epidermal growth factor receptor (EGFR) small molecule tyrosine kinase inhibitors, such as erlotinib and gefitinib, have expanded the treatment options for different tumor types. Dermatologic toxic effects are the most common side-effects of EGFR inhibitor therapy. They can profoundly affect the patient's quality of life. The aim of this study was to provide interdisciplinary expert recommendations on how to treat patients with skin reactions undergoing anti-EGFR treatment. An expert panel from Germany with expertise in medical oncology, dermatology or clinical pharmacology was convened to develop expert recommendations based on published peer-reviewed literature. The expert recommendations for the state-of-the-art treatment of skin reactions induced by EGFR inhibitor therapy include recommendations for diagnostics and grading as well as grade-specific and stage-adapted treatment approaches and preventive measures. It was concluded that EGFR-inhibitor-related dermatologic reactions should always be treated combining basic care of the skin and a specific therapy adapted to stage and grade of skin reaction. For grade 2 and above, specific treatment recommendations for early- and later-stage skin reactions induced by EGFR-inhibitor therapy were proposed. This paper presents a German national expert opinion for the treatment of skin reactions in patients receiving EGFR inhibitor therapy.

A variety of human monoclonal antibodies against epidermal growth factor receptor isolated from a phage antibody library.

Science.gov (United States)

Kurosawa, Gene; Kondo, Mariko; Kurosawa, Yoshikazu

2016-11-04

When the technology for constructing human antibody (Ab) libraries using a phage-display system was developed, many researchers in Ab-related fields anticipated that it would be widely applied to the development of pharmaceutical drugs against various diseases, including cancers. However, successful examples of such applications are very limited. Moreover, researchers who utilize phage-display technology now show divergent ways of thinking about phage Ab libraries. For example, there is debate about what should be the source of V H and V L genes for the construction of libraries to cover the whole repertoire of Abs present in the human body. In the immune system, the introduction of mutations into V genes followed by selection based on binding activity, termed Ab maturation, is required for the production of Abs exhibiting high affinity to the antigen (Ag). Therefore, introduction of mutations and selection are required for isolation of Abs with high affinity after isolation of clones from phage Ab libraries. We constructed a large human Ab library termed AIMS, developed a screening method termed ICOS, and succeeded in isolating many human monoclonal Abs (mAbs) that specifically and strongly bind to various tumor-associated Ags. Eight anti-EGFR mAbs were included, which we characterized. These mAbs showed various different activities against EGFR-expressing cancer cells. In this paper, we describe these data and discuss the possibility and necessity that the mAbs isolated from the AIMS library might be developed as therapeutic drugs against cancers without introduction of mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Evaluation of dry blood spot technique for quantification of an Anti-CD20 monoclonal antibody drug in human blood samples.

Science.gov (United States)

Lin, Yong-Qing; Zhang, Yilu; Li, Connie; Li, Louis; Zhang, Kelley; Li, Shawn

2012-01-01

To evaluate the dried blood spot (DBS) technique in ELISA quantification of larger biomolecular drugs, an anti-CD20 monoclonal antibody drug was used as an example. A method for the quantification of the anti-CD20 drug in human DBS was developed and validated. The drug standard and quality control samples prepared in fresh human blood were spotted on DBS cards and then extracted. A luminescent ELISA was used for quantification of the drug from DBS samples. The assay range of the anti-CD20 drug standards in DBS was 100-2500ng/mL. The intra-assay precision (%CV) ranged from 0.4% to 10.1%, and the accuracy (%Recovery) ranged from 77.9% to 113.9%. The inter assay precision (%CV) ranged from 5.9% to 17.4%, and the accuracy ranged from 81.5% to 110.5%. The DBS samples diluted 500 and 50-fold yielded recovery of 88.7% and 90.7%, respectively. The preparation of DBS in higher and lower hematocrit (53% and 35%) conditions did not affect the recovery of the drug. Furthermore, the storage stability of the anti-CD20 drug on DBS cards was tested at various conditions. It was found that the anti-CD20 drug was stable for one week in DBS stored at room temperature. However, it was determined that the stability was compro]mised in DBS stored at high humidity, high temperature (55°C), and exposed to direct daylight for a week, as well as for samples stored at room temperature and high humidity conditions for a month. Stability did not change significantly in samples that underwent 3 freeze/thaw cycles. Our results demonstrated a successful use of DBS technique in ELISA quantification of an anti-CD20 monoclonal antibody drug in human blood. The stability data provides information regarding sample storage and shipping for future clinical studies. It is, therefore, concluded that the DBS technique is applicable in the quantification of other large biomolecule drugs or biomarkers. Copyright © 2011 Elsevier Inc. All rights reserved.

Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody

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Donahue, Renee N.; Lepone, Lauren M.; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A.; Heery, Christopher R.; Gulley, James L.; Schlom, Jeffrey

2017-01-01

Background Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range...

Evaluation of monoclonal anti-A and anti-B and affinity-purified Ulex europaeus lectin I for forensic blood grouping.

Science.gov (United States)

Gaensslen, R E; Lee, H C; Carroll, J E

1984-01-01

Two different monoclonal anti-A and anti-B and several different affinity purified Ulex europaeus lectin I reagents were evaluated and compared with conventional anti-A and anti-B sera and Ulex anti-H for serologic properties, in inhibition tests with secretor salivas, and in elution tests with bloodstains. The monoclonal and purified reagents were found to be comparable to conventional ones, and accordingly suitable for forensic inhibition and elution procedures.

[Diagnostic and therapeutic use of human anti-D (Rho) monoclonal antibodies. Evaluation and perspectives].

Science.gov (United States)

Rouger, P; Goossens, D; Champomier, F; Tsikas, G; Liberge, G; Leblanc, J; Richard, C; Bailleul, C; Salmon, C

1985-12-01

Human monoclonal antibodies will be essential in medicine. They are valuable tools for biological diagnosis and therapeutics. Our model, human monoclonal antibodies directed against the Rhesus D antigen can be used for the determination of the Rhesus D phenotype and for the suppression of Rh(D) immunisation in women. These new products require new procedures of preparation, new regulations for the quality controls, which will be discussed in this paper.

Safety and efficacy of ofatumumab, a fully human monoclonal anti-CD20 antibody, in patients with relapsed or refractory B-cell chronic lymphocytic leukemia

DEFF Research Database (Denmark)

Coiffier, Bertrand; Lepretre, Stéphane; Pedersen, Lars Møller

2008-01-01

Safety and efficacy of the fully human anti-CD20 monoclonal antibody, ofatumumab, was analyzed in a multicenter dose-escalating study including 33 patients with relapsed or refractory chronic lymphocytic leukemia. Three cohorts of 3 (A), 3 (B), and 27 (C) patients received 4, once weekly, infusio...

Plant virus-resembling optical nano-materials conjugated with anti-EGFR for targeted cancer imaging

Science.gov (United States)

Gupta, Sharad; Wilder, Hailey; Rao, A. L. N.; Vullev, V. I.; Anvari, Bahman

2012-03-01

We recently reported the construction of a new type of optically active nano-particles composed of genome-depleted plant infecting brome mosaic virus (BMV) doped with indocyanine green (ICG), an FDA-approved chromophore . We refer to these constructs as optical viral ghosts (OVGs) since only the capsid protein (CP) subunits of BMV remain to encapsulate ICG. Herein, we covalently conjugated the surface of OVGs with anti-epidermal growth factor receptors (anti-EGFR) to target cancerous human bronchial epithelial cells (C-HBECs) in-vitro. Our preliminary results demonstrate the utility of conjugated OVGs for targeted imaging of cancer cells.

Generation and testing anti-influenza human monoclonal antibodies in a new humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO. IL-2Rγc KO. NOD).

Science.gov (United States)

Mendoza, Mirian; Ballesteros, Angela; Qiu, Qi; Pow Sang, Luis; Shashikumar, Soumya; Casares, Sofia; Brumeanu, Teodor-D

2018-02-01

Pandemic outbreaks of influenza type A viruses have resulted in numerous fatalities around the globe. Since the conventional influenza vaccines (CIV) provide less than 20% protection for individuals with weak immune system, it has been considered that broadly cross-neutralizing antibodies may provide a better protection. Herein, we showed that a recently generated humanized mouse (DRAGA mouse; HLA-A2. HLA-DR4. Rag1KO. IL-2Rgc KO. NOD) that lacks the murine immune system and expresses a functional human immune system can be used to generate cross-reactive, human anti-influenza monoclonal antibodies (hu-mAb). DRAGA mouse was also found to be suitable for influenza virus infection, as it can clear a sub-lethal infection and sustain a lethal infection with PR8/A/34 influenza virus. The hu-mAbs were designed for targeting a human B-cell epitope ( 180 WGIHHPPNSKEQ QNLY 195 ) of hemagglutinin (HA) envelope protein of PR8/A/34 (H1N1) virus with high homology among seven influenza type A viruses. A single administration of HA 180-195 specific hu-mAb in PR8-infected DRAGA mice significantly delayed the lethality by reducing the lung damage. The results demonstrated that DRAGA mouse is a suitable tool to (i) generate heterotype cross-reactive, anti-influenza human monoclonal antibodies, (ii) serve as a humanized mouse model for influenza infection, and (iii) assess the efficacy of anti-influenza antibody-based therapeutics for human use.

Two-site sandwich radioimmunoassay of human gamma interferon with monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Tanaka, E; Imai, M; Usuda, S; Tachibana, K; Okamoto, H; Ohike, Y; Nakamura, T; Miyakawa, Y; Mayumi, M [Jichi Medical School, Minamikawachi, Tochigi (Japan)

1985-03-18

Two monoclonal antibodies were raised against human gamma interferon (IFN-..gamma..) derived from E. coli harboring the recombinant cDNA for IFN-..gamma.., and one against a synthetic peptide representing its C-terminus amino acid sequence of 20 residues. The monoclonal antibody against the synthetic peptide reacted either with IFN-..gamma.. or the synthetic peptide. One monoclonal anti-IFN-..gamma.. did not react with the synthetic peptide, while the other showed a weak binding with the peptide. A 2-site '1-step' radioimmunoassay was developed. The assay was rapid with a sensitivity capable of detecting a few ng/ml of IFN-..gamma...

A Monoclonal Antibody against Wnt-1 Induces Apoptosis in Human Cancer Cells

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Biao He

2004-01-01

Full Text Available Aberrant activation of the Wingless-type (Wnt/β-catenin signaling pathway is associated with a variety of human cancers. Little is known regarding the role that Wnt ligands play in human carcinogenesis. To test whether a Wnt-1 signal is a survival factor in human cancer cells and thus may serve as a potential cancer therapeutic target, we investigated the effect of inhibition of Wnt-1 signaling in a variety of human cancer cell lines, including non small cell lung cancer, breast cancer, mesothelioma, and sarcoma. Both monoclonal antibody and RNA interference (RNAi were used to inhibit Wnt-1 signaling. We found that incubation of a monoclonal anti-Wnt-1 antibody induced apoptosis and caused downstream protein changes in cancer cells overexpressing Wnt-1. In contrast, apoptosis was not detected in cells lacking or having minimal Wnt-1 expression after the antibody incubation. RNAi targeting of Wnt-1 in cancer cells overexpressing Wnt-1 demonstrated similar downstream protein changes and induction of apoptosis. The antibody also suppressed tumor growth in vivo. Our results indicate that both monoclonal anti-Wnt-1 antibody and Wnt-1 siRNA inhibit Wnt-1 signaling and can induce apoptosis in human cancer cells. These findings hold promise as a novel therapeutic strategy for cancer.

Radiolabeled Humanized Anti-CD3 Monoclonal Antibody Visilizumab for Imaging Human T-Lymphocytes

NARCIS (Netherlands)

Malviya, Gaurav; D'Alessandria, Calogero; Bonanno, Elena; Vexler, Vladimir; Massari, Roberto; Trotta, Carlo; Scopinaro, Francesco; Dierckx, Rudi; Signore, Alberto

2009-01-01

Visilizumab is an IgG(2) humanized monoclonal antibody (mAb) characterized by non-Fc gamma R binding and specific to the CD3 antigen, expressed on more than 95% of circulating resting T-lymphocytes and on activated T-lymphocytes homing in inflamed tissues. We hypothesized that the use of a

Anti-idiotypes against a monoclonal anti-haloperidol antibody bind to dopamine receptor

International Nuclear Information System (INIS)

Elazar, Z.; Kanety, H.; Schreiber, M.; Fuchs, S.

1988-01-01

Anti-idiotypic antibodies were raised in rabbits by immunization with a monoclonal anti-haloperidol antibody. Some of these anti-idiotypic antibodies bind in a concentration dependent manner to bovine striatal membranes. Following affinity purification, these antibodies inhibit haloperidol binding to striatal membranes and deplete [ 3 H]-spiperone binding sites from a solubilized preparation of striatal membranes. It is thus concluded that these anti-idiotypic antibodies are an internal image of haloperidol and as such can interact with D 2 -dopamine receptors

EGFR, HER-2 and KRAS in canine gastric epithelial tumors: a potential human model?

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Rossella Terragni

Full Text Available Epidermal growth factor receptor (EGFR or HER-1 and its analog c-erbB-2 (HER-2 are protein tyrosine kinases correlated with prognosis and response to therapy in a variety of human cancers. KRAS mediates the transduction of signals between EGFR and the nucleus, and its mutation has been identified as a predictor of resistance to anti-EGFR drugs. In human oncology, the importance of the EGFR/HER-2/KRAS signalling pathway in gastric cancer is well established, and HER-2 testing is required before initiating therapy. Conversely, this pathway has never been investigated in canine gastric tumours. A total of 19 canine gastric epithelial neoplasms (5 adenomas and 14 carcinomas were retrospectively evaluated for EGFR/HER-2 immunohistochemical expression and KRAS mutational status. Five (35.7% carcinomas were classified as intestinal-type and 9 (64.3% as diffuse-type. EGFR was overexpressed (≥ 1+ in 8 (42.1% cases and HER-2 (3+ in 11 (57.9% cases, regardless of tumour location or biological behaviour. The percentage of EGFR-positive tumours was significantly higher in the intestinal-type (80% than in the diffuse-type (11.1%, p = 0.023. KRAS gene was wild type in 18 cases, whereas one mucinous carcinoma harboured a point mutation at codon 12 (G12R. EGFR and HER-2 may be promising prognostic and therapeutic targets in canine gastric epithelial neoplasms. The potential presence of KRAS mutation should be taken into account as a possible mechanism of drug resistance. Further studies are necessary to evaluate the role of dog as a model for human gastric cancer.

Molecular biomarkers of resistance to anti-EGFR treatment in metastatic colorectal cancer, from classical to innovation.

Science.gov (United States)

Giampieri, Riccardo; Scartozzi, Mario; Del Prete, Michela; Maccaroni, Elena; Bittoni, Alessandro; Faloppi, Luca; Bianconi, Maristella; Cecchini, Luca; Cascinu, Stefano

2013-11-01

Systematic dissection of the EGFR pathway was considered as the best way to identify putative markers of resistance to anti-EGFR therapies. This kind of approach leaves other, less known but by no means less important, putative mechanisms of resistance. We tried to shed some light on these mechanisms of resistance. We performed a research through Pubmed database of all published articles highlighting mechanisms of resistance to Cetuximab and Panitumumab based therapies, published in 2000-2012 period. We reviewed the "classical" molecular factors, extensively analyzed as predictive factors for efficacy to anti-EGFR therapy, such as K-ras, B-raf, and PI3K-mTOR-Akt, focusing on their predictive or prognostic value and on the controversial aspects of the biomarker analysis for clinical practice. On the second part we will then move on to other less known molecular markers, for the future understanding of biological mechanisms underlying anti-EGFR therapy resistance, such as non-canonical heterodimer candidates, microRNA, IGF1-IGF1R, HGF-cMET and secondary mutations of EGFR. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Secukinumab, a human anti-interleukin-17A monoclonal antibody, in patients with psoriatic arthritis (FUTURE 2): a randomised, double-blind, placebo-controlled, phase 3 trial

NARCIS (Netherlands)

McInnes, Iain B.; Mease, Philip J.; Kirkham, Bruce; Kavanaugh, Arthur; Ritchlin, Christopher T.; Rahman, Proton; van der Heijde, Désirée; Landewé, Robert; Conaghan, Philip G.; Gottlieb, Alice B.; Richards, Hanno; Pricop, Luminita; Ligozio, Gregory; Patekar, Manmath; Mpofu, Shephard; Bird, Paul; Hall, Stephen; Nash, Peter; Zochling, Jane; de Vlam, Kurt; Langenaken, Christine; Geusens, Piet; Beaulieu, Andre; Tremblay, Jean-Luc; McCarthy, Tim; Papp, Kim; Poulin, Yves; Cohen, Martin; Galatikova, Dagmar; Dokoupilova, Eva; Dvorak, Zdenek; Mann, Herman; Sieper, Joachim; Spieler, Wolfgang; Kurthen, Reiner; Braun, Juergen; Wollenhaupt, Juergen; Tony, Hans-Peter; Schuch, Florian; Schulze-Koops, Hendrik; Rech, Juergen; Leszczynski, Piotr; Adamski, Zygmunt; Szepietowski, Jacek; Tlustochowicz, Witold; Kaszuba, Andrzej; Szymanska, Malgorzata; Stanislav, Marina; Nesmeyanova, Olga; Vezikova, Natalia; Ershova, Olga; Izmozherova, Nadezda; Zotkin, Eugeny; Petrova, Marianna; Kastanayan, Alexander; Yakupova, Svetlana; Agafina, Alina; Asavatanabodee, Paijit; Suwannalai, Parawee; Kerrane, Jerome; Tahir, Hasan; McInnes, Iain; Edwards, Christopher; Chinoy, Hector; Marzo-Ortega, Helena; Kaul, Arvind; Sheeran, Thomas; Clunie, Gavin; Schechtman, Joy; Gaylis, Norman; Kaine, Jeffrey; Lawson, Jeffrey; El-Kadi, Hisham; Flint, Kathleen; Kivitz, Alan; Churchhill, Melvin; Sikes, David; Lowenstein, Mitchell; Halpert, Elias; Abdulky, Mary; Palmer, William; Codding, Christine; Legerton, Clarence; Singhal, Atul; Sunkureddi, Prashanth; Gough, William; Forman, Seth; Box, Jane; Khan, Mohamed; Barranco, Elizabeth

2015-01-01

Background Interleukin 17A is a proinflammatory cytokine that is implicated in the pathogenesis of psoriatic arthritis. We assessed the efficacy and safety of subcutaneous secukinumab, a human anti-interleukin-17A monoclonal antibody, in patients with psoriatic arthritis. Methods In this phase 3,

Characterization of monoclonal antibodies against human thyrotropin and use in an immunoradiometric assay and immunohistochemistry

International Nuclear Information System (INIS)

Benkirane, M.; Bon, D.; Bellot, F.; Prince, P.; Delori, P.; Hassoun, J.; Carayon, P.

1987-01-01

Monoclonal antibodies were prepared against human thyrotropin. 13 different antibodies were characterized. Ten antibodies were of the IgG1 subclass. The affinities of the antibodies were in the range 10 9 -10 11 mol -1 .l. Four of them were specific for hTSH and did not react with hLH, hFSH, hCG or αhCG. Four reacted with these hormones and recognized the α subunit of hCG. One cross-reacted only with HFSH. The remaining four antibodies recognized the holo-hTSH only, and thus were designated as anti-conformational determinants. Monoclonal antibodies reacting with different antigenic determinants on the hTSH molecule defined seven clusters. Two of them were used to develop a simplified two-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized on tubes (anti-βTSH) and another (anti-α) labelled with 125 I. This assay was highly specific and demonstrated a sensitivity level of 0.1 μIU/ml. Two monoclonal antibodies were used in immunohistochemistry and their quality and specificity was assessed in the detection of hTSH immunoreactivity in human pituitary biological sections. 20 refs.; 6 figs.; 2 tabs

PCR-based assays versus direct sequencing for evaluating the effect of KRAS status on anti-EGFR treatment response in colorectal cancer patients: a systematic review and meta-analysis.

Directory of Open Access Journals (Sweden)

Lianfeng Shan

Full Text Available The survival rate of colorectal cancer (CRC patients carrying wild-type KRAS is significantly increased by combining anti-EGFR monoclonal antibody (mAb with standard chemotherapy. However, conflicting data exist in both the wild-type KRAS and mutant KRAS groups, which strongly challenge CRC anti-EGFR treatment. Here we conducted a meta-analysis in an effort to provide more reliable information regarding anti-EGFR treatment in CRC patients.We searched full reports of randomized clinical trials using Medline, the American Society of Clinical Oncology (ASCO, and the European Society for Medical Oncology (ESMO. Two investigators independently screened the published literature according to our inclusive and exclusive criteria and the relative data were extracted. We used Review Manager 5.2 software to analyze the data.The addition of anti-EGFR mAb to standard chemotherapy significantly improved both progression-free survival (PFS and median overall survival (mOS in the wild-type KRAS group; hazard ratios (HRs for PFS and mOS were 0.70 [95% confidence interval (CI, 0.58-0.84] and 0.83 [95% CI, 0.75-0.91], respectively. In sub-analyses of the wild-type KRAS group, when PCR-based assays are employed, PFS and mOS notably increase: the HRs were 0.74 [95% CI, 0.62-0.88] and 0.87 [95% CI, 0.78-0.96], respectively. In sub-analyses of the mutant KRAS group, neither PCR-based assays nor direct sequencing enhance PFS or mOS.Our data suggest that PCR-based assays with high sensitivity and specificity allow accurate identification of patients with wild-type KRAS and thus increase PFS and mOS. Furthermore, such assays liberate patients with mutant KRAS from unnecessary drug side effects, and provide them an opportunity to receive appropriate treatment. Thus, establishing a precise standard reference test will substantially optimize CRC-targeted therapies.

Epidermal Growth Factor Receptor (EGFR gene copy number (GCN correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH and chromogenic in situ hybridization (CISH analysis

Directory of Open Access Journals (Sweden)

Scartozzi Mario

2009-08-01

Full Text Available Abstract Background K-RAS wild type colorectal tumors show an improved response rate to anti-EGFR monoclonal antibodies. Nevertheless 70% to 40% of these patients still does not seem to benefit from this therapeutic approach. FISH EGFR GCN has been previously demonstrated to correlate with clinical outcome of colorectal cancer treated with anti-EGFR monoclonal antibodies. CISH also seemed able to provide accurate EGFR GCN information with the advantage of a simpler and reproducible technique involving immunohistochemistry and light microscopy. Based on these findings we investigated the correlation between both FISH and CISH EGFR GCN and clinical outcome in K-RAS wild-type colorectal cancer treated with irinotecan-cetuximab. Methods Patients with advanced K-RAS wild-type, colorectal cancer receiving irinotecan-cetuximab after failure of irinotecan-based chemotherapy were eligible. A cut-off value for EGFR GCN of 2.6 and 2.12 for FISH and CISH respectively was derived from ROC curve analysis. Results Forty-four patients were available for analysis. We observed a partial remission in 9 (60% and 2 (9% cases with a FISH EGFR GCN ≥ 2.6 and Conclusion FISH and CISH EGFR GCN may both represent effective tools for a further patients selection in K-RAS wild-type colorectal cancer treated with cetuximab.

Cuban Monoclonal Antibodies for Radioimmunodiagnosis and Radioimmunotherapy of Cancer Diseases

International Nuclear Information System (INIS)

Casaco, A.

2009-01-01

The Centre of Molecular Immunology produces monoclonal antibodies for treating cancer diseases. We are mainly focus on two target systems; one is the epidermal growth factor receptor (EGF-R) because there is a tremendous relationship between the EGF/EGF-R system and several human tumours such as lung, head and neck, ovarian breast and brain cancers; the second one is the ganglioside system, the relevance of certain gangliosides in tumour growth and metastatic dissemination has been well documented, GM3(NeuGc) ganglioside is particularly interesting due to its restrictive expression in normal human tissues. Nimotuzumab (h-R3) is a humanized monoclonal antibody (mAb) that was obtained by complementarity-determining regions grafting of a murine mAb (ior egf/r3) to a human framework having remarkable antiproliferative, pro-apoptotic, and antiangiogenic effects. A Phase I clinical trial was performed to evaluate the toxicity and clinical effect of an intracavitary (intracerebral) administration of a single dose of nimotuzumab (h-R3) labelled with increasing doses of 188Re. All patients bearing astrocytomas grade III/IV should be treated previously with conventional therapies and have an EGF-R overexpression in the tumour, demonstrated by immunohistochemical study. Maximal tolerated dose was 3 mg of the h-R3 labelled with 10 mCi of 188 Re. The radioimmunoconjugate showed a high retention in the surgical created resection cavity and the brain adjacent tissues with a mean value of 85.5% of the injected dose one hour post-administration. This radioimmunoconjugate may be relatively safe and a promising therapeutic approach for treating high grade gliomas. GM3(NeuGc) ganglioside is particularly interesting due to its restrictive expression in normal human tissues according to immunohistochemical studies, using either polyclonal or monoclonal antibodies. But both immunohistochemical and biochemical methods have strongly suggested its over-expression in human breast and colon

Targeting EGFR-overexpressing tumor cells using Cetuximab-immunomicelles loaded with doxorubicin and superparamagnetic iron oxide

International Nuclear Information System (INIS)

Liao Chengde; Sun Qiquan; Liang, Biling; Shen Jun; Shuai Xintao

2011-01-01

Epidermal growth factor receptor (EGFR), a cellular transmembrane receptor, plays a key role in cell proliferation and is linked to a poor prognosis in various human cancers. In this study, we constructed Cetuximab-immunomicelles in which the anti-EGFR monoclonal antibody was linked to poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG–PCL) nanomicelles that were loaded with doxorubicin (DOX) and superparamagnetic iron oxide (SPIO). The specific interactions between EGFR-overexpressing tumor cells (A431) and immunomicelles were observed using confocal laser scanning microscopy (CLSM) and flow cytometry. Furthermore, the capacity of transporting SPIO into tumor cells using these immunomicelles was evaluated with a 1.5 T clinical magnetic resonance imaging (MRI) scanner. It was found that the acquired MRI T2 signal intensity of A431 cells that were treated with the SPIO-loaded and antibody-functionalized micelles decreased significantly. Using the thiazolyl blue tetrazolium bromide (MTT) assay, we also demonstrated that the immunomicelles inhibited cell proliferation more effectively than their nontargeting counterparts. Our results suggest that Cetuximab-immunomicelles are a useful delivery vehicle for DOX and SPIO to EGFR-overexpressing tumor cells in vitro and that Cetuximab-immunomicelles can serve as a MRI-visible and targeted drug delivery agent for better tumor imaging and therapy.

Bioactivity assays and application of 125I labeled human mouse chimeric anti-CD22 monoclonal antibody SM03

International Nuclear Information System (INIS)

Lu Pingping; Meng Zhiyun; Dou Guifang; Wu Yingliang; Wang Minwei

2008-01-01

To investigate the bioactivity and application of 125 I labeled human mouse chimeric monoclonal SM03, SM03 was labeled with 125 I using Indogen method. The labeled mixture was purified by Sephacryl S-300 HR separation chromospectry. The purity and concentration of separated fractions were determined by HPLC and Protein Assay Kit, respectively. Competitive binding method and ELISA method were used for bioactivity assays. 125 I-SM03 was applied to screen cell lines which express the most abundant CD22 antigen. The purity and recovery of 125 I-SM03 were >99% and >47%, respectively. The bioactivity of 125 I- SM03 and SM03 hasn't significant difference in statistics. Ramos cell line had the strongest special radioactivity when 125 I-SM03 bound with in Raji, Daudi and Ramos cell lines. Indogen method is a good way to label Human mouse chimeric anti-CD22 monoclonal antibody SM03 and the label will not affect the activity of SM03. The 125 I-SM03 not only can be used for detect agent, but also may be put into market for NHL therapy. (authors)

The anti-epidermal growth factor receptor (EGFR) monoclonal antibody, C225, enhances radiation-induced apoptosis in primary glioma cell lines through mediation of MAPK/JNK/p38 signaling pathways

International Nuclear Information System (INIS)

Chakravarti, A.; Noll, E.; Black, P.M.; Loeffler, J.S.

2001-01-01

Purpose: Increasing evidence suggests that signaling mediated by the epidermal growth factor receptor (EGFR) pathway contributes to radiation resistance. The anti-EGFR monoclonal antibody, C225, has been shown to enhance radiation response for several tumor types in preclinical models. Malignant gliomas are known to express, and quite frequently overexpress, EGFR. Our objectives in this study were to 1) Evaluate the efficacy of C225 as a radiation response modifier in EGFR-expressing glioma cell lines and to 2) Investigate the underlying molecular mechanisms mediating C225-induced enhancement of radiation response. Materials and Methods: Twelve EGFR-expressing glioma cells lines, established from patient tumors, were used for this study. Cells were incubated with C225, irradiated, and then evaluated for radiation response. Assays used to evaluate efficacy of C225-mediated radiosensitization included time-course apoptosis assays (Annexin V and TUNEL), viability assays (MTT), and clonogenic survival assays. The changes along MAPK (p44/p42)/JNK/p38-MAPK signal transduction pathways were then investigated using quantitative Western analysis with phospho-specific antibodies to determine the molecular mechanisms by which C225 mediates a given response. Results: C225 clearly enhanced radiation response for 7 of the 12 primary glioma cell lines studied. Enhancement of both immediate and delayed apoptotic responses was evident in these 7 responsive cell lines after C225 administration. The average apoptosis index at 6 hours post-RT+C225 for the 7 responsive lines was 9.5%, compared to 1.2% for the RT-only controls. A pattern of delayed apoptosis was evident in these 7 lines, with secondary apoptotic peaks (∼ 8.0%) occurring at 24 hours post-RT+C225. Time course viability measurements revealed a steady decrease in viable tumor cells in these responsive cell lines from 75% at 6 hours post-RT+C225 to 20% at 7 days. Clonogenic survival was also diminished in these 7 lines

Effect of producer cell line on functional activity of anti-D monoclonal antibodies destined for prevention of rhesus sensitization.

Science.gov (United States)

Olovnikova, N I; Ershler, M A; Belkina, E V; Nikolaeva, T L; Miterev, G Yu

2009-04-01

The ability of anti-D antibodies to cause antigen-specific immunosuppression depends on their interaction with low-affinity Fcgamma-receptors. Human monoclonal antibodies to D antigen of the rhesus system were investigated by antibody-dependent cytotoxicity assay in order to estimate their ability to induce hemolysis mediated by low-affinity Fcgamma receptors. We demonstrate that affinity of monoclonal antibodies to receptors of this type does not depend on primary structure of Fc-fragment, but depends on the producer cell line which expresses the antibodies. Monoclonal IgG1 antibodies interacting with FcgammaRIIa and FcgammaRIII lost this property, if they were secreted by human-mouse heterohybridoma, but not by human B-cell line. On the opposite, monoclonal antibodies that could not activate low-affinity Fcgamma receptors were highly active after human cells fusion with rat myeloma YB2/0. Hemolytic activity of IgG3 remained unchanged after fusion of human cells with rodent cells.

Epidermal Growth Factor Receptor (EGFR) gene copy number (GCN) correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH) and chromogenic in situ hybridization (CISH) analysis.

Science.gov (United States)

Scartozzi, Mario; Bearzi, Italo; Mandolesi, Alessandra; Pierantoni, Chiara; Loupakis, Fotios; Zaniboni, Alberto; Negri, Francesca; Quadri, Antonello; Zorzi, Fausto; Galizia, Eva; Berardi, Rossana; Biscotti, Tommasina; Labianca, Roberto; Masi, Gianluca; Falcone, Alfredo; Cascinu, Stefano

2009-08-27

K-RAS wild type colorectal tumors show an improved response rate to anti-EGFR monoclonal antibodies. Nevertheless 70% to 40% of these patients still does not seem to benefit from this therapeutic approach. FISH EGFR GCN has been previously demonstrated to correlate with clinical outcome of colorectal cancer treated with anti-EGFR monoclonal antibodies. CISH also seemed able to provide accurate EGFR GCN information with the advantage of a simpler and reproducible technique involving immunohistochemistry and light microscopy. Based on these findings we investigated the correlation between both FISH and CISH EGFR GCN and clinical outcome in K-RAS wild-type colorectal cancer treated with irinotecan-cetuximab. Patients with advanced K-RAS wild-type, colorectal cancer receiving irinotecan-cetuximab after failure of irinotecan-based chemotherapy were eligible. A cut-off value for EGFR GCN of 2.6 and 2.12 for FISH and CISH respectively was derived from ROC curve analysis. Forty-four patients were available for analysis. We observed a partial remission in 9 (60%) and 2 (9%) cases with a FISH EGFR GCN >or= 2.6 and CISH EGFR GCN >or= 2.12 and CISH EGFR GCN whereas it was 2.9 and 3.1 months in those with low FISH and CISH EGFR GCN (p = 0.04 and 0.02 respectively). FISH and CISH EGFR GCN may both represent effective tools for a further patients selection in K-RAS wild-type colorectal cancer treated with cetuximab.

Production and characterisation of monoclonal antibodies against native and disassembled human catalase

NARCIS (Netherlands)

Wiemer, E. A.; Ofman, R.; Middelkoop, E.; de Boer, M.; Wanders, R. J.; Tager, J. M.

1992-01-01

Catalase isolated from human erythrocytes was used to immunise mice, in order to generate hybridomas producing specific monoclonal antibodies to the enzyme. Hybridomas secreting anti-(catalase) antibodies were identified by a modified enzyme-linked immunosorbent assay (ELISA) using either

EGFR and Bcl-2 in gastric mucosa of children infected with Helicobacter pylori

Directory of Open Access Journals (Sweden)

Ewa Ryszczuk

2016-03-01

Full Text Available Aim: The aim of the study was to evaluate the expression of EGFR and Bcl-2 proteins as inhibitory markers of apoptosis in surface epithelial cells and gland cells of antral gastric mucosa in children infected with Helicobacter pylori according to the severity and activity of antral gastritis and to assess the correlation between the number of cells expressing EGFR and the number of cells expressing Bcl-2 in H. pylori infected children.Materials and methods: The study included 44 children: 68.2% with chronic gastritis and positive IgG against H. pylori, and 31.8% with functional disorders of the gastrointestinal tract and with normal IgG against H. pylori. The evaluation of EGFR expression in gastric mucosa was performed immunohistochemically using monoclonal mouse anti-EGFR antibody. The polyclonal antibody was used to determine the expression of anti-Bcl-2.Results: A significant increase in the number of cells expressing EGFR and Bcl-2 protein was found in the epithelial cells in severe as well as mild and moderate gastritis in the group of children infected with H. pylori. An increase in the number of cells expressing EGFR and Bcl-2 protein was also found in the epithelial cells in group I according to the activity of gastritis. There was a statistically significant positive correlation between the numbers of cells expressing EGFR and Bcl-2 in H. pylori infected children.Conclusion: Increased expression of EGFR and Bcl-2 proteins in the epithelial cells and a statistically significant positive correlation between the numbers of cells expressing EGFR and Bcl-2 in H. pylori infected children could suggest increased regeneration abilities of gastric mucosa.

Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

International Nuclear Information System (INIS)

Holers, V.M.; Kotzin, B.L.

1985-01-01

The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases

A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words.

Science.gov (United States)

Mirick, G R; Bradt, B M; Denardo, S J; Denardo, G L

2004-12-01

The United States Food and Drug Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ((90)yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ((131)I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) anti-CD20 MAbs for use in radioimmunotherapy (RIT) of non-Hodgkin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, assays were developed to determine HAGA (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to ''humanize'' MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words

International Nuclear Information System (INIS)

Mirick, G. R.; Bradt, B. M.; Denardo, S. J.; Denardo, G. L.

2004-01-01

The United States Food and Drugs Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ( 9 0yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ( 1 31I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) antiCD20 MAns for use in radioimmunotherapy (RIT) of non-Hodgikin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, essays were developed to determine HAGE (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to humanize MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades

Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

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Koushan Sineh sepehr

2013-02-01

Full Text Available Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells.

Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

Science.gov (United States)

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Abdolalizadeh, Jalal; Kazemi, Tohid; Aghebati Maleki, Ali; Sineh sepehr, Koushan

2013-01-01

Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells. PMID:24312838

Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

OpenAIRE

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Abdolalizadeh, Jalal; Kazemi, Tohid; Aghebati Maleki, Ali; Sineh sepehr, Koushan

2013-01-01

Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into t...

Upregulation of HLA Class I Expression on Tumor Cells by the Anti-EGFR Antibody Nimotuzumab

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Greta Garrido

2017-10-01

Full Text Available Defining how epidermal growth factor receptor (EGFR-targeting therapies influence the immune response is essential to increase their clinical efficacy. A growing emphasis is being placed on immune regulator genes that govern tumor – T cell interactions. Previous studies showed an increase in HLA class I cell surface expression in tumor cell lines treated with anti-EGFR agents. In particular, earlier studies of the anti-EGFR blocking antibody cetuximab, have suggested that increased tumor expression of HLA class I is associated with positive clinical response. We investigated the effect of another commercially available anti-EGFR antibody nimotuzumab on HLA class I expression in tumor cell lines. We observed, for the first time, that nimotuzumab increases HLA class I expression and its effect is associated with a coordinated increase in mRNA levels of the principal antigen processing and presentation components. Moreover, using 7A7 (a specific surrogate antibody against murine EGFR, we obtained results suggesting the importance of the increased MHC-I expression induced by EGFR-targeted therapies display higher in antitumor immune response. 7A7 therapy induced upregulation of tumor MHC-I expression in vivo and tumors treated with this antibody display higher susceptibility to CD8+ T cells-mediated lysis. Our results represent the first evidence suggesting the importance of the adaptive immunity in nimotuzumab-mediated antitumor activity. More experiments should be conducted in order to elucidate the relevance of this mechanism in cancer patients. This novel immune-related antitumor mechanism mediated by nimotuzumab opens new perspectives for its combination with various immunotherapeutic agents and cancer vaccines.

Sym004, a Novel EGFR Antibody Mixture, Can Overcome Acquired Resistance to Cetuximab1

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Iida, Mari; Brand, Toni M; Starr, Megan M; Li, Chunrong; Huppert, Evan J; Luthar, Neha; Pedersen, Mikkel W; Horak, Ivan D; Kragh, Michael; Wheeler, Deric L

2013-01-01

The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in a variety of human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for head and neck and colorectal cancer treatment, but many patients treated with cetuximab don't respond or eventually acquire resistance. To determine how tumor cells acquire resistance to cetuximab, we previously developed a model of acquired resistance using the non-small cell lung cancer line NCI-H226. These cetuximab-resistant (CtxR) cells exhibit increased steady-state EGFR expression secondary to alterations in EGFR trafficking and degradation and, further, retained dependence on EGFR signaling for enhanced growth potential. Here, we examined Sym004, a novel mixture of antibodies directed against distinct epitopes on the extracellular domain of EGFR, as an alternative therapy for CtxR tumor cells. Sym004 treatment of CtxR clones resulted in rapid EGFR degradation, followed by robust inhibition of cell proliferation and down-regulation of several mitogen-activated protein kinase pathways. To determine whether Sym004 could have therapeutic benefit in vivo, we established de novo CtxR NCI-H226 mouse xenografts and subsequently treated CtxR tumors with Sym004. Sym004 treatment of mice harboring CtxR tumors resulted in growth delay compared to mice continued on cetuximab. Levels of total and phospho-EGFR were robustly decreased in CtxR tumors treated with Sym004. Immunohistochemical analysis of these Sym004-treated xenograft tumors further demonstrated decreased expression of Ki67, and phospho-rpS6, as well as a modest increase in cleaved caspase-3. These results indicate that Sym004 may be an effective targeted therapy for CtxR tumors. PMID:24204198

Quantum-Dot-Based Theranostic Micelles Conjugated with an Anti-EGFR Nanobody for Triple-Negative Breast Cancer Therapy.

Science.gov (United States)

Wang, Yuyuan; Wang, Yidan; Chen, Guojun; Li, Yitong; Xu, Wei; Gong, Shaoqin

2017-09-13

A quantum-dot (QD)-based micelle conjugated with an anti-epidermal growth factor receptor (EGFR) nanobody (Nb) and loaded with an anticancer drug, aminoflavone (AF), has been engineered for EGFR-overexpressing cancer theranostics. The near-infrared (NIR) fluorescence of the indium phosphate core/zinc sulfide shell QDs (InP/ZnS QDs) allowed for in vivo nanoparticle biodistribution studies. The anti-EGFR nanobody 7D12 conjugation improved the cellular uptake and cytotoxicity of the QD-based micelles in EGFR-overexpressing MDA-MB-468 triple-negative breast cancer (TNBC) cells. In comparison with the AF-encapsulated nontargeted (i.e., without Nb conjugation) micelles, the AF-encapsulated Nb-conjugated (i.e., targeted) micelles accumulated in tumors at higher concentrations, leading to more effective tumor regression in an orthotopic triple-negative breast cancer xenograft mouse model. Furthermore, there was no systemic toxicity observed with the treatments. Thus, this QD-based Nb-conjugated micelle may serve as an effective theranostic nanoplatform for EGFR-overexpressing cancers such as TNBCs.

Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

Science.gov (United States)

Kirley, Terence L; Norman, Andrew B

2015-01-01

Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

Nanobiopolymer for direct targeting and inhibition of EGFR expression in triple negative breast cancer.

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Satoshi Inoue

Full Text Available Treatment options for triple negative breast cancer (TNBC are generally limited to cytotoxic chemotherapy. Recently, anti-epidermal growth factor receptor (EGFR therapy has been introduced for TNBC patients. We engineered a novel nanobioconjugate based on a poly(β-L-malic acid (PMLA nanoplatform for TNBC treatment. The nanobioconjugate carries anti-tumor nucleosome-specific monoclonal antibody (mAb 2C5 to target breast cancer cells, anti-mouse transferrin receptor (TfR antibody for drug delivery through the host endothelial system, and Morpholino antisense oligonucleotide (AON to inhibit EGFR synthesis. The nanobioconjugates variants were: (1 P (BioPolymer with AON, 2C5 and anti-TfR for tumor endothelial and cancer cell targeting, and EGFR suppression (P/AON/2C5/TfR, and (2 P with AON and 2C5 (P/AON/2C5. Controls included (3 P with 2C5 but without AON (P/2C5, (4 PBS, and (5 P with PEG and leucine ester (LOEt for endosomal escape (P/mPEG/LOEt. Drugs were injected intravenously to MDA-MB-468 TNBC bearing mice. Tissue accumulation of injected nanobioconjugates labeled with Alexa Fluor 680 was examined by Xenogen IVIS 200 (live imaging and confocal microscopy of tissue sections. Levels of EGFR, phosphorylated and total Akt in tumor samples were detected by western blotting. In vitro western blot showed that the leading nanobioconjugate P/AON/2C5/TfR inhibited EGFR synthesis significantly better than naked AON. In vivo imaging revealed that 2C5 increased drug-tumor accumulation. Significant tumor growth inhibition was observed in mice treated with the lead nanobioconjugate (1 [P = 0.03 vs. controls; P<0.05 vs. nanobioconjugate variant (2]. Lead nanobioconjugate (1 also showed stronger inhibition of EGFR expression and Akt phosphorylation than other treatments. Treatment of TNBC with the new nanobioconjugate results in tumor growth arrest by inhibiting EGFR and its downstream signaling intermediate, phosphorylated Akt. The nanobioconjugate

A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words

Energy Technology Data Exchange (ETDEWEB)

Mirick, G. R.; Bradt, B. M.; Denardo, S. J.; Denardo, G. L. [Calfornia Univ., Sacramento (United States). Davis Medical Center

2004-12-01

The United States Food and Drugs Administration (FDA) has approved unconjugated monoclonal antibodies (MAbs) for immunotherapy (IT) of B-cell lymphoma, breast cancer and acute myeloid leukemia. More recently, approval has been given for conjugated ZevalinTM ({sup 9}0yttrium ibritumomab tiuxetan, IDEC-Y2B8, Biogen Idec, Cambridge, MA) and BexxarTM ({sup 1}31I-tositumomab, Corixa, Corp., Seattle, WA and GlaxoSmithKline, Philadelphia, PA) antiCD20 MAns for use in radioimmunotherapy (RIT) of non-Hodgikin's lymphoma (NHL), thus redefining the standard care of cancer patients. Because of, and despite a lack of basis for concern about allergic reactions due to human antibody responses to these foreign proteins, essays were developed to determine HAGE (human anti-globulin antibody) levels that developed in patient sera following treatment with MAbs. Strategies were also devised to humanize MAbs and to temporarily block patient immune function with drugs in order to decrease the seroconversion rates, with considerable success. On the other hand, a survival advantage has been observed in some patients who developed a HAGA following treatment. This correlates with development of an anti-idiotype antibody cascade directed toward the MAbs used to treat these patients. What follows is a selective review of HAGA and its effect on cancer treatment over the past 2 decades.

Use of radiolabeled monoclonal anti-B1 antibody for B lymphocyte imaging in Rhesus monkeys

International Nuclear Information System (INIS)

Letvin, N.L.; Zalutsky, M.R.; Chalifoux, L.V.; Atkins, H.L.

1987-01-01

Imaging tissues rich in B lymphocytes in man using a radiolabeled monoclonal anti-B cell antibody would be extremely useful in the clinical staging of non-Hodgkins lymphomas. Studies were done in rhesus monkeys using radiolabeled monoclonal anti-B1 antibody to determine the feasibility of such an approach. Immunohistologic studies demonstrated that infused monoclonal anti-B1 binds in vivo with specificity to B cells in lymph nodes and spleen. The kinetics of clearance of 131 I-labeled anti-B1 were determined. The B lymphocyte-rich spleen could be readily visualized by gamma camera scanning without significant background and without the need for image intensification or blood background subtraction techniques. These data support the feasibility of using anti-B1 for staging B cell lymphomas in man. (author)

Three-site sandwich radioimmunoassay with monoclonal antibodies for a sensitive determination of human alpha-fetoprotein

International Nuclear Information System (INIS)

Nomura, M.; Imai, M.; Takahashi, K.; Kumakura, T.; Tachibana, K.; Aoyagi, S.; Usuda, S.; Nakamura, T.; Miyakawa, Y.; Mayumi, M.

1983-01-01

Utilizing monoclonal antibodies against human alpha-fetoprotein, 3 distinct antigenic determinants were identified. These antigenic determinants, provisionally designated a, b and c, were arranged in such a manner that the binding of one determinant with the corresponding antibody did not inhibit, or only barely inhibited the binding of antibodies directed to the other 2 determinants. Monoclonal antibodies with 3 different specificities were, therefore, applied to develop a sandwich-type solid-phase radioimmunoassay of the antigen in which wells were coated with anti-a, and radiolabeled anti-b together with radiolabeled anti-c was employed to detect the bound antigen. The 3-site sandwich radioimmunoassay involving 3 different determinants gave a higher sensitivity than 2-site assays in which only anti-b or anti-c was employed as a radiolabeled reagent, because the radioactivity of the 2 labeled antibodies was added on the antigen bound to immobilized anti-a. (Auth.)

Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

Science.gov (United States)

Ezzatifar, Fatemeh; Majidi, Jafar; Baradaran, Behzad; Aghebati Maleki, Leili; Abdolalizadeh, Jalal; Yousefi, Mehdi

2015-01-01

Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori. PMID:25789225

Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

Directory of Open Access Journals (Sweden)

Fatemeh Ezzatifar

2015-03-01

Full Text Available Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori.

Pharmacokinetics, biodistribution and dosimetry of 99mTc-labeled anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats

International Nuclear Information System (INIS)

Escobar, Normando Iznaga; Morales, Alejo Morales; Duconge, Jorge; Torres, Idania Caballero; Fernandez, Eduardo; Gomez, Jose A.

1998-01-01

The pharmacokinetics, biodistribution and dosimetry of 99m Tc-labeled anti-human epidermal growth factor receptor (anti-hEGF-r) humanized monoclonal antibody (MAb) R3 was investigated following intravenous injection in normal Wistar rats. Serum disappearance curves were best fit by a two-compartment model having a mean distribution half-life (t (1(2α)) ) of 0.250 h and a mean elimination (t (1(2β)) ) of 13.89 h. Among the various organs, a little accumulation of the radiolabeled antibody was found only in kidneys. Biodistribution and dosimetry studies in humans were performed by extrapolation of the animal data to humans. Absorbed dose to normal organs and the remainder of the whole body were estimated using the medical internal radiation dose formula, and dose contributions from radioactivity in transit through the gastrointestinal tract were estimated using a compartment model. Extrapolated values of radiation absorbed dose to normal organs in rads per millicurie administered were whole body, 0.0085; lower large intestine wall, 0.0898; small intestine, 0.0530; upper large intestine wall, 0.0731; and kidneys, 0.0455. The effective dose equivalent predicted was 0.0162 rem/mCi and the effective dose was found to be 0.015 rem/mCi. On the basis of the pharmacokinetics, biodistribution and internal radiation dosimetry information obtained in this study, a diagnostic phase I clinical trial with 99m Tc-labeled humanized MAb R3 conjugate in patients should be supported

Radiolabeled anti-EGFR-antibody improves local tumor control after external beam radiotherapy and offers theragnostic potential

International Nuclear Information System (INIS)

Koi, Lydia; Bergmann, Ralf; Brüchner, Kerstin; Pietzsch, Jens; Pietzsch, Hans-Jürgen; Krause, Mechthild

2014-01-01

Purpose: The effect of radioimmunotherapy (RIT) using the therapeutic radionuclide Y-90 bound to the anti-EGFR antibody cetuximab combined with external beam irradiation (EBRT) (EBRIT) on permanent local tumor control in vivo was examined. Methods: Growth delay was evaluated in three human squamous cell carcinoma models after RIT with [ 90 Y]Y-(CHX-A′′-DTPA) 4 -cetuximab (Y-90-cetuximab). The EBRT dose required to cure 50% of the tumors (TCD 50 ) for EBRT alone or EBRIT was evaluated in one RIT-responder (FaDu) and one RIT-non-responder (UT-SCC-5). EGFR expression and microenvironmental parameters were evaluated in untreated tumors, bioavailability was visualized by PET using ([ 86 Y]Y-(CHX-A′′-DTPA) 4 -cetuximab (Y-86-cetuximab) and biodistribution using Y-90-cetuximab. Results: In UT-SCC-8 and FaDu but not in UT-SCC-5 radiolabeled cetuximab led to significant tumor growth delay. TCD 50 after EBRT was significantly decreased by EGFR-targeted RIT in FaDu but not in UT-SCC-5. In contrast to EGFR expression, parameters of the tumor micromilieu and in particular the Y-90-cetuximab biodistribution or Y-86-cetuximab visualization in PET correlated with the responsiveness to RIT or EBRIT. Conclusion: EGFR-targeted EBRIT can improve permanent local tumor control compared to EBRT alone. PET imaging of bioavailability of labeled cetuximab appears to be a suitable predictor for response to EBRIT. This theragnostic approach should be further explored for clinical translation

Epidermal Growth Factor Receptor (EGFR) gene copy number (GCN) correlates with clinical activity of irinotecan-cetuximab in K-RAS wild-type colorectal cancer: a fluorescence in situ (FISH) and chromogenic in situ hybridization (CISH) analysis

International Nuclear Information System (INIS)

Scartozzi, Mario; Galizia, Eva; Berardi, Rossana; Biscotti, Tommasina; Labianca, Roberto; Masi, Gianluca; Falcone, Alfredo; Cascinu, Stefano; Bearzi, Italo; Mandolesi, Alessandra; Pierantoni, Chiara; Loupakis, Fotios; Zaniboni, Alberto; Negri, Francesca; Quadri, Antonello; Zorzi, Fausto

2009-01-01

K-RAS wild type colorectal tumors show an improved response rate to anti-EGFR monoclonal antibodies. Nevertheless 70% to 40% of these patients still does not seem to benefit from this therapeutic approach. FISH EGFR GCN has been previously demonstrated to correlate with clinical outcome of colorectal cancer treated with anti-EGFR monoclonal antibodies. CISH also seemed able to provide accurate EGFR GCN information with the advantage of a simpler and reproducible technique involving immunohistochemistry and light microscopy. Based on these findings we investigated the correlation between both FISH and CISH EGFR GCN and clinical outcome in K-RAS wild-type colorectal cancer treated with irinotecan-cetuximab. Patients with advanced K-RAS wild-type, colorectal cancer receiving irinotecan-cetuximab after failure of irinotecan-based chemotherapy were eligible. A cut-off value for EGFR GCN of 2.6 and 2.12 for FISH and CISH respectively was derived from ROC curve analysis. Forty-four patients were available for analysis. We observed a partial remission in 9 (60%) and 2 (9%) cases with a FISH EGFR GCN ≥ 2.6 and < 2.6 respectively (p = 0.002) and in 10 (36%) and 1 (6%) cases with a CISH EGFR GCN ≥ 2.12 and < 2.12 respectively (p = 0.03). Median TTP was 7.7 and 6.4 months in patients showing increased FISH and CISH EGFR GCN whereas it was 2.9 and 3.1 months in those with low FISH and CISH EGFR GCN (p = 0.04 and 0.02 respectively). FISH and CISH EGFR GCN may both represent effective tools for a further patients selection in K-RAS wild-type colorectal cancer treated with cetuximab

EGFR-targeted anti-cancer drugs in radiotherapy: Preclinical evaluation of mechanisms

International Nuclear Information System (INIS)

Baumann, Michael; Krause, Mechthild; Dikomey, Ekkehard; Dittmann, Klaus; Doerr, Wolfgang; Kasten-Pisula, Ulla; Rodemann, H. Peter

2007-01-01

Preclinical and clinical results indicate that the EGFR can mediate radioresistance in different solid human tumours. Combination of radiotherapy and EGFR inhibitors can improve local tumour control compared to irradiation alone and has been introduced into clinical radiotherapy practice. So far several mechanisms have been identified in preclinical studies to contribute to improved local tumour control after radiation combined with EGFR inhibitors. These include direct kill of cancer stem cells by EGFR inhibitors, cellular radiosensitization through modified signal transduction, inhibition of repair of DNA damage, reduced repopulation and improved reoxygenation during fractionated radiotherapy. Effects and mechanisms may differ for different classes of EGFR inhibitors, for different tumours and for normal tissues. The mechanisms underlying this heterogeneity are currently poorly understood, and predictive assays are not available yet. Importantly, mechanisms and predictors for the combined effects of radiation with EGFR inhibitors appear to be considerably different to those for application of EGFR inhibitors alone or in combination with chemotherapy. Therefore to further evaluate the efficacy and mechanisms of EGFR-inhibition in combined treatments, radiotherapy-specific preclinical research strategies, which include in vivo experiments using local tumour control as an endpoint, as well as animal studies on normal tissue toxicity are needed

Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: Fine mapping and escape mutant analysis

NARCIS (Netherlands)

Marissen, W.E.; Kramer, R.A.; Rice, A.; Weldon, W.C.; Niezgoda, M.; Faber, M.; Slootstra, J.W.; Meloen, R.H.; Clijsters-van der Horst, M.; Visser, T.J.; Jongeneelen, M.; Thijsse, S.; Throsby, M.; Kruif, de J.; Rupprecht, C.E.; Dietzschold, B.; Goudsmit, J.; Bakker, A.B.H.

2005-01-01

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We

Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: fine mapping and escape mutant analysis

NARCIS (Netherlands)

Marissen, Wilfred E.; Kramer, R. Arjen; Rice, Amy; Weldon, William C.; Niezgoda, Michael; Faber, Milosz; Slootstra, Jerry W.; Meloen, Rob H.; Clijsters-van der Horst, Marieke; Visser, Therese J.; Jongeneelen, Mandy; Thijsse, Sandra; Throsby, Mark; de Kruif, John; Rupprecht, Charles E.; Dietzschold, Bernhard; Goudsmit, Jaap; Bakker, Alexander B. H.

2005-01-01

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We

Effects of the EGFR Inhibitor Erlotinib on Magnesium Handling

NARCIS (Netherlands)

Dimke, Henrik; van der Wijst, Jenny; Alexander, Todd R.; Meijer, Inez M. J.; Mulder, Gemma M.; van Goor, Harry; Tejpar, Sabine; Hoenderop, Joost G.; Bindels, Rene J.

A mutation in pro-EGF causes isolated hypomagnesemia, and monoclonal antibodies targeting the extracellular domain of the EGF receptor (EGFR) affect epithelial Mg2+ transport. The effect of the EGFR tyrosine kinase inhibitor erlotinib on Mg2+ homeostasis, however, remains unknown. Here, we injected

Effective therapeutic approach for head and neck cancer by an engineered minibody targeting the EGFR receptor.

Directory of Open Access Journals (Sweden)

Young Pil Kim

Full Text Available Cetuximab, a chimeric monoclonal antibody developed for targeting the Epidermal Growth Factor Receptor (EGFR, has been intensively used to treat cancer patients with metastatic colorectal cancer and head and neck cancer. Intact immunoglobulin G (IgG antibody like cetuximab, however, has some limitations such as high production cost and low penetration rate from vasculature into solid tumor mass due to its large size. In attempt to overcome these limitations, we engineered cetuximab to create single chain variable fragments (scFv-CH3; Minibody that were expressed in bacterial system. Among three engineered minibodies, we found that MI061 minibody, which is composed of the variable heavy (VH and light (VL region joined by an 18-residue peptide linker, displays higher solubility and better extraction properties from bacterial lysate. In addition, we validated that purified MI061 significantly interferes ligand binding to EGFR and blocks EGFR's phosphorylation. By using a protein microarray composed of 16,368 unique human proteins covering around 2,400 plasma membrane associated proteins such as receptors and channels, we also demonstrated that MI061 only recognizes the EGFR but not other proteins as compared with cetuximab. These results indicated that engineered MI061 retains both binding specificity and affinity of cetuximab for EGFR. Although it had relatively short half-life in serum, it was shown to be highly significant anti-tumor effect by inhibiting ERK pathway in A431 xenograft model. Taken together, our present study provides compelling evidence that engineered minibody is more effective and promising agent for in vivo targeting of solid tumors.

Production of a germline-humanized cetuximab scFv and evaluation of its activity in recognizing EGFR- overexpressing cancer cells.

Science.gov (United States)

Banisadr, Arsham; Safdari, Yaghoub; Kianmehr, Anvarsadat; Pourafshar, Mahdieh

2018-04-03

The aim of this study was to produce a humanized single chain antibody (scFv) as a potential improved product design to target EGFR (Epidermal Growth Factor Receptor) overexpressing cancer cells. To this end, CDR loops of cetuximab (an FDA-approved anti-EGFR antibody) were grafted on framework regions derived from type 3 (VH3 and VL3 kappa) human germline sequences to obtain recombinant VH and VL domainslinked together with a flexible linker [(Gly 4 Ser) 3 ] to form a scFv. Codon optimized synthetic gene encoding the scFv (with NH2-VH-linker-VL-COOH orientation) was expressed in E. coli Origami™ 2(DE3) cells and the resultant scFv purified by using Ni-NTA affinity chromatography. The scFv, called cet.Hum scFv, was evaluated in ELISA and immunoblot to determine whether it can recognize EGFR. The scFv was able to recognize EGFR over-expressing cancer cells (A-431) but failed to detect cancer cells with low levels of EGFR (MCF-7 cells). Although the affinity of the scFv forA-431 cells was 9 fold lower than that of cetuximab, it was strong enough to recognize these cells. Considering its ability to bind EGFR molecules, the scFv may exhibit a potential application for the detection of EGFR-overexpressing cancer cells.

Internal radiation dosimetry, pharmacokinetics and biodistribution of the 99mTc labeled ior egf/r3 monoclonal antibody

International Nuclear Information System (INIS)

Torres, L.A.; Perera, A.; Ramos, M.; Hernandez, A.; Iznaga, N.; Solano, M.E.; Alvarez, I.; Rodriguez, J.L.

2001-01-01

The aim of this work was to assess the internal radiation dosimetry, human pharmacokinetics and biodistribution of the 99m Tc-labeled murine monoclonal antibody (MAb) ior egf/r3, used for diagnosis of epithelial tumors. Five patients were included in this study. Multiple blood and urine samples were collected and sequential anterior and posterior whole-body scintigraphies up to 24 hr post-injection were acquired from all patients. The internal radiation dosimetry was estimated using the methods developed by the Medical Internal Radiation Dosimetry (MIRD) committee. Raw data were computed from operations between scintigraphic images and regions of interest (ROI). The residence times of the activity on the source organs were computed to assess the absorbed dose by 24 target organs. The dosimetric results showed that liver, gallbladder and spleen received the higher absorbed dose. The computed mean values were 0.69 mGy/MBq, 0.19mGy/MBq and 0.37 mGy/MBq, respectively. The mean value of effective dose was 1,2E-01 mSv/MBq and the effective equivalent dose was 9,2E-02 mSv/MBq. The pharmacokinetics and biodistribution results showed that this compound has a biexponential plasmatic and blood clearance with a rapid biodistribution phase and a slower elimination phase. This compound was excreted by the urinary and hepatobiliary systems. Liver was the principal target organ of this product showing a great retention of the MAb. These dosimetric results have allowed to use the ior egf/r3 kit in a safe and controlled way. (author)

Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

OpenAIRE

Fatemeh Ezzatifar; Jafar Majidi; Behzad Baradaran; Leili Aghebati Maleki; Jalal Abdolalizadeh; Mehdi Yousefi

2015-01-01

Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies again...

Monoclonal anti-elastin antibody labelled with technetium-99m

International Nuclear Information System (INIS)

Oliveira, Marcia B.N. de; Silva, Claudia R. da; Araujo, Adriano C. de; Bernardo Filho, Mario; Porto, Luis Cristovao M.S.; Gutfilen, Bianca; Souza, J.E.Q.; Frier, Malcolm

1999-01-01

Technetium-99m ( 99m Tc) is widely employed in nuclear medicine due to its desirable physical, chemical and biological properties. Moreover, it is easily available and normally is inexpensive. A reducing agent is necessary to label cells and molecules with 99m Tc and stannous chloride (Sn C L 2 ) is usually employed. Elastin is the functional protein component of the elastic fiber and it is related with some diseases such as arteriosclerosis, pulmonary emphysema and others. The present study refers to the preparation of the 99m Tc labeled monoclonal anti-elastin antibody. The monoclonal antibody was incubated with an excess of 2-iminothiolane. The free thiol groups created, were capable of binding with the reduced technetium. Labeling was an exchange reaction with 99m Tc-glucoheptonate. The labeled preparation was left at 4 deg C for one hour. Then, it was passed through a Sephadex G50 column. Various fractions were collected and counted. A peak corresponding to the radiolabeled antibody was obtained. Stability studies of the labelled anti-elastin were performed at 0,3 6, 24 hours, at both 4 deg C or room temperature. The biodistribution pattern of the 99m Tc-anti-elastin was studied in healthy male Swiss mice. The immunoreactivity was also determined. An useful labeled-anti-elastin was obtained to future immunoscintigraphic investigations. (author)

Radioimmunodetection of colorectal cancer, using anti-CEA monoclonal antibodies

International Nuclear Information System (INIS)

Murayama, Hiroki; Watanabe, Tadashi; Tadokoro, Masanori; Takagi, Hiroshi; Sakuma, Sadayuki; Sakamoto, Junichi.

1989-01-01

Aiming at radioimmunodetection of colorectal cancer, anti-CEA monoclonal antibodies (CEA102) were produced by immunization with purified CEA. CEA102 showed high specificity with clorectal cancer by mixed hemadsorption assay and immunoperoxidase technique. The antigen detected by CEA102 was confirmed to be carcinoembryonic antigen (CEA) and its molecular weight was estimated to be ca. 180,000 by biochemical analysis. The in vivo study using nude mice grafted a human colorectal cancer or a human malignant melanoma showed greater accumulation of 125 I-labeled CEA102 in CEA-positive colorectal cancer than in nude mouse tissues and CEA-negative malignant melanoma. Moreover we successfully obtained scans with good localization of the grafted colorectal cancer on FCR (Fuji Computed Radiography). Using 131 I-labeled CEA102 liver metastasis in the patient with colorectal cancer was successfully detected by external scanning with γ-camera. These results suggest that radiolabeled CEA102 is useful for the detection of colorectal cancer. (author)

Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer

Directory of Open Access Journals (Sweden)

Chen H

2013-10-01

Full Text Available Hongwei Chen,1,* Liya Wang,1,2,* Qiqi Yu,1,2 Weiping Qian,3 Diana Tiwari,1 Hong Yi,4 Andrew Y Wang,5 Jing Huang,1,2 Lily Yang,3 Hui Mao1,2 1Department of Radiology and Imaging Sciences, 2Center for Systems Imaging, 3Department of Surgery, Emory University School of Medicine, 4Robert Apkarian Electron Microscopy Core, Emory University, Atlanta, GA, 5Ocean NanoTech LLC, Springdale, AK, USA *These authors contributed equally to this work Abstract: Antifouling magnetic iron oxide nanoparticles (IONPs coated with block copolymer poly(ethylene oxide-block-poly(γ-methacryloxypropyltrimethoxysilane (PEO-b-PγMPS were investigated for improving cell targeting by reducing nonspecific uptake. Conjugation of a HER2 antibody, Herceptin®, or a single chain fragment (ScFv of antibody against epidermal growth factor receptor (ScFvEGFR to PEO-b-PγMPS-coated IONPs resulted in HER2-targeted or EGFR-targeted IONPs (anti-HER2-IONPs or ScFvEGFR-IONPs. The anti-HER2-IONPs bound specifically to SK-BR-3, a HER2-overexpressing breast cancer cell line, but not to MDA-MB-231, a HER2-underexpressing cell line. On the other hand, the ScFvEGFR-IONPs showed strong reactivity with MDA-MB-231, an EGFR-positive human breast cancer cell line, but not with MDA-MB-453, an EGFR-negative human breast cancer cell line. Transmission electron microscopy revealed internalization of the receptor-targeted nanoparticles by the targeted cancer cells. In addition, both antibody-conjugated and non-antibody-conjugated IONPs showed reduced nonspecific uptake by RAW264.7 mouse macrophages in vitro. The developed IONPs showed a long blood circulation time (serum half-life 11.6 hours in mice and low accumulation in both the liver and spleen. At 24 hours after systemic administration of ScFvEGFR-IONPs into mice bearing EGFR-positive breast cancer 4T1 mouse mammary tumors, magnetic resonance imaging revealed signal reduction in the tumor as a result of the accumulation of the targeted IONPs

The combination of anti-KIR monoclonal antibodies with anti-PD-1/PD-L1 monoclonal antibodies could be a critical breakthrough in overcoming tumor immune escape in NSCLC

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He YY

2018-04-01

Full Text Available Yayi He,1,2,* Sangtian Liu,1,* Jane Mattei,3 Paul A Bunn Jr,2 Caicun Zhou,1 Daniel Chan2 1Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University Medical School Cancer Institute, Tongji University School of Medicine, Shanghai, China; 2Division of Medical Oncology, Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA; 3Oncology Department, Moinhos de Vento Hospital, Porto Alegre, Brazil *These authors contributed equally to this work Background: The anti-programmed death-1 (PD-1/programmed death ligand-1 (PD-L1 monoclonal antibody has a good effect in the treatment of non-small cell lung cancer (NSCLC, but not all PD-1/PD-L1 positive patients can get benefit from it. Compensatory expression of other immune checkpoints may be correlated with the poor efficacy of anti-PD-1/PD-L1 monoclonal antibodies. The inhibitory human leukocyte antigen (HLA/killer cell Ig-like receptor (KIR can effectively block the killing effect of natural killer (NK cells on tumors. Our previous studies have confirmed that high expression of KIR was correlated with poor prognosis of NSCLC. Inhibitory KIR expression was positively correlated with the expression of PD-1. Methods: The expressions of KIR 2D (L1, L3, L4, S4 (BC032422/ADQ31987/NP_002246/NP_036446, Abcam and PD-1 (NAT 105, Cell marque proteins was assessed by immunohis­tochemistry. Results: The expression of inhibitory KIR in tumor cells or tumor infiltrating lymphocytes (TILs is associated with PD-1 expression. Among PD-1 positive patients, 76.3% were KIR 2D (L1, L3, L4, S4 positive on tumor cells, and 74.6% were KIR 2D (L1, L3, L4, S4 positive on TILs. We compared the expression of inhibitory KIR before and after treatment with nivolumab in 11 patients with NSCLC. We found that five (45.5% patients had positive expression of inhibitory KIR in tumor tissue after being treated with anti-PD-1 monoclonal antibodies, two of whom exhibited a significant

[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

Science.gov (United States)

Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

2012-06-01

To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

Immunohistochemical Analysis of Inflammatory Rheumatoid Synovial Tissues Using Anti-Human Podoplanin Monoclonal Antibody Panel.

Science.gov (United States)

Suzuki, Tomoto; Takakubo, Yuya; Oki, Hiroharu; Liu, Xing; Honma, Ryusuke; Naganuma, Yasushi; Goodman, Stuart B; Kaneko, Mika K; Kato, Yukinari; Takagi, Michiaki

2018-02-01

Podoplanin (PDPN) is a transmembrane sialoglycoprotein, which is expressed in several normal tissues and malignant tumors. Although PDPN expression in rheumatoid arthritis (RA) has been reported, the role of PDPN in RA and other arthritic conditions has not been fully elucidated. In this study, we examined PDPN expression in inflammatory synovial tissues using an anti-human PDPN (hPDPN) monoclonal antibody (mAb) panel to select the most useful one for evaluation of synovitis. Synovial tissue samples were obtained from 11 RA patients and 9 osteoarthritis (OA) patients undergoing joint surgery. PDPN-positive cells were immunostained by a panel of PDPN mAbs (NZ-1, LpMab-3, LpMab-7, LpMab-10, LpMab-12, LpMab-13, and LpMab-17), followed by cell grading of inflammation and cell counting of PDPN-positivity by a quantitative analyzer. Immunohistochemistry showed that PDPN was markedly expressed in both macrophage-like type A and fibroblast-like type B lining cells of the hyperplastic synovial lining cell layer, and macrophages and fibroblasts in the stroma of RA. Among anti-PDPN mAbs, LpMab-12 showed the highest score. In inflammatory OA synovium, PDPN expression was also detectable. Although LpMab-12 also showed the highest score in OA, the difference was not statistically significant. The inflammatory synovitis score of RA was significantly higher than that of OA. PDPN was expressed in inflammatory lining cells and sublining stroma of RA and OA synovium. In the seven anti-hPDPN antibodies examined, LpMab-12 was the most stainable antibody for PDPN in RA synovitis. Thus, LpMab-12 for PDPN has a possible and promising specific biomarker for evaluating synovitis in RA and inflammatory OA.

A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3

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Leili Aghebati Maleki

2013-08-01

Full Text Available Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG. Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases.

A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3

Science.gov (United States)

Aghebati Maleki, Leili; Baradaran, Behzad; Abdolalizadeh, Jalal; Ezzatifar, Fatemeh; Majidi, Jafar

2013-01-01

Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity with IgM & IgA by ELISA and confirmed by immunoblotting. The subclasses of the selected mAbs were determined. The best clone was injected intraperitoneally to some pristane-injected mice. Anti-IgG mAb was purified from the animals' ascitic fluid by Ion exchange chromatography and then, mAb was conjugated with HRP. Results: In the present study, over than 50 clones were obtained that 1 clone had optical density over than 3. We named this clone as supermonoclone which was selected for limiting dilution. The result of the immunoblotting, showed sharp band in IgG position and did not show any band in IgM&IgA position. Conclusion: Based on the findings of this study, the conjugated monoclonal antibody could have application in diagnosis of infectious diseases like Toxoplasmosis, Rubella and IgG class of other infectious and non- infectious diseases. PMID:24312857

Cellular Immunotherapy for Carcinoma Using Genetically Modified EGFR-Specific T Lymphocytes

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Xikun Zhou

2013-05-01

Full Text Available Epidermal growth factor receptor (EGFR is overexpressed in a variety of human malignancies, including pancreatic cancer, breast cancer, colon cancer, and non-small cell lung cancer. Overexpression of EGFR is a predictive marker of therapeutic response and several lines of evidence suggest that EGFR is an excellent target for tumor therapy. However, the effective antitumor capacity of EGFR-specific T cells against EGFR-overexpressing tumor cells has not been fully elucidated. In our previous study, we identified an anti-EGFR single-chain variable fragment (scFv with specific and high affinity after screening by ribosome display. In this study, the anticancer potential of anti-EGFR scFv was investigated on the basis of cell-targeted therapy. A chimeric antigen receptor (CAR targeting EGFR was constructed and expressed on the cell membrane of T lymphocytes. These CAR-modified T cells demonstrated antitumor efficacy both in vitro and in vivo. In addition, the safety evaluation showed that CAR-modified lymphocytes have no or very minimal acute systemic toxicity. Taken together, our study provided the experimental basis for clinical application of genetically engineered lymphocytes; moreover, we also evaluate a new and interesting cell therapy protocol.

A fundamental study of immunoscintigraphy with sup 131 I-labeled anti-CA 19-9 and anti-CEA monoclonal antibodies; Imaging of tumor-bearing mice by IMACIS-1 and cell ELISA with human tumor cells

Energy Technology Data Exchange (ETDEWEB)

Nogami, Toshihiko; Miura, Hiroshi; Ohmi, Shoichi; Kazahaya, Yasuhiro [CIS DIAGNOSTIC K.K., Chiba (Japan)

1990-05-01

A study was made on 2 types of {sup 131}I-labeled anti-CA 19-9 and anti-CEA mouse monoclonal antibodies (IMACIS-1) against human cancer related antigen as to their usefulness in radioimmunoimaging. Tumor-bearing nude mice were used for comparison. The transplanted tumors (SW948, COLO 201) were clearly visualized 48-72 hours after administration of IMACIS-1. Tumor/blood ratio 72 hours after administration: 8.69 in COLO 201 and 5.70 in SW948, showing ca. 10-15 times as high as those in PC-3 and HEp-2. IMACIS-1 therefore is considered useful in radioimmunoimaging of cancer. Analysis was made by in vitro cell ELISA. As a result, both of the cells specifically reacted with anti-CA 19-9 but not anti-CEA. (author).

{sup 99m}Tc-labeled chimeric anti-NCA 95 antigranulocyte monoclonal antibody for bone marrow imaging

Energy Technology Data Exchange (ETDEWEB)

Sarwar, M.; Higuchi, Tetsuya; Tomiyoshi, Katsumi [Gunma Univ., Maebashi (Japan). School of Medicine] [and others

1998-09-01

Chimeric mouse-human antigranulocyte monoclonal antibody (ch MAb) against non-specific cross-reacting antigen (NCA-95) was labeled with {sup 99m}Tc (using a direct method) and {sup 125}I (using the chloramine T method), and its binding to human granulocytes and LS-180 colorectal carcinoma cells expressing carcinoembryonic antigen on their surfaces, cross-reactive with anti-NCA-95 chimeric monoclonal antibody, increased in proportion to the number of cells added and reached more than 80% and 90%, respectively. In biodistribution studies, {sup 99m}Tc and {sup 125}I-labeled ch anti-NCA-95 MAb revealed high tumor uptake, and the tumor-to-blood ratio was 2.9 after 24 hours. The tumor-to-normal-organ ratio was also more than 3.0 in all organs except for the tumor-to-kidney ratio. Scintigrams of athymic nude mice confirmed the results of biodistribution studies that showed higher radioactivity in tumor and kidney of the mice administered with {sup 99m}Tc-labeled ch MAb. A normal volunteer injected with {sup 99m}Tc-labeled ch anti-NCA-95 antigranulocyte MAb showed clear bone marrow images, and a patient with aplastic anemia revealed irregular uptake in his lumbar spine, suggesting its utility for bone marrow scintigraphy and for the detection of hematological disorders, infections, and bone metastasis. (author)

Chemovirotherapy for head and neck squamous cell carcinoma with EGFR-targeted and CD/UPRT-armed oncolytic measles virus.

Science.gov (United States)

Zaoui, K; Bossow, S; Grossardt, C; Leber, M F; Springfeld, C; Plinkert, P K; Kalle, C von; Ungerechts, G

2012-03-01

First-line treatment of recurrent and/or refractory head and neck squamous cell carcinoma (HNSCC) is based on platinum, 5-fluorouracil (5-FU) and the monoclonal antiEGFR antibody cetuximab. However, in most cases this chemoimmunotherapy does not cure the disease, and more than 50% of HNSCC patients are dying because of local recurrence of the tumors. In the majority of cases, HNSCC overexpress the epidermal growth factor receptor (EGFR), and its presence is associated with a poor outcome. In this study, we engineered an EGFR-targeted oncolytic measles virus (MV), armed with the bifunctional enzyme cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT). CD/UPRT converts 5-fluorocytosine (5-FC) into the chemotherapeutic 5-FU, a mainstay of HNSCC chemotherapy. This virus efficiently replicates in and lyses primary HNSCC cells in vitro. Arming with CD/UPRT mediates efficient prodrug activation with high bystander killing of non-infected tumor cells. In mice bearing primary HNSCC xenografts, intratumoral administration of MV-antiEGFR resulted in statistically significant tumor growth delay and prolongation of survival. Importantly, combination with 5-FC is superior to virus-only treatment leading to significant tumor growth inhibition. Thus, chemovirotherapy with EGFR-targeted and CD/UPRT-armed MV is highly efficacious in preclinical settings with direct translational implications for a planned Phase I clinical trial of MV for locoregional treatment of HNSCC.

Labeling an anti-CD20 monoclonal antibody with 90Y

International Nuclear Information System (INIS)

Perera Pintado, Alejandro; Leyva Montaña, René; Prats Capote, Anaís; Góngora Bravo, Magdiel; Alberti Ramírez, Alejandro; León, Mariela; Hernández González, Ignacio; Dorvignit, Denise

2016-01-01

Lymphomas are among the 10 leading causes of death, both in Cuba and in the world, with an increasing incidence in recent years. Follicular lymphoma low-grade (indolent) is one of the most common in the Western world, representing 1/3 of all non-Hodgkin lymphomas (NHL). More than 90% of patients present with disseminated disease at diagnosis and generally have a slow evolution and good response to conventional treatment; but radically changed its forecast to relapse, resistance to therapeutic and histologic transformation can occur. The monoclonal antibody therapy has been a promising therapeutic. In this respect CD20 antigen it has been considered one of the most attractive targets in the therapy of follicular B cell lymphoma This is expressed in more than 90% of cases, while not present in stem cells and lines progenitors. Despite the success of immunotherapy, the relapse rate is still considerable. In order to increase the cytotoxic potential of immunotherapy, marked with beta emitting radionuclides alpha particles or monoclonal antibodies are used today. Despite encouraging results in patients with non-Hodgkin lymphomas refractory to other treatments, the extremely high costs of these commercial radiopharmaceuticals have greatly limited its application, even in the first world. A sustainable alternative is the marking of other anti-CD20 monoclonal antibodies, so researchers from several countries have concentrated their efforts on rituximaby other similar antibodies labeled with therapeutic radionuclides, as a possible cost-effectively to more problem. Today in Cuba it has an electrolytic generator 90 Sr- 90 Y Isotope Center, which ensures the availability of the radionuclide. In addition, the chimeric MAb rituximab is applied as part of the therapy of NHL in its health system and, recently, the Center for Molecular Immunology has obtained a chimeric monoclonal anti-CD20 antibody biosimilar rituximab, which is in phase clinical trial; which opens prospects for

Pharmacokinetics, biodistribution and dosimetry of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor humanized monoclonal antibody R3 in rats

Energy Technology Data Exchange (ETDEWEB)

Escobar, Normando Iznaga; Morales, Alejo Morales; Duconge, Jorge; Torres, Idania Caballero; Fernandez, Eduardo; Gomez, Jose A

1998-01-01

The pharmacokinetics, biodistribution and dosimetry of {sup 99m}Tc-labeled anti-human epidermal growth factor receptor (anti-hEGF-r) humanized monoclonal antibody (MAb) R3 was investigated following intravenous injection in normal Wistar rats. Serum disappearance curves were best fit by a two-compartment model having a mean distribution half-life (t{sub (1(2{alpha}}{sub ))}) of 0.250 h and a mean elimination (t{sub (1(2{beta}}{sub ))}) of 13.89 h. Among the various organs, a little accumulation of the radiolabeled antibody was found only in kidneys. Biodistribution and dosimetry studies in humans were performed by extrapolation of the animal data to humans. Absorbed dose to normal organs and the remainder of the whole body were estimated using the medical internal radiation dose formula, and dose contributions from radioactivity in transit through the gastrointestinal tract were estimated using a compartment model. Extrapolated values of radiation absorbed dose to normal organs in rads per millicurie administered were whole body, 0.0085; lower large intestine wall, 0.0898; small intestine, 0.0530; upper large intestine wall, 0.0731; and kidneys, 0.0455. The effective dose equivalent predicted was 0.0162 rem/mCi and the effective dose was found to be 0.015 rem/mCi. On the basis of the pharmacokinetics, biodistribution and internal radiation dosimetry information obtained in this study, a diagnostic phase I clinical trial with {sup 99m}Tc-labeled humanized MAb R3 conjugate in patients should be supported.

Signal integration: a framework for understanding the efficacy of therapeutics targeting the human EGFR family

Science.gov (United States)

Shepard, H. Michael; Brdlik, Cathleen M.; Schreiber, Hans

2008-01-01

The human EGFR (HER) family is essential for communication between many epithelial cancer cell types and the tumor microenvironment. Therapeutics targeting the HER family have demonstrated clinical success in the treatment of diverse epithelial cancers. Here we propose that the success of HER family–targeted monoclonal antibodies in cancer results from their ability to interfere with HER family consolidation of signals initiated by a multitude of other receptor systems. Ligand/receptor systems that initiate these signals include cytokine receptors, chemokine receptors, TLRs, GPCRs, and integrins. We further extrapolate that improvements in cancer therapeutics targeting the HER family are likely to incorporate mechanisms that block or reverse stromal support of malignant progression by isolating the HER family from autocrine and stromal influences. PMID:18982164

Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2017-12-01

Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

Anti-respiratory syncytial virus (RSV) G monoclonal antibodies reduce lung inflammation and viral lung titers when delivered therapeutically in a BALB/c mouse model.

Science.gov (United States)

Caidi, Hayat; Miao, Congrong; Thornburg, Natalie J; Tripp, Ralph A; Anderson, Larry J; Haynes, Lia M

2018-06-01

RSV continues to be a high priority for vaccine and antiviral drug development. Unfortunately, no safe and effective RSV vaccine is available and treatment options are limited. Over the past decade, several studies have focused on the role of RSV G protein on viral entry, viral neutralization, and RSV-mediated pathology. Anti-G murine monoclonal antibody (mAb) 131-2G treatment has been previously shown to reduce weight loss, bronchoalveolar lavage (BAL) cell number, airway reactivity, and Th2-type cytokine production in RSV-infected mice more rapidly than a commercial humanized monoclonal antibody (mAb) against RSV F protein (Palivizumab). In this study, we have tested two human anti-RSV G mAbs, 2B11 and 3D3, by both prophylactic and therapeutic treatment for RSV in the BALB/c mouse model. Both anti-G mAbs reduced viral load, leukocyte infiltration and IFN-γ and IL-4 expression in cell-free BAL supernatants emphasizing the potential of anti-G mAbs as anti-inflammatory and antiviral strategies. Published by Elsevier B.V.

Novel Clostridium difficile Anti-Toxin (TcdA and TcdB Humanized Monoclonal Antibodies Demonstrate In Vitro Neutralization across a Broad Spectrum of Clinical Strains and In Vivo Potency in a Hamster Spore Challenge Model.

Directory of Open Access Journals (Sweden)

Hongyu Qiu

Full Text Available Clostridium difficile (C. difficile infection (CDI is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA, and cytotoxin, toxin B (TcdB, which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4 and anti-TcdB (CANmAbB4 and CANmAbB1 antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively in a hamster gastrointestinal infection (GI model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI.

Novel anti-c-Mpl monoclonal antibodies identified multiple differentially glycosylated human c-Mpl proteins in megakaryocytic cells but not in human solid tumors.

Science.gov (United States)

Zhan, Jinghui; Felder, Barbara; Ellison, Aaron R; Winters, Aaron; Salimi-Moosavi, Hossein; Scully, Sheila; Turk, James R; Wei, Ping

2013-06-01

Thrombopoietin and its cognate receptor, c-Mpl, are the primary molecular regulators of megakaryocytopoiesis and platelet production. To date the pattern of c-Mpl expression in human solid tumors and the distribution and biochemical properties of c-Mpl proteins in hematopoietic tissues are largely unknown. We have recently developed highly specific mouse monoclonal antibodies (MAb) against human c-Mpl. In this study we used these antibodies to demonstrate the presence of full-length and truncated human c-Mpl proteins in various megakaryocytic cell types, and their absence in over 100 solid tumor cell lines and in the 12 most common primary human tumor types. Quantitative assays showed a cell context-dependent distribution of full-length and truncated c-Mpl proteins. All forms of human c-Mpl protein were found to be modified with extensive N-linked glycosylation but different degrees of sialylation and O-linked glycosylation. Of note, different variants of full-length c-Mpl protein exhibiting differential glycosylation were expressed in erythromegakaryocytic leukemic cell lines and in platelets from healthy human donors. This work provides a comprehensive analysis of human c-Mpl mRNA and protein expression on normal and malignant hematopoietic and non-hematopoietic cells and demonstrates the multiple applications of several novel anti-c-Mpl antibodies.

Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

Science.gov (United States)

Rubin, I; Lykkegaard, S; Olsen, A A; Selmer, J; Ballegaard, M

1988-01-01

Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

Application of four anti-human interferon-alpha monoclonal antibodies for immunoassay and comparative analysis of natural interferon-alpha mixtures

International Nuclear Information System (INIS)

Andersson, G.; Lundgren, E.; Ekre, H.P.

1991-01-01

Four different mouse monoclonal antibodies to human interferon-alpha (IFN-alpha) were evaluated for application in quantitative and comparative analysis of natural IFN-alpha mixtures. Binding to IFN-alpha subtypes in solution revealed individual reactivity patterns. These patterns changed if the IFN-alpha molecules were immobilized either passively to a surface or bound by another antibody. Also, substitution of a single amino acid in IFN-alpha 2 affected the binding, apparently by altering the conformation. Isoelectric focusing of three natural IFN-alpha preparations from different sources, followed by immunoblotting, resulted in individual patterns with each of the four mAbs and also demonstrated variation in the composition of the IFN-alpha preparations. None of the mAbs was subtype specific, but by combining the different mAbs, and also applying polyclonal anti-human IFN-alpha antibodies, it was possible to design sensitive sandwich ELISAs with broad or more limited IFN-alpha subtype specificity

Nuclear EGFR as a molecular target in cancer

International Nuclear Information System (INIS)

Brand, Toni M.; Iida, Mari; Luthar, Neha; Starr, Megan M.; Huppert, Evan J.; Wheeler, Deric L.

2013-01-01

The epidermal growth factor receptor (EGFR) has been one of the most targeted receptors in the field of oncology. While anti-EGFR inhibitors have demonstrated clinical success in specific cancers, most patients demonstrate either intrinsic or acquired resistance within one year of treatment. Many mechanisms of resistance to EGFR inhibitors have been identified, one of these being attributed to alternatively localized EGFR from the cell membrane into the cell’s nucleus. Inside the nucleus, EGFR functions as a co-transcription factor for several genes involved in cell proliferation and angiogenesis, and as a tyrosine kinase to activate and stabilize proliferating cell nuclear antigen and DNA dependent protein kinase. Nuclear localized EGFR is highly associated with disease progression, worse overall survival in numerous cancers, and enhanced resistance to radiation, chemotherapy, and the anti-EGFR therapies gefitinib and cetuximab. In this review the current knowledge of how nuclear EGFR enhances resistance to cancer therapeutics is discussed, in addition to highlighting ways to target nuclear EGFR as an anti-cancer strategy in the future

Durvalumab: an investigational anti-PD-L1 monoclonal antibody for the treatment of urothelial carcinoma

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Faiena I

2018-01-01

Full Text Available Izak Faiena,1,2 Amy L Cummings,3 Anna M Crosetti,3 Allan J Pantuck,1,2 Karim Chamie,1,2 Alexandra Drakaki1–3 1Department of Urology, 2Institute of Urologic Oncology, 3Department of Medicine, Division of Hematology and Oncology, David Geffen School of Medicine at University of California, Los Angeles, CA, USA Abstract: Our expanding knowledge of immunotherapy for solid tumors has led to an explosion of clinical trials aimed at urothelial carcinoma. The primary strategy is centered on unleashing the immune system by releasing the inhibitory signals propagated by programmed cell death-1 (PD-1 and its ligand programmed cell death ligand-1 (PD-L1. Many antibody constructs have been developed to block these interactions and are used in clinical trials. The Food and Drug Administration has already approved a number of checkpoint inhibitors such as anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4 monoclonal antibodies including ipilimumab; anti-PD-1 monoclonal antibodies including nivolumab and pembrolizumab; anti-PD-L1 antibodies including atezolizumab, avelumab, and durvalumab. One of the latest inhibitors is durvalumab, which is a high-affinity human immunoglobulin G1 kappa monoclonal antibody and blocks the interaction of PD-L1 with PD-1 and CD80. Currently, there are a number of ongoing trials in advanced urothelial carcinoma both using durvalumab monotherapy and in combination with other targeted therapies. In addition, durvalumab is being investigated in the non-muscle-invasive urothelial carcinoma, which is centered around intravenous formulations. These exciting developments have added a significant number of therapies in a previously limited treatment landscape. Keywords: durvalumab, checkpoint inhibitors, metastatic urothelial carcinoma

Itolizumab – a humanized anti-CD6 monoclonal antibody with a better side effects profile for the treatment of psoriasis

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Menon R

2015-04-01

Full Text Available Roshni Menon, Brinda G David Department of Dermatology, Venereology and Leprosy, Sri Venkateshwaraa Medical College Hospital and Research Centre, Ariyur, Pondicherry, India Abstract: Management of psoriasis is a challenge to the treating physician. The chronic inflammatory state of psoriasis with exacerbations and remissions necessitate “on-and-off” treatment schedules. The safety profiles of drugs and tolerability issues for patients are important factors to be considered during treatment. Various biological agents targeting T-cells and the inflammatory cytokines are available for systemic treatment of psoriasis. However, major causes of concern while using these drugs are risk of susceptibility to infection and development of anti-drug antibodies, which will affect the pharmacokinetic properties, efficacy, and safety profile of the drug. Itolizumab, a humanized anti-CD6 monoclonal antibody, is a new molecule that acts by immunomodulating the CD6 molecule. CD6 is a co-stimulatory molecule required for optimal T-cell stimulation by the antigen-presenting cells. This step is crucial in T-cell proliferation to form Th1 and Th17 cells, which play a major role in the pathogenesis of psoriasis. This article deals with the properties of Itolizumab and its role in the treatment of psoriasis. Based on the available published data, Itolizumab seems to have a better adverse effects profile and at the same time comparatively less efficacy when compared to other biological agents available for treating psoriasis. Larger studies with longer duration are required to clearly depict the long-term side effects profile. Keywords: Itolizumab, CD6, psoriasis, monoclonal antibody, biologicals 

Comparison of an anti-rabies human monoclonal antibody combination with human polyclonal anti-rabies immune globulin

NARCIS (Netherlands)

Goudsmit, Jaap; Marissen, Wilfred E.; Weldon, William C.; Niezgoda, Michael; Hanlon, Cathleen A.; Rice, Amy B.; Kruif, John de; Dietzschold, Bernhard; Bakker, Alexander B. H.; Rupprecht, Charles E.

2006-01-01

The World Health Organization estimates human mortality from endemic canine rabies to be 55,000 deaths/year. Limited supply hampers the accessibility of appropriate lifesaving treatment, particularly in areas where rabies is endemic. Anti-rabies antibodies are key to protection against lethal

Rapid Visualization of Human Tumor Xenografts through Optical Imaging with a Near-Infrared Fluorescent Anti–Epidermal Growth Factor Receptor Nanobody

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Sabrina Oliveira

2012-01-01

Full Text Available Given that overexpression of the epidermal growth factor receptor (EGFR is found in many types of human epithelial cancers, noninvasive molecular imaging of this receptor is of great interest. A number of studies have employed monoclonal antibodies as probes; however, their characteristic long half-life in the bloodstream has encouraged the development of smaller probes. In this study, an anti-EGFR nanobody-based probe was developed and tested in comparison with cetuximab for application in optical molecular imaging. To this aim, the anti-EGFR nanobody 7D12 and cetuximab were conjugated to the near-infrared fluorophore IRDye800CW. 7D12-IR allowed the visualization of tumors as early as 30 minutes postinjection, whereas with cetuximab-IR, no signal above background was observed at the tumor site. Quantification of the IR-conjugated proteins in the tumors revealed ≈ 17% of injected dose per gram 2 hours after injection of 7D12-IR, which was significantly higher than the tumor uptake obtained 24 hours after injection of cetuximab-IR. This difference is associated with the superior penetration and distribution of 7D12-IR within the tumor. These results demonstrate that this anti-EGFR nanobody conjugated to the NIR fluorophore has excellent properties for rapid preclinical optical imaging, which holds promise for its future use as a complementary diagnostic tool in humans.

EGFR gene copy number as a prognostic marker in colorectal cancer patients treated with cetuximab or panitumumab: a systematic review and meta analysis.

Directory of Open Access Journals (Sweden)

Zheng Jiang

Full Text Available The epidermal growth factor receptor (EGFR gene copy number (GCN has been previously demonstrated to correlate with the clinical outcome of colorectal cancer (CRC treated with anti-EGFR monoclonal antibodies (mAbs, although it remains controversial. We conducted a systematic review and meta-analysis to assess EGFR GCN as a potential biomarker of survival for patients with advanced CRC receiving treatment with anti-EGFR mAbs.We systematically identified articles investigating EGFR GCN by fluorescent or chromogenic in situ hybridization or other detection techniques in patients with metastatic CRC treated with panitumumab or cetuximab, (last search: 10 August 2012. Eligible studies had to report on overall survival (OS, progression-free survival (PFS or time-to-progression (TTP, stratified by EGFR GCN. Summary hazard ratios (HRs were calculated using random-effects models.Among 13 identified studies, 10 (776 patients, 302 with increased GCN, 8 (893 patients, 282 with increased GCN and 3 (149 patients, 66 with increased GCN were eligible for the OS, PFS and TTP meta-analyses, respectively. Increased EGFR GCN was associated with increased OS (HR = 0.62; 95% CI 0.50-0.77; P<0.001, PFS (HR = 0.65; 95% CI 0.47-0.89; P = 0.008 but not TTP (HR = 0.71; 95% CI 0.44-1.14; P = 0.157. It was also shown that EGFR GCN is independent of other factors such as KRAS status. Among those populations received second-line or higher treatment, increased EGFR GCN was strongly associated with improved survival (for OS, HR = 0.60; 95% CI 0.47-0.75; P<0.001; for PFS, HR = 0.59; 95% CI 0.47-0.75; P<0.001, whereas it did not influence survival in patients that received first-line therapy.Among the anti-EGFR-treated patients, increased EGFR GCN appears to be associated with improved survival outcomes. The effect on survival appears to be related to patients receiving the line of treatment.

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

Science.gov (United States)

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

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

Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-α

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Ceran Ceyhan

2012-10-01

Full Text Available Abstract Background One-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-α (TNF-α. Methods Mice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-α, were tested for their effects on breast cancer cell proliferation. Results We produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-α, antibodies acted synergistically on SK-BR-3 cells

EFFICACY EVALUATION OF A MONOCLONAL ANTIBODY AGAINST THE EPIDERMAL GROWTH FACTORS RECEPTOR IN THE MODEL OF SUBCUTANEOUS XENOGRAFT IN IMMUNODEFICIENT MICE

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Ya. Yu. Ustyugov

2015-01-01

Full Text Available This article presents the results of the comparative antitumor efficacy study of two test articles of therapeutic humanized monoclonal antibodies against epidermal growth factor receptor (EGFR manufactured by Russian biopharmaceutical company CJSC “Biocad” and the commercial drug “Erbitux®” (Merck, Germany in subcutaneous xenografts model using human epidermoid carcinoma A431NS cell line. EGFR overexpression in epithelial tumor cells is a commonly known fact that determines use of this receptor as a target for therapeutic monoclonal antibodies. The basic mechanism of action of such drugs is blocking of epithelial cells proliferation through competitive binding to EGFR. Evaluation of tumor growth dynamics in immunodeficient (Nu/Nu mice was performed during in vivo experiment using two parameters: tumor growth index and tumor growth inhibition (TGI, %. The results received with used study design show that antitumor effects of the test articles manufactured by CJSC “Biocad” and the commercial comparator drug “Erbitux®” estimated by values of TGI and tumor growth index are comparable.

Enzyme-linked immunosorbent assay for total sennosides using anti-sennside A and anti-sennoside B monoclonal antibodies.

Science.gov (United States)

Morinaga, Osamu; Uto, Takuhiro; Sakamoto, Seiichi; Tanaka, Hiroyuki; Shoyama, Yukihiro

2009-01-01

Total sennosides concentration is a very important factor when rhubarb and senna will be used as crude drugs. However, one-step analytical technique for total sennosides has not been reported except HPLC. An enzyme-linked immunosorbent assay (ELISA) for total sennosides concentration by using the combination of anti-sennoside A (SA) and anti-sennoside B (SB) monoclonal antibodies (MAbs) in a single assay has been investigated. Total sennosides concentration in rhubarb and senna samples determined by newly developed assay system showed good agreement with those analyzed by ELISA using anti-SA MAb and anti-SB MAb, respectively.

Demonstration of monoclonal anti-carcinoembryonic antigen (CEA) antibody internalization by electron microscopy, western blotting and radioimmunoassay.

Science.gov (United States)

Tsaltas, G; Ford, C H; Gallant, M

1992-01-01

One of the important factors affecting the action of monoclonal antibodies (Mabs) or immunoconjugates on tumour sites depends on whether the Mab is internalized by the cancer cells in question. The underexplored subject of internalization is discussed in this paper, and a number of in vitro techniques for investigating internalization are evaluated, using a model which consists of a well characterized anti-carcinoembryonic antigen (anti-CEA) Mab and a number of CEA expressing human cancer cell lines. Employing two alternative radiolabeling assays, evidence for internalization of the anti-CEA Mab by a CEA-positive colorectal cancer cell line (LS174T) was obtained throughout the time intervals examined (5 min to 150 min). Electronmicroscopy employing horseradish-peroxidase labeled anti-CEA Mab and control antibody permitted direct visualization of anti-CEA Mab-related staining in intracellular compartments of a high CEA-expressor human colorectal cell line (SKCO1). Finally Western blots of samples derived from cytosolic and membrane components of solubilized cells from lung and colonic cancer cell lines provided evidence for internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 minutes. Internalized anti-CEA was detected in all CEA expressing cell lines (LS174T, SKCO1, BENN) but not in the case of a very low CEA expressor line (COLO 320).

Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

Science.gov (United States)

Abbott, Christina; Huang, Guo; Ellison, Aaron R; Chen, Ching; Arora, Taruna; Szilvassy, Stephen J; Wei, Ping

2010-04-01

Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.

Expression and clinical value of EGFR in human meningiomas

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Magnus B. Arnli

2017-03-01

Full Text Available Background Meningiomas are common intracranial tumors in humans that frequently recur despite having a predominantly benign nature. Even though these tumors have been shown to commonly express EGFR/c-erbB1 (epidermal growth factor receptor, results from previous studies are uncertain regarding the expression of either intracellular or extracellular domains, cellular localization, activation state, relations to malignancy grade, and prognosis. Aims This study was designed to investigate the expression of the intracellular and extracellular domains of EGFR and of the activated receptor as well as its ligands EGF and TGFα in a large series of meningiomas with long follow-up data, and investigate if there exists an association between antibody expression and clinical and histological data. Methods A series of 186 meningiomas consecutively operated within a 10-year period was included. Tissue microarrays were constructed and immunohistochemically analyzed with antibodies targeting intracellular and extracellular domains of EGFR, phosphorylated receptor, and EGF and TGFα. Expression levels were recorded as a staining index (SI. Results Positive immunoreactivity was observed for all antibodies in most cases. There was in general high SIs for the intracellular domain of EGFR, phosphorylated EGFR, EGF, and TGFα but lower for the extracellular domain. Normal meninges were negative for all antibodies. Higher SIs for the phosphorylated EGFR were observed in grade II tumors compared with grade I (p = 0.018. Survival or recurrence was significantly decreased in the time to recurrence analysis (TTR with high SI-scores of the extracellular domain in a univariable survival analysis (HR 1.152, CI (1.036–1.280, p = 0.009. This was not significant in a multivariable analysis. Expression of the other antigens did not affect survival. Conclusion EGFR is overexpressed and in an activated state in human meningiomas. High levels of ligands also support this

Ofatumumab, a human anti-CD20 monoclonal antibody, for treatment of rheumatoid arthritis with an inadequate response to one or more disease-modifying antirheumatic drugs: results of a randomized, double-blind, placebo-controlled, phase I/II study

DEFF Research Database (Denmark)

Østergaard, Mikkel; Baslund, Bo; Rigby, William

2010-01-01

To investigate the safety and efficacy of ofatumumab, a novel human anti-CD20 monoclonal antibody (mAb), in patients with active rheumatoid arthritis (RA) whose disease did not respond to > or = 1 disease-modifying antirheumatic drug....

Serological analysis of human anti-human antibody responses in colon cancer patients treated with repeated doses of humanized monoclonal antibody A33.

Science.gov (United States)

Ritter, G; Cohen, L S; Williams, C; Richards, E C; Old, L J; Welt, S

2001-09-15

Mouse monoclonal antibody A33 (mAb A33) recognizes a M(r) 43,000 cell surface glycoprotein (designated A33) expressed in human colonic epithelium and colon cancer but absent from most other normal tissues. In patients, mAb A33 localizes with high specificity to colon cancer and is retained for up to 6 weeks in the cancer but cleared rapidly from normal colon (5-6 days). As a carrier of (125)I or (131)I, mAb A33 has shown antitumor activity. Induction of strong human anti-mouse antibody (immunoglobulin; HAMA) responses in patients, however, limits the use of the murine mAb A33 to very few injections. A humanized version of this antibody (huAb A33) has been prepared for Phase I and II clinical studies in patients with colon cancer. In those studies, immunogenicity of huAb A33 has been monitored using a novel, highly sensitive BIACORE method, which allows measurement of human anti-human antibodies (HAHAs) without the use of secondary reagents. We found that 63% (26 of 41) of the patients treated with repeated doses of huAb A33 developed HAHAs against a conformational antigenic determinant located in the V(L) and V(H) regions of huAb A33. Detailed serological analysis showed two distinct types of HAHAs. HAHA of type I (49% of patients) was characterized by an early onset with peak HAHA levels after 2 weeks of treatment, which declined with ongoing huAb A33 treatment. HAHA of type II (17% of patients) was characterized by a typically later onset of HAHA than in type I and by progressively increasing HAHA levels with each subsequent huAb A33 administration. Colon cancer patients with type I HAHAs did not develop infusion-related adverse events. In contrast, HAHA of type II was indicative of infusion-related adverse events. By using this new method, we were able to distinguish these two types of HAHAs in patients while on antibody treatment, allowing patients to be removed from study prior to the onset of severe infusion-related adverse events.

A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

International Nuclear Information System (INIS)

Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen

2015-01-01

The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice

A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

Energy Technology Data Exchange (ETDEWEB)

Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen, E-mail: srrshurology@163.com

2015-08-14

The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.

Application of monoclonal antibodies for diagnosis and treatment of human digestive cancer

International Nuclear Information System (INIS)

Otsuji, Eigo

2007-01-01

Radioimmunoscintigraphic applications of monoclonal antibodies (Mabs) for noninvasive detection and visualization of target tumors have grown immensely, and it suggests that Mabs can reach specifically to the targeted tumors in the human body. Radionuclides, cytotoxic drugs and anti-cancer drugs can be coupled to these specific MAbs to detect the extent of disease and/or to treat the tumors. Many of such immunoconjugates were studied for targeting therapy for cancer in animal experiments and some of them have applied to human. In this paper, we described the existing status of application of Mabs for diagnosis and immunotargeting therapy of digestive cancers. (author)

Nimotuzumab enhances temozolomide?induced growth suppression of glioma cells expressing mutant EGFR in vivo

OpenAIRE

Nitta, Yusuke; Shimizu, Saki; Shishido?Hara, Yukiko; Suzuki, Kaori; Shiokawa, Yoshiaki; Nagane, Motoo

2016-01-01

Abstract A mutant form of epidermal growth factor receptor (EGFR), EGFRvIII, is common in glioblastoma (GBM) and confers enhanced tumorigenic activity and drug resistance. Nimotuzumab, an anti?EGFR antibody, has shown preclinical and clinical activity to GBM, but its specific activity against EGFRvIII has not been fully investigated. Human glioma U87MG or LNZ308 cells overexpressing either wild?type (wt) EGFR or EGFRvIII were treated with nimotuzumab, temozolomide, or both. Expression and pho...

Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

Science.gov (United States)

Nakanishi, Takeshi; Maru, Takamitsu; Tahara, Kazuhiro; Sanada, Hideaki; Umetsu, Mitsuo; Asano, Ryutaro; Kumagai, Izumi

2013-02-01

We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

Radioiodination of monoclonal antibody intact anti-CEA

International Nuclear Information System (INIS)

Okada, H.; Souza, I.T.T.; Silva, C.P.G.

1990-11-01

The purpose of this study is to examine a convenient system that can be used to iodinate monoclonal antibodies which is rapid, simple, efficient and reproducible, and which can be accomplished in radiopharmaceutical laboratories. It is important to remember that antibodies are sensitive biochemicals, subject to losses of the activity that is essential to their mode of action, namely the ability to bind specific antigen. The advent of solid phase iodination agents has greatly expanded the range of gentle iodination techniques available for iodinating sensitive biological materials. The agent most widely used is the Iodogen (1,3,4,6 tetrachloro-3a-6a diphenylglycoluril) method. Anti-CEA 4C sub(11) IgG sub(2a,k) (prepared in the Ludwig Institute-Sao Paulo-Brazil ) is used as model to evaluate the Iodogen methodology. The miniature chromatographic system, also rapid, accurate, simple, efficient was elaborated to determine the labelling efficiency incorporation of iodine into immunoglobulin, and the radiochemical purity of sup(131)I-anti-CEA. (author)

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

Science.gov (United States)

Alper, O; De Santis, M L; Stromberg, K; Hacker, N F; Cho-Chung, Y S; Salomon, D S

2000-11-15

Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness. Copyright 2000 Wiley-Liss, Inc.

Long-term survival after a favorable response to anti-EGFR antibody plus chemotherapy to treat bone marrow metastasis: a case report of KRAS-wildtype rectal cancer

Directory of Open Access Journals (Sweden)

Nakamura S

2017-02-01

Full Text Available Sho Nakamura, Tadahisa Fukui, Shuhei Suzuki, Hiroyuki Takeda, Kaname Watanabe, Takashi Yoshioka Department of Clinical Oncology, Yamagata University Faculty of Medicine, Yamagata, Japan Abstract: Bone marrow metastasis is a rare consequence of colorectal cancer that results in a poor prognosis; few reports describe a favorable response to doublet chemotherapy combined with targeted therapy, which is currently the standard treatment. We experienced a case where anti-epidermal growth factor receptor (EGFR antibody produced a marked anti-tumor response to bone marrow metastasis that led to long-term survival. A 51-year-old man was diagnosed with a primary KRAS-wildtype rectal cancer with multiple metastases, including the bone marrow. Disease control was achieved for 10.8 months following chemotherapy with a modified FOLFOX6 regimen combined with an anti-EGFR antibody. He died of cancer 22.7 and 16.6 months after disease onset and first-line chemotherapy, respectively. This case shows that early tumor shrinkage and deepness of response to the anti-EGFR antibody were observed even in a patient with bone marrow metastasis. Anti-EGFR antibody therapy should therefore be considered even when a patient’s medical condition appears to be poor owing to bone marrow metastasis. Moreover, tumors that are likely to be sensitive to chemotherapy, such as RAS-wildtype colorectal cancers, can be considered for anti-EGFR antibody therapy even if the patient is considered unfit for chemotherapy. Keywords: colorectal cancer, anti-epidermal growth factor receptor antibody, molecular targeted therapies, disseminated intravascular coagulation, standard of care

Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody.

Science.gov (United States)

Donahue, Renee N; Lepone, Lauren M; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A; Heery, Christopher R; Gulley, James L; Schlom, Jeffrey

2017-01-01

Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range of human tumor cells, and clinical studies have demonstrated anti-tumor activity versus a range of human cancers. This study was designed to investigate the effect on immune cell subsets in the peripheral blood of cancer patients prior to and following multiple administrations of avelumab. One hundred twenty-three distinct immune cell subsets in the peripheral blood of cancer patients ( n  = 28) in a phase I trial were analyzed by flow cytometry prior to and following one, three, and nine cycles of avelumab. Changes in soluble (s) CD27 and sCD40L in plasma were also evaluated. In vitro studies were also performed to determine if avelumab would mediate ADCC of PBMC. No statistically significant changes in any of the 123 immune cell subsets analyzed were observed at any dose level, or number of doses, of avelumab. Increases in the ratio of sCD27:sCD40L were observed, suggesting potential immune activation. Controlled in vitro studies also showed lysis of tumor cells by avelumab versus no lysis of PBMC from five donors. These studies demonstrate the lack of any significant effect on multiple immune cell subsets, even those expressing PD-L1, following multiple cycles of avelumab. These results complement prior studies showing anti-tumor effects of avelumab and comparable levels of adverse events with avelumab versus other anti-PD-1/PD-L1 MAbs. These studies provide the rationale to further exploit the potential ADCC mechanism of action of avelumab as well as other human IgG1 checkpoint

Evaluación de los reactivos hemoclasificadores monoclonales cubanos HEMO CIM ANTI-A y HEMO CIM ANTI-B en pacientes VIH/SIDA Evaluation of the Cuban anti-A Hemo CIM and anti-B Hemo CIM monoclonal hemoclassifiers in HIV/AIDS patients

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Ananidia Rivero

2000-12-01

Full Text Available Se describe un estudio realizado con los hemoclasificadores monoclonales cubanos Hemo CIM anti-A y anti-B producidos por el Centro de Inmunología Molecular (CIM y comercializados por CIMAB S.A, para ser evaluados en pacientes VIH/SIDA. Se analizaron un total de 130 pacientes en el Servicio de Transfusiones del Instituto "Pedro Kourí". Se utilizaron en paralelo los antisueros policlonales de producción nacional (Laboratorios Betera y los reactivos monoclonales comerciales producidos y donados por International Blood Group Reference Laboratory; Bristol, UK. Se demostró que los reactivos Hemo CIM anti-A y Hemo CIM anti-B se comportaron de forma similar a sus homólogos comerciales y policlonales. Al comparar la efectividad de la aglutinación para los 3 reactivos pudimos comprobar que con el reactivo monoclonal, Hemo CIM anti-A y anti-B se obtuvieron los mejores resultados expresados en crucesA study conducted with the Cuban anti-A and anti-B Hemo CIM monoclonal hemoclassifiers produced by the Center of Molecular Immunology (CMI and commercialized by CIMAB S.A. to be evaluated in HIV/AIDS patients is described. 130 patients were analyzed at the Service of Transfusions of "Pedro Kouri" Institute. The polyclonal antisera of national production (Betera Laboratories and the commercial monoclonal reagents produced and donated by the International Blood Group Reference Laboratory; Bristol, UK, were used at the same time. It was proved that the anti-A Hemo CIM and anti-B Hemo CIM reagents behaved similarly to their commercial and polyclonal homologues. On comparing the effectiveness of the agglutination for the 3 reagents, we were able to verify that the best results expressed in crosses were obtained with the anti-A and anti-B Hemo CIM monoclonal reagents

Study on in vivo examination of tumors of epithelial origin for the monoclonales antibodies ior c5, ior egf/r3 and humanized hr3, by using inmunogammagraphy technique

International Nuclear Information System (INIS)

Ramos Suzarte, Mayra; Iznaga Escobar, Normando; Torres Gemil, Olga; Rodriguez Obaya, Teresita; Perez Rodriguez, Rolando; Rodriguez Mesa, Nelson; Oliva Gonzalez, Juan P.; Perera Pintado, Alejandro; Torres Aroche, Leonel; Batista Cuellar, Juan; Gonzalez Lazo, Nery; Cruz, Teresa; Leonard, Ibrahim; Neninger, Elia

2003-01-01

The present work is aimed at demonstrating that humanized hR3 and ior egf/r3, ior c5, ior cea-1 murine monoclonal antibodies (mAbs) can identify primary neoplasia lesions, Their post-surgical received and metastasis by using immunography, Results showed that above mentioned mAbs are useful for the diagnosis of epithelial tumors, their metastasis and post-surgical received. this technique becomes a concept prove for further use of mAbs in cancer therapy

Enhancement of retroviral infection in vitro by anti-Le(y) IgG: reversal by humanization of monoclonal mouse antibody

DEFF Research Database (Denmark)

Hansen, J E; Sørensen, A M; Arendrup, M

1993-01-01

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...

Itolizumab – a humanized anti-CD6 monoclonal antibody with better side effects profile for the treatment of psoriasis [Corrigendum

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Menon R

2015-06-01

Full Text Available Menon R, David BG. Clinical, Cosmetic and Investigational Dermatology. 2015;8:215–22.On page 215, please note correspondence should have been listed as:   Roshni Menon, D II/17,JIPMER Campus, Dhanvanthri Nagar,Pondicherry, India 605006Tel +91 944 320 8140Email roshnijagdish@gmail.com.On page 215, the first sentence of the Introduction was “Psoriasis is a chronic inflammatory disease of the skin characterized by exacerbations and remissions affecting 1%–3% of the world’s population, and approximately 20% of patients have moderate to severe disease.1,2” however should have been “Psoriasis is a chronic inflammatory disease of the skin characterized by exacerbations and remissions affecting 1%–3% of the world’s population. Approximately 20% of patients have moderate to severe disease.1,2”On page 217, 219, and 221 the running header was “Itolizumab – aCD6 monoclonal antibody for the treatment of psoriasis” however should have been “Itolizumab – a humanized anti CD6 monoclonal antibody for the treatment of psoriasis”.On page 218, Table 1, the second column heading was listed as “Anand et al25 n=40 (moderate–severe psoriasis” however should have been “Anand et al25 n=40/32 weeks (moderate–severe psoriasis”.Read the original article 

Micrometastatic cancer cells in bone marrow: in vitro detection with anti-cytokeratin and in vivo labeling with anti-17-1A monoclonal antibodies

International Nuclear Information System (INIS)

Schlimok, G.; Funke, I.; Holzmann, B.

1987-01-01

The detection of early micrometastasis or disseminated single tumor cells poses a problem for conventional diagnosis procedures. Using a panel of monoclonal antibodies against cytokeratin and the 17-1A epithelial antigen the authors identified immunocytochemically tumor cells in bone marrow of patients with breast cancer and colorectal cancer at the time of surgery of the primary tumor. Monoclonal antibody CK2, recognizing the human cytokeratin component 18 in simple epithelia, appeared to be the most suitable reagent because of its negative reaction with bone marrow samples of the noncarcinoma patients. Its specificity was further demonstrated in a double-marker staining procedure using an anti-leukocyte common antigen monoclonal antibody (T200) as counterstain. A comparative analysis showed that immunocytology was clearly superior to conventional cytology and histology. In 9.5-20.5% of patients without distant metastasis, tumor cells could be detected in bone marrow. They found a significant correlation between tumor cells in bone marrow and conventional risk factors, such as distant metastasis or lymph node involvement. In a first approach toward immunotherapy they demonstrated in 3 patients that infused monoclonal antibody 17-1A can label single tumor cells in bone marrow in vivo. They then used this single approach to follow up on 7 patients undergoing 17-1A therapy in an adjuvant clinical trial

Anti-EGFR-iRGD recombinant protein conjugated silk fibroin nanoparticles for enhanced tumor targeting and antitumor efficiency

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Bian X

2016-05-01

Full Text Available Xinyu Bian,* Puyuan Wu,* Huizi Sha, Hanqing Qian, Qing Wang, Lei Cheng, Yang Yang, Mi Yang, Baorui LiuComprehensive Cancer Center of Drum-Tower Hospital, Medical School of Nanjing University, Clinical Cancer Institute of Nanjing University, Nanjing, People’s Republic of China*These authors contributed equally to this workAbstract: In this study, we report a novel kind of targeting with paclitaxel (PTX-loaded silk fibroin nanoparticles conjugated with iRGD–EGFR nanobody recombinant protein (anti-EGFR-iRGD. The new nanoparticles (called A-PTX-SF-NPs were prepared using the carbodiimide-mediated coupling procedure and their characteristics were evaluated. The cellular cytotoxicity and cellular uptake of A-PTX-SF-NPs were also investigated. The results in vivo suggested that NPs conjugated with the recombinant protein exhibited more targeting and anti-neoplastic property in cells with high EGFR expression. In the in vivo antitumor efficacy assay, the A-PTX-SF-NPs group showed slower tumor growth and smaller tumor volumes than PTX-SF-NPs in a HeLa xenograft mouse model. A real-time near-infrared fluorescence imaging study showed that A-PTX-SF-NPs could target the tumor more effectively. These results suggest that the anticancer activity and tumor targeting of A-PTX-SF-NPs were superior to those of PTX-SF-NPs and may have the potential to be used for targeted delivery for tumor therapies. Keywords: EGFR, nanobody, iRGD, recombinant protein, targeting drug carriers, antitumor efficiency

Humanization and characterization of an anti-ricin neutralization monoclonal antibody.

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Wei-Gang Hu

Full Text Available Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9 variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with K(D of 1.63 nM, comparable to its parental murine D9 (2.55 nM. In a mouse model, intraperitoneal (i.p. administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.

Expression of epidermal growth factor receptors in human endometrial carcinoma

DEFF Research Database (Denmark)

Nyholm, H C; Nielsen, Anette Lynge; Ottesen, B

1993-01-01

Little data exist on the expression of epidermal growth factor receptors (EGF-Rs) in human endometrial cancer. EGF-R status was studied in 65 patients with endometrial carcinomas and in 26 women with nonmalignant postmenopausal endometria, either inactive/atrophic endometrium or adenomatous...... hyperplasia. EGF-R was identified on frozen tissue sections by means of an indirect immunoperoxidase technique with a monoclonal antibody against the external domain of the EGF-R. Seventy-one percent of the carcinomas expressed positive EGF-R immunoreactivity. In general, staining was most prominent...

The predictive value of KRAS, NRAS, BRAF, PIK3CA and PTEN for anti-EGFR treatment in metastatic colorectal cancer

DEFF Research Database (Denmark)

Therkildsen, Christina; Bergmann, Troels K; Henrichsen-Schnack, Tine

2014-01-01

BACKGROUND: In metastatic colorectal cancer, mutation testing for KRAS exon 2 is widely implemented to select patients with wild-type tumors for treatment with the monocloncal anti-EGFR antibodies cetuximab and panitumumab. The added predictive value of additional biomarkers in the RAS-RAF-MAPK a......BACKGROUND: In metastatic colorectal cancer, mutation testing for KRAS exon 2 is widely implemented to select patients with wild-type tumors for treatment with the monocloncal anti-EGFR antibodies cetuximab and panitumumab. The added predictive value of additional biomarkers in the RAS...

Therapeutic potential of an anti-high mobility group box-1 monoclonal antibody in epilepsy.

Science.gov (United States)

Zhao, Junli; Wang, Yi; Xu, Cenglin; Liu, Keyue; Wang, Ying; Chen, Liying; Wu, Xiaohua; Gao, Feng; Guo, Yi; Zhu, Junming; Wang, Shuang; Nishibori, Masahiro; Chen, Zhong

2017-08-01

Brain inflammation is a major factor in epilepsy, and the high mobility group box-1 (HMGB1) protein is known to contribute significantly to the generation of seizures. Here, we investigated the therapeutic potential of an anti-HMGB1 monoclonal antibody (mAb) in epilepsy. anti-HMGB1 mAb attenuated both acute seizure models (maximal electroshock seizure, pentylenetetrazole-induced and kindling-induced), and chronic epilepsy model (kainic acid-induced) in a dose-dependent manner. Meanwhile, the anti-HMGB1 mAb also attenuated seizure activities of human brain slices obtained from surgical resection from drug-resistant epilepsy patients. The mAb showed an anti-seizure effect with a long-term manner and appeared to be minimal side effects at even very high dose (no disrupted physical EEG rhythm and no impaired basic physical functions, such as body growth rate and thermoregulation). This anti-seizure effect of mAb results from its inhibition of translocated HMGB1 from nuclei following seizures, and the anti-seizure effect was absent in toll-like receptor 4 knockout (TLR4 -/- ) mice. Interestingly, the anti-HMGB1 mAb also showed a disease-modifying anti-epileptogenetic effect on epileptogenesis after status epileptics, which is indicated by reducing seizure frequency and improving the impaired cognitive function. These results indicate that the anti-HMGB1 mAb should be viewed as a very promising approach for the development of novel therapies to treat refractory epilepsy. Copyright © 2017 Elsevier Inc. All rights reserved.

Lymphocyte antibody-dependent cytotoxicity test for evaluation of clinical role of monoclonal anti-D-antibodies for prevention of rhesus sensitization.

Science.gov (United States)

Olovnikova, N I; Belkina, E V; Nikolaeva, T L; Miterev, G Yu; Chertkov, I L

2006-01-01

Monoclonal antibodies to D antigen were studied in the reaction of antibody-dependent cytotoxicity for evaluation of the possibility of using these antibodies for preventing rhesus sensitization. High hemolytic activity of four anti-D-monoclonal antibodies in the antibody-dependent cytotoxicity test, mediated by their interaction with FcgammaRI, and the capacity to accelerate elimination of D+ erythrocytes from circulation did not provide the immunosuppressive effect. It was hypothesized that monoclonal antibodies for prevention of rhesus sensitization should interact with FcgammaRIII on lymphocytes. These monoclonal antibodies are extremely rare: only 4 of 125 studied antibodies mediated hemolysis in the antibody-dependent cytotoxicity test with lymphocytes, while all polyclonal anti-D-preparations exhibited this activity.

The importance of tumor marker titers for the indication of immunoscintigraphy with monoclonal antibodies anti-CEA and anti-CA 19.9

International Nuclear Information System (INIS)

Bouvier, J.F.; Charrie, A.; Fleury-Goyon, M.C.; Chauvot, P. et; Lahneche, B.E.

1986-01-01

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)

A view on EGFR-targeted therapies from the oncogene-addiction perspective.

Science.gov (United States)

Perez, Rolando; Crombet, Tania; de Leon, Joel; Moreno, Ernesto

2013-01-01

Tumor cell growth and survival can often be impaired by inactivating a single oncogen- a phenomenon that has been called as "oncogene addiction." It is in such scenarios that molecular targeted therapies may succeed. among known oncogenes, the epidermal growth factor receptor (EGFR) has become the target of different cancer therapies. So far, however, the clinical benefit from EGFR-targeted therapies has been rather limited. a critical review of the large amount of clinical data obtained with anti-EGFR agents, carried out from the perspective of the oncogene addiction concept, may help to understand the causes of the unsatisfactory results. In this article we intend to do such an exercise taking as basis for the analysis a few case studies of anti-EGFR agents that are currently in the clinic. There, the "EGFR addiction" phenomenon becomes apparent in high-responder patients. We further discuss how the concept of oncogene addiction needs to be interpreted on the light of emerging experimental evidences and ideas; in particular, that EGFR addiction may reflect the interconnection of several cellular pathways. In this regard we set forth several hypotheses; namely, that requirement of higher glucose uptake by hypoxic tumor cells may reinforce EGFR addiction; and that chronic use of EGFR-targeted antibodies in EGFR-addicted tumors would induce stable disease by reversing the malignant phenotype of cancer stem cells and also by sustaining an anti-tumor T cell response. Finally, we discuss possible reasons for the failure of certain combinatorial therapies involving anti-EGFR agents, arguing that some of these agents might produce either a negative or a positive trans-modulation effect on other oncogenes. It becomes evident that we need operational definitions of EGFR addiction in order to determine which patient populations may benefit from treatment with anti-EGFR drugs, and to improve the design of these therapies.

A view on EGFR-targeted therapies from the oncogene-addiction perspective

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Rolando ePerez

2013-04-01

Full Text Available Tumor cell growth and survival can often be impaired by inactivating a single oncogen – a phenomenon that has been called as 'oncogene addiction'. It is in such scenarios that molecular targeted therapies may succeed. Among known oncogenes, the epidermal growth factor receptor (EGFR has become the target of different cancer therapies. So far, however, the clinical benefit from EGFR-targeted therapies has been rather limited. A critical review of the large amount of clinical data obtained with anti-EGFR agents, carried out from the perspective of the oncogene addiction concept, may help to understand the causes of the unsatisfactory results. In this article we intend to do such an exercise taking as basis for the analysis a few case studies of anti-EGFR agents that are currently in the clinic. There, the 'EGFR addiction' phenomenon becomes apparent in high-responder patients. We further discuss how the concept of oncogene addiction needs to be interpreted on the light of emerging experimental evidences and ideas; in particular, that EGFR addiction may reflect the interconnection of several cellular pathways. In this regard we set forth several hypotheses; namely, that requirement of higher glucose uptake by hypoxic tumor cells may reinforce EGFR addiction; and that chronic use of EGFR-targeted antibodies in EGFR-addicted tumors would induce stable disease by reversing the malignant phenotype of cancer stem cells and also by sustaining an anti-tumor T cell response. Finally, we discuss possible reasons for the failure of certain combinatorial therapies involving anti-EGFR agents, arguing that some of these agents might produce either a negative or a positive trans-modulation effect on other oncogenes. It becomes evident that we need operational definitions of EGFR addiction in order to determine which patient populations may benefit from treatment with anti-EGFR drugs, and to improve the design of these therapies.

Randomized phase III study of panitumumab with fluorouracil, leucovorin, and irinotecan (FOLFIRI) compared with FOLFIRI alone as second-line treatment in patients with metastatic colorectal cancer

NARCIS (Netherlands)

Peeters, Marc; Price, Timothy Jay; Cervantes, Andrés; Sobrero, Alberto F.; Ducreux, Michel; Hotko, Yevhen; André, Thierry; Chan, Emily; Lordick, Florian; Punt, Cornelis J. A.; Strickland, Andrew H.; Wilson, Gregory; Ciuleanu, Tudor-Eliade; Roman, Laslo; van Cutsem, Eric; Tzekova, Valentina; Collins, Simon; Oliner, Kelly S.; Rong, Alan; Gansert, Jennifer

2010-01-01

Panitumumab is a fully human anti-epidermal growth factor receptor (EGFR) monoclonal antibody that improves progression-free survival (PFS) in chemotherapy-refractory metastatic colorectal cancer (mCRC). This trial evaluated the efficacy and safety of panitumumab plus fluorouracil, leucovorin, and

Crossreactivity of boar sperm monoclonal antibodies with human ...

African Journals Online (AJOL)

Monoclonal antibodies against the head (H mabs) and tail (Tmabs) of boar spermatozoa were produced. Spermatozoa from boar, stallion, bull, human, ram, goat and rabbit were independently incubated with the monoclonal antibodies and later stained by immunofluorescence method. There were positive reactions of the ...

Effect on renal function of an iso-osmolar contrast agent in patients with monoclonal gammopathies

International Nuclear Information System (INIS)

Preda, Lorenzo; Agazzi, Alberto; Martinelli, Giovanni; Raimondi, Sara; Lanfranchi, Carla Federica; Passerini, Rita; Calvetta, Albania; Bellomi, Massimo

2011-01-01

To assess the safety of the non-ionic iso-osmolar contrast agent iodixanol on renal function in patients with monoclonal gammopathies undergoing CT. We explored the effect of iodixanol on renal function in 30 patients with monoclonal gammopathies and 20 oncological patients with a normal electrophoretic profile (control group). The parameters used to estimate renal function were: serum creatinine, eGFR (determined 24 h before and 48 h after the administration of iodixanol), and urinary excretion of Neutrophil Gelatinase-Associated Lipocalin (NGAL) determined 2 h and 24 h after. Serum creatinine was also determined 1 month after the administration of iodixanol. No significant increase in serum creatinine values were observed in the monoclonal gammopathies group and in 19/20 patients in the control group. Only 1 patient in the control group developed a transient contrast agent-induced nephropathy. We found no statistically significant difference between the two groups regarding the percentage variation from baseline values of serum creatinine, creatinine clearance, NGAL 2 h after, and eGFR. Whereas NGAL at 24 h showed a statistically significant increase in patients with Monoclonal gammopathies. The use of iodixanol appears to be safe in patients with monoclonal gammopathies and an eGFR ≥ 60 ml/min/1.73 mq. (orig.)

HER2 induced EMT and tumorigenicity in breast epithelial progenitor cells is inhibited by coexpression of EGFR.

Science.gov (United States)

Ingthorsson, S; Andersen, K; Hilmarsdottir, B; Maelandsmo, G M; Magnusson, M K; Gudjonsson, T

2016-08-11

The members of the epidermal growth factor receptor (EGFR) kinase family are important players in breast morphogenesis and cancer. EGFR2/HER2 and EGFR expression have a prognostic value in certain subtypes of breast cancer such as HER2-amplified, basal-like and luminal type B. Many clinically approved small molecular inhibitors and monoclonal antibodies have been designed to target HER2, EGFR or both. There is, however, still limited knowledge on how the two receptors are expressed in normal breast epithelium, what effects they have on cellular differentiation and how they participate in neoplastic transformation. D492 is a breast epithelial cell line with stem cell properties that can undergo epithelial to mesenchyme transition (EMT), generate luminal- and myoepithelial cells and form complex branching structures in three-dimensional (3D) culture. Here, we show that overexpression of HER2 in D492 (D492(HER2)) resulted in EMT, loss of contact growth inhibition and increased oncogenic potential in vivo. HER2 overexpression, furthermore, inhibited endogenous EGFR expression. Re-introducing EGFR in D492(HER2) (D492(HER2/EGFR)) partially reversed the mesenchymal state of the cells, as an epithelial phenotype reappeared both in 3D cultures and in vivo. The D492(HER2/EGFR) xenografts grow slower than the D492(HER2) tumors, while overexpression of EGFR alone (D492(EGFR)) was not oncogenic in vivo. Consistent with the EGFR-mediated epithelial phenotype, overexpression of EGFR drove the cells toward a myoepithelial phenotype in 3D culture. The effect of two clinically approved anti-HER2 and EGFR therapies, trastuzumab and cetuximab, was tested alone and in combination on D492(HER2) xenografts. While trastuzumab had a growth inhibitory effect compared with untreated control, the effect of cetuximab was limited. When administered in combination, the growth inhibitory effect of trastuzumab was less pronounced. Collectively, our data indicate that in HER2-overexpressing D492

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

Directory of Open Access Journals (Sweden)

Titis Sekar Humani

2017-12-01

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

Potency of a human monoclonal antibody to diphtheria toxin relative to equine diphtheria anti-toxin in a guinea pig intoxication model.

Science.gov (United States)

Smith, Heidi L; Cheslock, Peter; Leney, Mark; Barton, Bruce; Molrine, Deborah C

2016-08-17

Prompt administration of anti-toxin reduces mortality following Corynebacterium diphtheriae infection. Current treatment relies upon equine diphtheria anti-toxin (DAT), with a 10% risk of serum sickness and rarely anaphylaxis. The global DAT supply is extremely limited; most manufacturers have ceased production. S315 is a neutralizing human IgG1 monoclonal antibody to diphtheria toxin that may provide a safe and effective alternative to equine DAT and address critical supply issues. To guide dose selection for IND-enabling pharmacology and toxicology studies, we dose-ranged S315 and DAT in a guinea pig model of diphtheria intoxication based on the NIH Minimum Requirements potency assay. Animals received a single injection of antibody premixed with toxin, were monitored for 30 days, and assigned a numeric score for clinical signs of disease. Animals receiving ≥ 27.5 µg of S315 or ≥ 1.75 IU of DAT survived whereas animals receiving ≤ 22.5 µg of S315 or ≤ 1.25 IU of DAT died, yielding a potency estimate of 17 µg S315/IU DAT (95% CI 16-21) for an endpoint of survival. Because some surviving animals exhibited transient limb weakness, likely a systemic sign of toxicity, DAT and S315 doses required to prevent hind limb paralysis were also determined, yielding a relative potency of 48 µg/IU (95% CI 38-59) for this alternate endpoint. To support advancement of S315 into clinical trials, potency estimates will be used to evaluate the efficacy of S315 versus DAT in an animal model with antibody administration after toxin exposure, more closely modeling anti-toxin therapy in humans.

Anti-CEA monoclonal antibody in the diagnosis of colorectal, lung and ovarian carcinoma

International Nuclear Information System (INIS)

Jiang, N.; Lu, B.; Lu, X.; Sha, X.; Yue, D.

2000-01-01

This study evaluated the diagnostic value of radioimmnoimaging (RII) with 99 Tc labeled monoclonal antibody C50, raised originally against carcinoembryonic antigen (anti-CEA) in various tumors. 152 pathologically confirmed patients with a tumor were imaged prior to surgery with an anti-CEA monoclonal antibody labeled with 99 Tc. There were 115 patients with ovarian carcinoma, 26 patients with colorectal carcinoma and 11 patients with lung carcinoma. Images were acquired at 3-6 h post injection and were analyzed by the double blind method. Images of patients with ovarian cancer were compared with B-ultrasound images. Immunohistochemical staining was performed on all cases of colorectal cancer. All RII images demonstrated excellent contrast, clear lesions, and no serious toxic or other side reactions occurred. Transient chills and fever were observed in 3 cases. This study showed a sensitivity=88.2%, specificity=83.2%, and an accuracy=4.0%. The smallest lesion size detected was 2 x 2 cm. The total combined lesion detection rate for primary, metastatic, and recurrence lesions was 84.4%. We conclude that 99 Tc labeled anti-CEA MoAb C50 can be used in the diagnosis of colorectal carcinoma, ovarian carcinoma, and lung carcinoma

EGFR and KRAS mutation coexistence in lung adenocarcinomas

Directory of Open Access Journals (Sweden)

Vitor Manuel Leitão de Sousa

2015-04-01

Full Text Available Lung cancer is one of the most common causes of cancer deaths. The development of EGFR targeted therapies, including monoclonal antibodies and tyrosine kinase inhibitors have generated an interest in the molecular characterization of these tumours. KRAS mutations are associated with resistance to EGFR TKIs. EGFR and KRAS mutations have been considered as mutually exclusive. This paper presents three bronchial-pulmonary carcinomas, two adenocarcinomas and one pleomorphic sarcomatoid carcinoma, harboring EGFR and KRAS mutations. Case 1 corresponded to an adenocarcinoma with EGFR exon 21 mutation (L858R and KRAS codon 12 point mutation (G12V; case 2, a  mucinous adenocarcinoma expressed coexistence of EGFR exon 21 mutation (L858R and KRAS codon 12 point mutation (G12V; and case 3 a sarcomatoid carcinoma with EGFR exon 19 deletion – del 9bp and KRAS codon 12 point mutation (G12C - cysteine. Based on our experience and on the literature, we conclude that EGFR and KRAS mutations can indeed coexist in the same bronchial-pulmonary carcinoma, either in the same histological type or in different patterns. The biological implications of this coexistence are still poorly understood mainly because these cases are not frequent or currently searched. It is therefore necessary to study larger series of cases with the two mutations to better understand the biological, clinical and therapeutic implications.

Antitumor Efficacy of Dual Blockade of EGFR Signaling by Osimertinib in Combination With Selumetinib or Cetuximab in Activated EGFR Human NCLC Tumor Models.

Science.gov (United States)

Della Corte, Carminia Maria; Ciaramella, Vincenza; Cardone, Claudia; La Monica, Silvia; Alfieri, Roberta; Petronini, Pier Giorgio; Malapelle, Umberto; Vigliar, Elena; Pepe, Francesco; Troncone, Giancarlo; Castellone, Maria Domenica; Troiani, Teresa; Martinelli, Erika; Ciardiello, Fortunato; Morgillo, Floriana

2018-03-08

Osimertinib showed great clinical efficacy for activated-EGFR NCLC patient treatment. The aim of this work was to test the efficacy of a complete EGFR-inhibition by osimertinib plus the monoclonal antibody cetuximab or the MEK1/2-inhibitor selumetinib in EGFR-mutated NCLC in vivo models. We evaluated combinations of osimertinib plus selumetinib/cetuximab in HCC827 (E746-A759del/T790M-), H1975 (L858R/T790M+), and PC9-T790M (E746-A759del /T790M+) xenografts in second-line therapy after the development of resistance to osimertinib, and in first-line therapy, and we explored mechanisms of resistance to these treatments. The addition of selumetinib or cetuximab to osimertinib in second-line therapy reverted the sensibility to osimertinib in the majority of mice, with a response rate (RR) of 50% to 80%, and a median progression-free survival (mPFS) of first- plus second-line of therapy of 28 weeks. The early use of combinations in first-line therapy increased the RR to 90%, with an mPFS not reached in all combination arms in the three xenografts models, with a statistically significant superiority (p < 0.005) as compared to osimertinib, achieving in first-line therapy an mPFS time of 17 to 18 weeks. Moreover, in ex vivo primary cell cultures obtained from osimertinib plus selumetinib-resistant tumors, we found Hedgehog pathway activation and we showed that therapy with an SMO inhibitor plus osimertinib and selumetinib inhibited proliferation and migratory and invasive properties of resistant cells. We showed that a dual vertical EGFR blockade with osimertinib plus selumetinib/cetuximab is a novel effective therapeutic option in EGFR-mutated NCLC and that hedgehog pathway activation and its interplay with MAPK is involved in resistance to these combination treatments. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody.

Science.gov (United States)

Fox, D A; Hussey, R E; Fitzgerald, K A; Acuto, O; Poole, C; Palley, L; Daley, J F; Schlossman, S F; Reinherz, E L

1984-09-01

By using a murine monoclonal antibody produced against an IL 2-dependent human T cell line, we defined a T lineage-specific molecule, termed Ta1, that is expressed strongly on activated T lymphocytes of both the T4 and T8 subsets, as well as on T cell lines and clones, but only weakly on a fraction of resting T cells. SDS-PAGE analysis of immunoprecipitates from 125I-labeled, activated T cells demonstrates a single major band of apparent m.w. 105 KD under both reducing and nonreducing conditions. Unlike anti-IL 2 receptor antibodies, anti-Ta1 does not inhibit T cell proliferative responses to mitogen, antigen, or IL 2-containing medium. Moreover, anti-Ta1 has no effect on T cell-mediated cytotoxicity. Ta1 appears to be a novel human T cell-specific activation antigen that may serve as a useful marker of T cell activation in human disease.

Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines

Energy Technology Data Exchange (ETDEWEB)

Carrasco-Garcia, Estefania; Saceda, Miguel [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad de Investigacion, Hospital General Universitario de Elche, 03203 Elche (Alicante) (Spain); Grasso, Silvina; Rocamora-Reverte, Lourdes; Conde, Mariano; Gomez-Martinez, Angeles [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Garcia-Morales, Pilar [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad de Investigacion, Hospital General Universitario de Elche, 03203 Elche (Alicante) (Spain); Ferragut, Jose A. [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Martinez-Lacaci, Isabel, E-mail: imlacaci@umh.es [Instituto de Biologia Molecular y Celular, Universidad Miguel Hernandez, 03202 Elche (Alicante) (Spain); Unidad AECC de Investigacion Traslacional en Cancer, Hospital Universitario Virgen de la Arrixaca, 30120 Murcia (Spain)

2011-06-10

Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G{sub 1} arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G{sub 1} arrest. This G{sub 1} arrest was associated with up-regulation of p27{sup kip1}, whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G{sub 1} arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 {Delta}EGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.

Effect of cetuximab and fractionated irradiation on tumour micro-environment.

NARCIS (Netherlands)

Santiago, A.; Eicheler, W.; Bussink, J.; Rijken, P.F.J.W.; Yaromina, A.; Beuthien-Baumann, B.; Kogel, A.J. van der; Baumann, M.; Krause, M.

2010-01-01

BACKGROUND AND PURPOSE: Previous experiments have shown that application of the anti-EGFR monoclonal antibody C225 (cetuximab) improves local tumour control after irradiation in FaDu human squamous cell carcinoma (hSCC) due to the combined effect of decreased repopulation and improved reoxygenation.

HER2 induced EMT and tumorigenicity in breast epithelial progenitor cells is inhibited by coexpression of EGFR

OpenAIRE

Ingthorsson, Saevar; Andersen, K; Hilmarsdóttir, Bylgja; Mælandsmo, Gunhild M; Magnusson, Magnus Karl; Gudjonsson, Thorarinn

2015-01-01

The members of the epidermal growth factor receptor (EGFR) kinase family are important players in breast morphogenesis and cancer. EGFR2/HER2 and EGFR expression have a prognostic value in certain subtypes of breast cancer such as HER2-amplified, basal-like and luminal type B. Many clinically approved small molecular inhibitors and monoclonal antibodies have been designed to target HER2, EGFR or both. There is, however, still limited knowledge on how the two receptors are expressed in normal ...

Enavatuzumab, a Humanized Anti-TWEAK Receptor Monoclonal Antibody, Exerts Antitumor Activity through Attracting and Activating Innate Immune Effector Cells

Directory of Open Access Journals (Sweden)

Shiming Ye

2017-01-01

Full Text Available Enavatuzumab is a humanized IgG1 anti-TWEAK receptor monoclonal antibody that was evaluated in a phase I clinical study for the treatment of solid malignancies. The current study was to determine whether and how myeloid effector cells were involved in postulated mechanisms for its potent antitumor activity in xenograft models. The initial evidence for a role of effector cells was obtained in a subset of tumor xenograft mouse models whose response to enavatuzumab relied on the binding of Fc of the antibody to Fcγ receptor. The involvement of effector cells was further confirmed by immunohistochemistry, which revealed strong infiltration of CD45+ effector cells into tumor xenografts in responding models, but minimal infiltration in nonresponders. Consistent with the xenograft studies, human effector cells preferentially migrated toward in vivo-responsive tumor cells treated by enavatuzumab in vitro, with the majority of migratory cells being monocytes. Conditioned media from enavatuzumab-treated tumor cells contained elevated levels of chemokines, which might be responsible for enavatuzumab-triggered effector cell migration. These preclinical studies demonstrate that enavatuzumab can exert its potent antitumor activity by actively recruiting and activating myeloid effectors to kill tumor cells. Enavatuzumab-induced chemokines warrant further evaluation in clinical studies as potential biomarkers for such activity.

Improved radioimaging and tumor localization with monoclonal F(ab')2

International Nuclear Information System (INIS)

Wahl, R.L.; Parker, C.W.; Philpott, G.W.

1983-01-01

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

A Neutralizing Anti-gH/gL Monoclonal Antibody Is Protective in the Guinea Pig Model of Congenital CMV Infection

Science.gov (United States)

Auerbach, Marcy R.; Yan, Donghong; Vij, Rajesh; Hongo, Jo-Anne; Nakamura, Gerald; Vernes, Jean-Michel; Meng, Y. Gloria; Lein, Samantha; Chan, Pamela; Ross, Jed; Carano, Richard; Deng, Rong; Lewin-Koh, Nicholas; Xu, Min; Feierbach, Becket

2014-01-01

Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2–1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective [1]–[3]. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans. PMID:24722349

Radiobromination of humanized anti-HER2 monoclonal antibody trastuzumab using N-succinimidyl 5-bromo-3-pyridinecarboxylate, a potential label for immunoPET

Energy Technology Data Exchange (ETDEWEB)

Mume, Eskender [Organic Chemistry, Department of Chemistry, Uppsala University, S-751 24 Uppsala (Sweden); Orlova, Anna [Affibody AB, S-161 02 Bromma (Sweden); Malmstroem, Per-Uno [Division of Urology, Department of Surgical Sciences, Uppsala University, S-751 85 Uppsala (Sweden); Lundqvist, Hans [Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, S-751 85 Uppsala (Sweden); Sjoeberg, Stefan [Organic Chemistry, Department of Chemistry, Uppsala University, S-751 24 Uppsala (Sweden); Tolmachev, Vladimir [Unit of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, S-751 85 Uppsala (Sweden)]. E-mail: vladimir.tolmachev@bms.uu.se

2005-08-01

Combining the specificity of radioimmunoscintigraphy and the high sensitivity of PET in an in vivo detection technique could improve the quality of nuclear diagnostics. Positron-emitting nuclide {sup 76}Br (T {sub 1/2}=16.2 h) might be a possible candidate for labeling monoclonal antibodies (mAbs) and their fragments, provided that the appropriate labeling chemistry has been established. For internalizing antibodies, such as the humanized anti-HER2 monoclonal antibody, trastuzumab, radiobromine label should be residualizing, i.e., ensuring that radiocatabolites are trapped intracellularly after the proteolytic degradation of antibody. This study evaluated the chemistry of indirect radiobromination of trastuzumab using N-succinimidyl 5-(tributylstannyl)-3-pyridinecarboxylate. Literature data indicated that the use of this method provided residualizing properties for iodine and astatine labels on some antibodies. An optimized 'one-pot' procedure produced an overall labeling efficiency of 45.5{+-}1.2% over 15 min. The bromine label was stable under physiological and denaturing conditions. The labeled trastuzumab retained its capacity to bind specifically to HER2-expressing SKOV-3 ovarian carcinoma cells in vitro (immunoreactivity more than 75%). However, in vitro cell test did not demonstrate that the radiobromination of trastuzumab using N-succinimidyl 5-bromo-3-pyridinecarboxylate improves cellular retention of radioactivity in comparison with the use of N-succinimidyl 4-bromobenzoate.

Pharmacokinetics and immunogenicity investigation of a human anti-interleukin-17 monoclonal antibody in non-naïve cynomolgus monkeys.

Science.gov (United States)

Han, Chao; Gunn, George R; Marini, Joseph C; Shankar, Gopi; Han Hsu, Helen; Davis, Hugh M

2015-05-01

The pharmacokinetics (PK) of biologic therapeutics, especially monoclonal antibodies (mAbs), in monkeys generally presents the most relevant predictive PK information for humans. However, human mAbs, xenogeneic proteins to monkeys, are likely to be immunogenic. Monkeys previously treated with a human mAb (non-naïve) may have developed antidrug antibodies (ADAs) that cross-react with another test mAb in subsequent studies. Unlike PK studies for small-molecule therapeutics, in which animals may be reused, naïve monkeys have been used almost exclusively for preclinical PK studies of biologic therapeutics to avoid potential pre-existing immunologic cross-reactivity issues. The propensity and extent of pre-existing ADAs have not been systematically investigated to date. In this study, the PK and immunogenicity of mAb A, a human anti-human interkeukin-17 mAb, were investigated in a colony of 31 cynomolgus monkeys previously exposed to other human mAbs against different targets. We screened the monkeys for pre-existing antibodies to mAb A prior to the PK study and showed that 44% of the monkeys had pre-existing cross-reactive antibodies to mAb A, which could affect the PK characterization of the antibody. In the subcolony of monkeys without measurable pre-existing ADAs, PK and immunogenicity of mAb A were successfully characterized. The impact of ADAs on mAb A PK was also demonstrated in the monkeys with pre-existing ADAs. Here we report the results and propose a pragmatic approach for the use of non-naïve monkeys when conducting PK studies of biologic therapeutics. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Human pharmacokinetics, biodistribution and dosimetry of 99m Tc labelled monoclonal antibody ior egf/r3 in patients with tumors of epithelial origin: preliminary results

International Nuclear Information System (INIS)

Iznaga-Escobar, Normando E.; Morales, Alejo; Ramos, Mayra; Perez, Niuvis; Torres, Leonel A.; Alavarez, Ivette; Rodriguez, Nelson; Fraxedas, Roberto; Rodriguez, Oscar; Stabin, Michael G.

1997-01-01

Human pharmacokinetics, biodistribution and internal radiation dosimetry to normal organs and total body of 99m Tc-labeled monoclonal antibody ior egf/r 3 was investigated following intravenous injection in 5 patients. Following administration, blood and urine samples were collected from 4 patients up to 24 hr after injection Pharmacokinetics obtained from whole blood radioactivity showed blood disappearance described most properly by a biexponential model with a mean distribution half-life value of 0.14±0.02 hr and elimination half-life value of 31.0±13.6 hr. Whole body anterior and posterior scans were obtained at 10 min, 1,3,5 and 24 hr after injection. ROIs were drawn over the heart, liver spleen and bladder to measure the activity in the source organs. Time-activity curves for each source organ were fitted to mono- or biexponential functions by non-linear least squares regression using the flexible polyhedral method and integrated to determine organ residence times. The mean absorbed dose to the whole body and various normal organs were then estimated from residence times and from blood and urine samples using the MIRD method. The effective dose equivalent (EDE) and effective dose (ED) were calculated. Estimates of radiation absorbed dose to normal organs in rads/mCi administered (mean ± SD, n=4) were: whole body, 0.0185± 0.0023, gallbladder wall, 0.0755± 0.00761, spleen, 0.0637± 0.0167 and liver, 0.276± 0.029. The effective dose equivalent and effective dose estimates for adults were 0.039± 0.008 and 0.028± 0.004 rem/m Ci administered. (author). 15 refs., 3 figs., 3 tabs

Serological blind spots for variants of human IgG3 and IgG4 by a commonly used anti-immunoglobulin reagent.

Science.gov (United States)

Howie, Heather L; Delaney, Meghan; Wang, Xiaohong; Er, Lay See; Vidarsson, Gestur; Stegmann, Tamara C; Kapp, Linda; Lebedev, Jenna N; Wu, Yanyun; AuBuchon, James P; Zimring, James C

2016-12-01

Human immunoglobulin G (IgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4), and it is also now appreciated that there are genetic variations within IgG subtypes (called isoallotypes). Twenty-nine different isoallotypes have been described, with 7, 4, 15, and 3 isoallotypes described for IgG1, IgG2, IgG3, and IgG4, respectively. The reactivity of anti-IgG with different isoallotypes has not been characterized. A novel monoclonal anti-K antibody (PugetSound Monoclonal Antibody 1 [PUMA1]) was isolated and sequenced, and a panel of PUMA1 variants was expressed, consisting of the 29 known IgG isoallotypes. The resulting panel of antibodies was preincubated with K-positive red blood cells (RBCs) and then subjected to testing with currently approved anti-IgG by flow cytometry, solid phase systems, gel cards, and tube testing. A US Food and Drug Administration (FDA)-approved monoclonal anti-IgG (gamma-clone) failed to recognize 2 of 15 IgG3 isoallotypes (IgG3-03 and IgG3-13) and 3 of 3 IgG4 isoallotypes (IgG4-01, IgG4-02, and IgG4-03). In contrast, an FDA-approved rabbit polyclonal anti-IgG recognized each of the known human IgG isoallotypes. These findings demonstrate "blind spots" in isoalloantibody detection by a monoclonal anti-IgG. If a patient has anti-RBC antibodies predominantly of an IgG3 subtype (the IgG3-03 and/or IgG3-13 variety), then it is possible that a clinically significant alloantibody would be missed. IgG-03 and IgG-13 have an estimated frequency of 1% to 3% in Caucasian populations and 20% to 30% in certain African populations. Nonreactivity with IgG4 is a known characteristic of this monoclonal anti-IgG, but IgG4 isoallotypes have not been previously reported. © 2016 AABB.

Safety, Pharmacokinetics, Immunogenicity, and Biodistribution of (186)Re-Labeled Humanized Monoclonal Antibody BIWA 4 (Bivatuzumab( in Patients with Early-Stage Breast Cancer.

NARCIS (Netherlands)

Koppe, M.; Schaijk, F. van; Roos, J.C.; Leeuwen, P.; Heider, K.H.; Kuthan, H.; Bleichrodt, R.P.

2004-01-01

The aim of this prospective study was to evaluate the safety, pharmacokinetics, immunogenicity, and biodistribution of (186)Re-labeled humanized anti-CD44v6 monoclonal antibody (MAb( BIWA 4 (Bivatuzumab( in 9 patients with early-stage breast cancer. Radioimmunoscintigraphy (RIS( was performed within

Rituximab chimeric anti-CD20 monoclonal antibody treatment for adult refractory idiopathic thrombocytopenic purpura

DEFF Research Database (Denmark)

Braendstrup, Peter; Bjerrum, Ole W; Nielsen, Ove J

2005-01-01

. Recent studies have shown that rituximab, a chimeric anti-CD20 monoclonal antibody, is useful in the treatment of these patients, with overall response rates of about 50%. Most published reports have included a small number patients including case reports. The present study reports the results...

Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.

Science.gov (United States)

Lee, Gregory; Ge, Bixia

2010-07-01

As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations.

Combination of two anti-CD5 monoclonal antibodies synergistically induces complement-dependent cytotoxicity of chronic lymphocytic leukaemia cells.

Science.gov (United States)

Klitgaard, Josephine L; Koefoed, Klaus; Geisler, Christian; Gadeberg, Ole V; Frank, David A; Petersen, Jørgen; Jurlander, Jesper; Pedersen, Mikkel W

2013-10-01

The treatment of chronic lymphocytic leukaemia (CLL) has been improved by introduction of monoclonal antibodies (mAbs) that exert their effect through secondary effector mechanisms. CLL cells are characterized by expression of CD5 and CD23 along with CD19 and CD20, hence anti-CD5 Abs that engage secondary effector functions represent an attractive opportunity for CLL treatment. Here, a repertoire of mAbs against human CD5 was generated and tested for ability to induce complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) both as single mAbs and combinations of two mAbs against non-overlapping epitopes on human CD5. The results demonstrated that combinations of two mAbs significantly increased the level of CDC compared to the single mAbs, while no enhancement of ADCC was seen with anti-CD5 mAb combinations. High levels of CDC and ADCC correlated with low levels of Ab-induced CD5 internalization and degradation. Importantly, an anti-CD5 mAb combination enhanced CDC of CLL cells when combined with the anti-CD20 mAbs rituximab and ofatumumab as well as with the anti-CD52 mAb alemtuzumab. These results suggest that an anti-CD5 mAb combination inducing CDC and ADCC may be effective alone, in combination with mAbs against other targets or combined with chemotherapy for CLL and other CD5-expressing haematological or lymphoid malignancies. © 2013 John Wiley & Sons Ltd.

Nebulized Anti-IL-13 Monoclonal Antibody Fab' Fragment Reduces Allergen-Induced Asthma

OpenAIRE

Hacha, Jonathan; Tomlinson, K; Maertens, Ludovic; Paulissen, Geneviève; Rocks, Natacha; Foidart, Jean-Michel; Noël, Agnès; Palframan, R; Guéders, Maud; Cataldo, Didier

2012-01-01

Rationale: Interleukin-13 (IL-13) is a prototypic Th2 cytokine and a central mediator of the complex cascade of events leading to asthmatic phenotype. Indeed, IL-13 plays key roles in IgE synthesis, bronchial hyperresponsiveness, mucus hypersecretion, subepithelial fibrosis and eosinophil infiltration. Objectives: We assessed the potential efficacy of inhaled anti-IL-13 monoclonal antibody Fab' fragment on allergen-induced airway inflammation, hyperresponsiveness and remodeling in an experime...

A mathematical model of a recombinant humanized anti-cocaine monoclonal antibody's effects on cocaine pharmacokinetics in mice.

Science.gov (United States)

Wetzel, Hanna N; Zhang, Tongli; Norman, Andrew B

2017-09-01

A recombinant humanized anti-cocaine monoclonal antibody (mAb), h2E2, is at an advanced stage of pre-clinical development as an immunotherapy for cocaine abuse. It is hypothesized that h2E2 binds to and sequesters cocaine in the blood. A three-compartment model of the effects of h2E2 on cocaine's distribution was constructed. The model assumes that h2E2 binds to cocaine and that the h2E2-cocaine complex does not enter the brain but distributes between the central and peripheral compartments. Free cocaine is eliminated from both the central and peripheral compartments, and h2E2 and the h2E2-cocaine complex are eliminated from the central compartment only. This model was tested against a new dataset measuring cocaine concentrations in the brain and plasma over 1h in the presence and absence of h2E2. The mAb significantly increased plasma cocaine concentrations with a concomitant significant decrease in brain concentration. Plasma concentrations declined over the 1-hour sampling period in both groups. With a set of parameters within reasonable physiological ranges, the three-compartment model was able to qualitatively and quantitatively simulate the increased plasma concentration in the presence of the antibody and the decreased peak brain concentration in the presence of antibody. Importantly, the model explained the decline in plasma concentrations over time as distribution of the cocaine-h2E2 complex into a peripheral compartment. This model will facilitate the targeting of ideal mAb PK/PD properties thus accelerating the identification of lead candidate anti-drug mAbs. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of a Novel Humanized Anti-CD20 Antibody with Potent Anti-Tumor Activity against Non-Hodgkin's Lymphoma

Directory of Open Access Journals (Sweden)

Haifeng Zhang

2013-09-01

Full Text Available Background: Rituximab, a mouse Fab and human Fc chimeric antibody, has been widely used to treat Non-Hodgkin's lymphoma (NHL. However, only 48% of patients respond to the treatment and complete response rate is below 10%. Also, immunogenicity was reported in 17-20% patients receiving the treatment, making it unsuitable for long term diseases such as autoimmune disorders. It has been a hot research field to “humanize” rituximab toward improved efficacy and reduced immunogenicity. Methods: In this study, an advanced antibody humanization technology was applied to the sequence of the anti-CD20 antibody 2B8, its sequence of which was based on the original murine monoclonal antibody of rituximab in Roche. The complementarity-determining regions (CDRs of the humanized antibodies were further optimized through computer-aided molecular dock. Results: Five novel humanized anti-CD20 antibodies 1-5(1635, 1534, 3637, 1634 and 1536 were generated and their immunogenicity was significantly decreased when compared to rituximab. The novel humanized anti-CD20 antibodies 1-5 retained the binding activity of their murine counterpart, as demonstrated by the fluorescence-activated cell-sorting analysis (FACS. When compared to rituximab, the humanized antibodies still have the similar properties on both complement-dependent cytotoxicity (CDC and antibody-dependent cell-mediated cytotoxicity (ADCC. Furthermore, its anti-tumor efficacy in xenograft model is comparable to that of rituximab. Conclusion: The humanized anti-CD20 antibodies 1-5 have lower immunogenicity than rituximab. And at the same time, they still retain the anti-tumor effect both in vitro and vivo.

The Use of EGFR Tyrosine Kinase Inhibitors in EGFR Wild-Type Non-Small-Cell Lung Cancer.

Science.gov (United States)

Stinchcombe, Thomas E

2016-04-01

The objective response rate and progression-free survival observed with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) in patients with metastatic epidermal growth factor receptor (EGFR) wild-type non-small cell lung cancer (NSCLC) are modest. The adverse events associated with EGFR TKIs are manageable but they must be considered in the context of the limited efficacy. The development of anti-PD-1 immunotherapy as second-line therapy has reduced the role of EGFR TKIs in EGFR wild-type NSCLC. Recently, there has been increased recognition of the benefit of the earlier integration of palliative care and symptom management, and this is reasonable alternative to treatment with an EGFR TKI for many patients. My practice pattern for patients with EGFR wild-type NSCLC is platinum-based chemotherapy as first-line therapy, immunotherapy as second-line therapy, and single-agent chemotherapy as third-line therapy for patients with preserved performance status who want to pursue further therapy. Only a small proportion of patients are eligible for fourth-line therapy, and I prefer to enroll them in clinical trials rather than use EGFR TKIs. I suspect that the use of EGFR TKIs in clinical use and as a comparator arm for clinical trials will continue to decline over the next several years.

Inhibition of radiation-induced EGFR nuclear import by C225 (Cetuximab) suppresses DNA-PK activity

International Nuclear Information System (INIS)

Dittmann, Klaus; Mayer, Claus; Rodemann, Hans-Peter

2005-01-01

Background and purpose: Inhibition of EGFR-function can induce radiosensitization in tumor cells. Purpose of our investigation was to identify the possible molecular mechanism of radiosensitization following treatment with anti-EGFR-antibody C225 (Cetuximab). Materials and methods: The effect of C225 on radiation response was determined in human cell lines of bronchial carcinoma (A549) and breast adenoma cells (MDA MB 231). The molecular effects of C225 on EGFR-function after irradiation were analyzed applying western blotting, immune-precipitation and kinase assays. Effects on DNA-repair were detected by quantification of γ-H2AX positive foci 24 h after irradiation. Results: The EGFR specific antibody C225 induced radiosensitization in A549 and also in MDA MB 231 cells. Radiosensitization in A549 was associated with blockage of radiation-induced EGFR transport into the nucleus, and immobilized the complex of EGFR with DNA-dependent protein kinase (DNA-PK) in the cytoplasm. As a consequence radiation-induced DNA-PK activation was abolished, a process that is essential for DNA-repair after radiation exposure. Likewise C225 treatment increased the residual amount of γ-H2AX-positive foci 24 h after irradiation in A549 and in MDA MB 231 cells. Conclusions: Our results suggest that irradiation induced DNA-PK activation-essential for DNA repair-may be hampered specifically by use of the anti-EGFR-antibody C225. This process is associated with radiosensitization

A novel anti-GPC3 monoclonal antibody (YP7) | Center for Cancer Research

Science.gov (United States)

Glypican-3 (GPC3) is an emerging therapeutic target in hepatoma. A novel anti-GPC3 monoclonal antibody (YP7) has been generated through a combination of peptide immunization and high-throughput flow cytometry screening. YP7 binds cell-surface-associated GPC3 with high affinity and exhibits significant hepatoma xenograft growth inhibition in nude mice. The new antibody may have

Anti-Mycobacterium leprae monoclonal antibodies cross-react with human skin: an alternative explanation for the immune responses in leprosy

NARCIS (Netherlands)

Naafs, B.; Kolk, A. H.; Chin A Lien, R. A.; Faber, W. R.; van Dijk, G.; Kuijper, S.; Stolz, E.; van Joost, T.

1990-01-01

A panel of 17 mouse monoclonal antibodies (MoAb) raised against Mycobacterium leprae (M. leprae) antigens was used to detect antigenic determinants in normal human skin. An indirect immunoperoxidase technique was used. Eight of the MoAb detected epidermal antigens similar to patterns well known for

Immunohistochemical Examination of Novel Rat Monoclonal Antibodies against Mouse and Human Podoplanin

International Nuclear Information System (INIS)

Kaji, Chiaki; Tsujimoto, Yuta; Kato Kaneko, Mika; Kato, Yukinari; Sawa, Yoshihiko

2012-01-01

This study aims to develop new monoclonal antibodies (mAbs) against mouse and human podoplanin. Rats were immunized with synthetic peptides, corresponding to amino acids 38–51 of mouse podoplanin or human podoplanin which is 100% homologous to the same site of monkey podoplanin; anti-mouse podoplanin mAb PMab-1 (IgG 2a ) and anti-human mAb NZ-1.2 (IgG 2a ) were established. In immunocytochemistry, the mouse melanoma B16-F10 and mouse podoplanin (mPDPN)-expressed CHO transfectant were stained by PMab-1; human lymphatic endothelial cells (LEC) and human podoplanin (hPDPN)-expressed squamous cell carcinoma HSC3 transfectant, were stained by NZ-1.2. Western-blot analysis detected an about 40-kDa protein in CHO-mPDPN and B16-F10 by PMab-1, and in HSC3-hPDPN and LEC by NZ-1.2. In frozen sections, PMab-1 reacted with mouse kidney, pulmonary alveoli, pulmonary pleura, and salivary gland myoepithelial cells while NZ-1.2 reacted to the human salivary gland myoepithelial cells. The immunostaining of paraffin-embedded sections also showed the reaction of PMab-1 or NZ-1.2 to the mouse or monkey kidney glomerulus, pulmonary alveoli, and lung lymphatic vessels. These results indicate that the two novel rat mAbs to the mouse and human/monkey podoplanin are useful for Western-blot and immunostaining of somatic tissues on paraffin-embedded sections as well as frozen sections

Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

Directory of Open Access Journals (Sweden)

Felix Hart

2017-05-01

Full Text Available IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich and hematological (CD20 cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

Radiolabelled monoclonal antibodies against alpha-fetoprotein for in vivo localization of human hepatocellular carcinoma by immunotomoscintigraphy

International Nuclear Information System (INIS)

Bergmann, J.F.; Lumbroso, J.D.; Manil, L.; Saccavini, J.C.; Rougier, P.; Assicot, M.; Mathieu, A.; Bellet, D.; Bohuon, C.

1987-01-01

Two high affinity monoclonal antibodies, designated AF01 and AF04, directed against distinct epitopes of human alpha-fetoprotein (AFP) and the Fab fragments of one of them, were labelled with 131 I and injected into 18 patients with AFP producing hepatocellular carcinoma (HCC) in order to carry out imaging studies by tomoscintigraphy. Twelve patients were injected with whole antibody, only three of seven patients injected with AF01 and two of five patients injected with AF04 had a positive scan. In contrast, five out of six patients injected with labelled Fab fragments of AF04 had positive imaging. These results confirm that tumour imaging of HCC using 131 I labelled monoclonal antibody against AFP is feasible. Moreover, utilization of tomoscintigraphy in place of linear scintigraphy and Fab fragments instead of whole immunoglobulin may improve the sensitivity of radioimmunolocalization. This technique provides useful information on the in vivo distribution of monoclonal antibodies directed against AFP and on the practicability of the eventual therapeutic use of anti-AFP antibodies in HCC. (orig.)

Development and characterization of anti-glycopeptide monoclonal antibodies against human podoplanin, using glycan-deficient cell lines generated by CRISPR/Cas9 and TALEN.

Science.gov (United States)

Kaneko, Mika K; Nakamura, Takuro; Honma, Ryusuke; Ogasawara, Satoshi; Fujii, Yuki; Abe, Shinji; Takagi, Michiaki; Harada, Hiroyuki; Suzuki, Hiroyoshi; Nishioka, Yasuhiko; Kato, Yukinari

2017-02-01

Human podoplanin (hPDPN), which binds to C-type lectin-like receptor-2 (CLEC-2), is involved in platelet aggregation and cancer metastasis. The expression of hPDPN in cancer cells or cancer-associated fibroblasts indicates poor prognosis. Human lymphatic endothelial cells, lung-type I alveolar cells, and renal glomerular epithelial cells express hPDPN. Although numerous monoclonal antibodies (mAbs) against hPDPN are available, they recognize peptide epitopes of hPDPN. Here, we generated a novel anti-hPDPN mAb, LpMab-21. To characterize the hPDPN epitope recognized by the LpMab-21, we established glycan-deficient CHO-S and HEK-293T cell lines, using the CRISPR/Cas9 or TALEN. Flow cytometric analysis revealed that the minimum hPDPN epitope, in which sialic acid is linked to Thr76, recognized by LpMab-21 is Thr76-Arg79. LpMab-21 detected hPDPN expression in glioblastoma, oral squamous carcinoma, and seminoma cells as well as in normal lymphatic endothelial cells. However, LpMab-21 did not react with renal glomerular epithelial cells or lung type I alveolar cells, indicating that sialylation of hPDPN Thr76 is cell-type-specific. LpMab-21 combined with other anti-hPDPN antibodies that recognize different epitopes may therefore be useful for determining the physiological function of sialylated hPDPN. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Characterization of the Anti-Bovine Podoplanin Monoclonal Antibody PMab-44.

Science.gov (United States)

Yamada, Shinji; Honma, Ryusuke; Kaneko, Mika K; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Takagi, Michiaki; Konnai, Satoru; Kato, Yukinari

2017-06-01

A type I transmembrane sialoglycoprotein podoplanin (PDPN) is expressed in several normal cells, including podocytes of the kidney, type I alveolar cells of the lung, and lymphatic endothelial cells. We recently produced an anti-bovine PDPN (bovPDPN) monoclonal antibody (mAb), PMab-44, by immunizing mice with recombinant proteins of bovPDPN. In this study, we determined the critical epitope of PMab-44 for the recognition of bovPDPN using many deletion mutants and point mutants of bovPDPN. Flow cytometric analyses revealed that the epitope of PMab-44 was Glu46-Thr50, which corresponds to platelet aggregation-stimulating (PLAG) domain-3. The important amino acids in the PMab-44 epitope were determined to be Glu46, Tyr48, and Thr50. Western blot analysis also confirmed these results, indicating that the PLAG domain of bovPDPN is also important in immunogenicity for producing useful anti-PDPN mAbs.

Development of an Anti-HER2 Monoclonal Antibody H2Mab-139 Against Colon Cancer.

Science.gov (United States)

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Kato, Yukinari

2018-02-01

Human epidermal growth factor receptor 2 (HER2) expression has been reported in several cancers, such as breast, gastric, lung, pancreatic, and colorectal cancers. HER2 is overexpressed in those cancers and is associated with poor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits for patients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-139 (IgG 1 , kappa) and investigated it against colon cancers using flow cytometry, western blot, and immunohistochemical analyses. Flow cytometry analysis revealed that H 2 Mab-139 reacted with colon cancer cell lines, such as Caco-2, HCT-116, HCT-15, HT-29, LS 174T, COLO 201, COLO 205, HCT-8, SW1116, and DLD-1. Although H 2 Mab-139 strongly reacted with LN229/HER2 cells on the western blot, we did not observe a specific signal for HER2 in colon cancer cell lines. Immunohistochemical analyses revealed sensitive and specific reactions of H 2 Mab-139 against colon cancers, indicating that H 2 Mab-139 is useful in detecting HER2 overexpression in colon cancers using flow cytometry and immunohistochemical analyses.

Radioimmunodetection of human leukemia with anti-interleukin-2 receptor antibody in severe combined immunodeficiency mice

International Nuclear Information System (INIS)

Hosono, Makoto; Takaori-Kondo, Akifumi; Zheng-Sheng, Yao; Kobayashi, Hisataka; Hosono, Masako N.; Sakahara, Harumi; Imada, Kazunori; Okuma, Minoru; Uchiyama, Takashi; Konishi, Junji

1995-01-01

Anti-Tac monoclonal antibody recognizes human interleukin-2 receptor, which is overexpressed in leukemic cells of most adult T-cell leukemia (ATL) patients. To examine the potency of anti-Tac for targeting of ATL, biodistributions of intravenously administered 125 I- and 111 In-labeled anti-Tac were examined in severe combined immunodeficiency (SCID) mice inoculated with ATL cells. Significant amounts of radiolabeled anti-Tac were found in the spleen and thymus. The trafficking of ATL cells in SCID mice was detected using 111 In-oxine-labeled ATL cells. These results were coincident with the histologically confirmed infiltration of ATL cells. The radiolabeled anti-Tac seemed potent for targeting of ATL

LpMab-23: A Cancer-Specific Monoclonal Antibody Against Human Podoplanin.

Science.gov (United States)

Yamada, Shinji; Ogasawara, Satoshi; Kaneko, Mika K; Kato, Yukinari

2017-04-01

Human podoplanin (hPDPN), the ligand of C-type lectin-like receptor-2, is involved in cancer metastasis. Until now, many monoclonal antibodies (mAbs) have been established against hPDPN. However, it is still difficult to develop a cancer-specific mAb (CasMab) against hPDPN because the protein sequence of hPDPN expressed in cancer cells is the same as that in normal cells. Herein, we report LpMab-23 of the mouse IgG 1 subclass, a novel CasMab against hPDPN. In an immunohistochemical analysis, LpMab-23 reacted with tumor cells of human oral cancer, but did not react with normal cells such as lymphatic endothelial cells (LECs). In contrast, LpMab-17, another anti-hPDPN mAb, reacted with both tumor cells and LECs. Furthermore, flow cytometric analysis revealed that LpMab-23 reacted with hPDPN-expressing cancer cell lines (LN319, RERF-LC-AI/hPDPN, Y-MESO-14/hPDPN, and HSC3/hPDPN) but showed little reaction with normal cells (LECs and HEK-293T), although another anti-hPDPN mAb, LpMab-7, reacted with both hPDPN-expressing cancer cells and normal cells, indicating that LpMab-23 is a CasMab against hPDPN.

Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

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Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Oshita, Masatoshi; Ideno, Shoji [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Yunoki, Mikihiro [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Kuhara, Motoki [Ina Laboratory, Medical and Biological Laboratories Corporation, Ltd., Ina, Nagano 396-0002 (Japan); Yamamoto, Naomasa [Department of Biochemistry, School of Pharmaceutical Sciences, Ohu University, Koriyama, Fukushima 963-8611 (Japan); Okuno, Yoshinobu [Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa 768-0061 (Japan); Ikuta, Kazuyoshi, E-mail: ikuta@biken.osaka-u.ac.jp [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan)

2009-09-11

Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

International Nuclear Information System (INIS)

Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki; Oshita, Masatoshi; Ideno, Shoji; Yunoki, Mikihiro; Kuhara, Motoki; Yamamoto, Naomasa; Okuno, Yoshinobu; Ikuta, Kazuyoshi

2009-01-01

Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

TP53, STK11 and EGFR Mutations Predict Tumor Immune Profile and the Response to anti-PD-1 in Lung Adenocarcinoma.

Science.gov (United States)

Biton, Jerome; Mansuet-Lupo, Audrey; Pécuchet, Nicolas; Alifano, Marco; Ouakrim, Hanane; Arrondeau, Jennifer; Boudou-Rouquette, Pascaline; Goldwasser, Francois; Leroy, Karen; Goc, Jeremy; Wislez, Marie; Germain, Claire; Laurent-Puig, Pierre; Dieu-Nosjean, Marie-Caroline; Cremer, Isabelle; Herbst, Ronald; Blons, Hélène F; Damotte, Diane

2018-05-15

By unlocking anti-tumor immunity, antibodies targeting programmed cell death 1 (PD-1) exhibit impressive clinical results in non-small cell lung cancer, underlining the strong interactions between tumor and immune cells. However, factors that can robustly predict long-lasting responses are still needed. We performed in depth immune profiling of lung adenocarcinoma using an integrative analysis based on immunohistochemistry, flow-cytometry and transcriptomic data. Tumor mutational status was investigated using next-generation sequencing. The response to PD-1 blockers was analyzed from a prospective cohort according to tumor mutational profiles and to PD-L1 expression, and a public clinical database was used to validate the results obtained. We showed that distinct combinations of STK11 , EGFR and TP53 mutations, were major determinants of the tumor immune profile (TIP) and of the expression of PD-L1 by malignant cells. Indeed, the presence of TP53 mutations without co-occurring STK11 or EGFR alterations ( TP53 -mut/ STK11 - EGFR -WT), independently of KRAS mutations, identified the group of tumors with the highest CD8 T cell density and PD-L1 expression. In this tumor subtype, pathways related to T cell chemotaxis, immune cell cytotoxicity, and antigen processing were up-regulated. Finally, a prolonged progression-free survival (PFS: HR=0.32; 95% CI, 0.16-0.63, p <0.001) was observed in anti-PD-1 treated patients harboring TP53 -mut/ STK11 - EGFR -WT tumors. This clinical benefit was even more remarkable in patients with associated strong PD-L1 expression. Our study reveals that different combinations of TP53 , EGFR and STK11 mutations , together with PD-L1 expression by tumor cells, represent robust parameters to identify best responders to PD-1 blockade. Copyright ©2018, American Association for Cancer Research.

KRAS mutation: should we test for it, and does it matter?

Science.gov (United States)

Roberts, Patrick J; Stinchcombe, Thomas E

2013-03-10

Lung cancer is the leading cause of cancer mortality in the United States and worldwide. Previously, lung cancer was simplistically divided into non-small-cell lung cancer (NSCLC) and small-cell lung cancer. However, in the last decade, we have gone from a simplistic binary system of classifying lung cancer to defining NSCLC on the basis of molecular subsets. KRAS mutations represent the most common molecular change in NSCLC. The presence of KRAS mutation has been shown to be associated with a poor prognosis in NSCLC, but this is of little clinical utility. More important is determining the clinical utility of KRAS mutational analysis for predicting benefit of chemotherapy, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), anti-EGFR monoclonal antibodies, or other novel therapeutics. Current data does not support the routine use of KRAS mutational analysis for predicting chemotherapy benefit. Additionally, there was significant interest in using KRAS status to select patients for EGFR TKI and anti-EGFR monoclonal antibodies. However, the EGFR mutational status has demonstrated significant predictive value in the selection of patients for EGFR TKI therapy and is now the preferred test. An association between KRAS mutational status and benefit of anti-EGFR monoclonal antibodies has not been demonstrated in NSCLC. Here we review, in the context of NSCLC, the underlying biology of KRAS mutations, the predictive value of KRAS mutations for therapeutic intervention, and the integration of KRAS mutational testing into our current clinical paradigms.

Prolonged skin graft survival by administration of anti-CD80 monoclonal antibody with cyclosporin A

NARCIS (Netherlands)

Ossevoort, MA; Lorre, K; Boon, L; van den Hout, Y; de Boer, M; De Waele, P; Jonker, M; VandeVoorde, A

Costimulation via the B7/CD28 pathway is an important signal for the activation of T cells. Maximal inhibition of T-cell activation and the induction of alloantigen-specific nonresponsiveness in vitro was achieved using anti-CD80 monoclonal antibody (mAb) in combination with cyclosporin A (CsA).

Monoclonal antibodies for radioimmunodetection of tumours and for targeting

International Nuclear Information System (INIS)

Baldwin, R.W.; Embleton, M.J.; Pimm, M.V.

1983-01-01

A monoclonal antibody 791T/36 prepared against human osteogenic sarcoma has been used to detect primary and metastatic colorectal carcinomas by external imaging of patients following injection of 131 I-labelled antibody. In 10 of 11 patients radiolabelled 791T/36 antibody localized in tumours, the tumour/non tumour ratio of radioactivity ranging from 1.5:1 to 8.1. 791T/36 antibody was also evaluated for its potential for targeting anti-tumour agents including cytotoxic drugs (Vindesine) and immunomodulating agents (interferon). Vindesine-791T/36 conjugates were preferentially cytotoxic in vitro for target cells expressing the 791T/36 anti-body defined antigen. Also interferon conjugated to 791T/36 antibody, like free interferon activated peripheral blood natural killer cell activity. These in vitro tests together with related studies on antibody localization in vivo indicate the potential of monoclonal antibody targeting of anti-tumour agents

A Unique Report: Development of Super Anti-Human IgG Monoclone with Optical Density Over Than 3

OpenAIRE

Aghebati Maleki, Leili; Baradaran, Behzad; Abdolalizadeh, Jalal; Ezzatifar, Fatemeh; Majidi, Jafar

2013-01-01

Purpose: Monoclonal antibodies and related conjugates are key reagents used in biomedical researches as well as, in treatment, purification and diagnosis of infectious and non- infectious diseases. Methods: Balb/c mice were immunized with purified human IgG. Spleen cells of the most immune mouse were fused with SP2/0 in the presence of Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. Then, the sample was assessed for cross-reactivity ...

Acquired resistance to EGFR inhibitors: mechanisms and prevention strategies

International Nuclear Information System (INIS)

Viloria-Petit, Alicia M.; Kerbel, Robert S.

2004-01-01

Potent and specific, or relatively specific, inhibitors of epidermal growth factor receptor (EGFR) signaling, including monoclonal antibodies and small molecular weight compounds, have been successfully developed. Both types of agent have been found to have significant antitumor activity, especially when used in combination with radio- hormone- and chemotherapy in preclinical studies. Because of the potentiation of the conventional drug activity in these combination settings, inhibitors of EGFR signaling have often been referred to as sensitizers for chemotherapy or radiation, as well as drug resistance reversal agents. Phase II clinical trials in head-and-neck as well as lung cancer suggested this concept of chemosensitization might translate into the clinic, but this remains to be definitively proven in randomized, double-blind Phase III trials. Given the extensive preclinical literature on EGFR blocking drugs and the advanced clinical development of such agents, it is surprising that the possibility of development of acquired resistance to the EGFR inhibitors themselves, a common clinical problem with virtually all other currently used anticancer drugs, remains a largely unexplored subject of investigation. Here we summarize some of the possible mechanisms that can result in acquired resistance to EGFR-targeting drugs. Alternative combination therapies to circumvent and delay this problem are suggested

Propolin C Inhibited Migration and Invasion via Suppression of EGFR-Mediated Epithelial-to-Mesenchymal Transition in Human Lung Cancer Cells

Directory of Open Access Journals (Sweden)

Jih-Tung Pai

2018-01-01

Full Text Available Controlling lung cancer cell migration and invasion via epithelial-to-mesenchymal transition (EMT through the regulation of epidermal growth factor receptor (EGFR signaling pathway has been demonstrated. Searching biological active phytochemicals to repress EGFR-regulated EMT might prevent lung cancer progression. Propolis has been used as folk medicine in many countries and possesses anti-inflammatory, antioxidant, and anticancer activities. In this study, the antimigration and anti-invasion activities of propolin C, a c-prenylflavanone from Taiwanese propolis, were investigated on EGFR-regulated EMT signaling pathway. Cell migration and invasion activities were dose-dependently suppressed by noncytotoxic concentration of propolin C. Downregulations of vimentin and snail as well as upregulation of E-cadherin expressions were through the inhibition of EGFR-mediated phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt and extracellular signal-regulated kinase (ERK signaling pathway in propolin C-treated cells. In addition, EGF-induced migration and invasion were suppressed by propolin C-treated A549 lung cancer cells. No significant differences in E-cadherin expression were observed in EGF-stimulated cells. Interestingly, EGF-induced expressions of vimentin, snail, and slug were suppressed through the inhibition of PI3K/Akt and ERK signaling pathway in propolin C-treated cells. Inhibition of cell migration and invasion by propolin C was through the inhibition of EGF/EGFR-mediated signaling pathway, followed by EMT suppression in lung cancer.

EGFR is not a major driver for osteosarcoma cell growth in vitro but contributes to starvation and chemotherapy resistance.

Science.gov (United States)

Sevelda, Florian; Mayr, Lisa; Kubista, Bernd; Lötsch, Daniela; van Schoonhoven, Sushilla; Windhager, Reinhard; Pirker, Christine; Micksche, Michael; Berger, Walter

2015-11-02

Enhanced signalling via the epidermal growth factor receptor (EGFR) is a hallmark of multiple human carcinomas. However, in recent years data have accumulated that EGFR might also be hyperactivated in human sarcomas. Aim of this study was to investigate the influence of EGFR inhibition on cell viability and its interaction with chemotherapy response in osteosarcoma cell lines. We have investigated a panel of human osteosarcoma cell lines regarding EGFR expression and downstream signalling. To test its potential applicability as therapeutic target, inhibition of EGFR by gefitinib was combined with osteosarcoma chemotherapeutics and cell viability, migration, and cell death assays were performed. Osteosarcoma cells expressed distinctly differing levels of functional EGFR reaching in some cases high amounts. Functionality of EGFR in osteosarcoma cells was proven by EGF-mediated activation of both MAPK and PI3K/AKT pathway (determined by phosphorylation of ERK1/2, AKT, S6, and GSK3β). The EGFR-specific inhibitor gefitinib blocked EGF-mediated downstream signal activation. At standard in vitro culture conditions, clinically achievable gefitinib doses demonstrated only limited cytotoxic activity, however, significantly reduced long-term colony formation and cell migration. In contrast, under serum-starvation conditions active gefitinib doses were distinctly reduced while EGF promoted starvation survival. Importantly, gefitinib significantly supported the anti-osteosarcoma activities of doxorubicin and methotrexate regarding cell survival and migratory potential. Our data suggest that EGFR is not a major driver for osteosarcoma cell growth but contributes to starvation- and chemotherapy-induced stress survival. Consequently, combination approaches including EGFR inhibitors should be evaluated for treatment of high-grade osteosarcoma patients.

Generation, characterization and in vivo biological activity of two distinct monoclonal anti-PEG IgMs

International Nuclear Information System (INIS)

Hashimoto, Yosuke; Shimizu, Taro; Mima, Yu; Abu Lila, Amr S.; Ishida, Tatsuhiro; Kiwada, Hiroshi

2014-01-01

PEGylation, the attachment of polyethylene glycol (PEG) to nanocarriers and proteins, is a widely accepted approach to improving the in vivo efficacy of the non-PEGylated products. However, both PEGylated liposomes and PEGylated proteins reportedly trigger the production of specific antibodies, mainly IgM, against the PEG moiety, which possibly leads to a reduction in safety and therapeutic efficacy of the PEGylated products. In the present study, two monoclonal anti-PEG IgMs — HIK-M09 via immunization with an intravenous injection of PEGylated liposomes (SLs) and HIK-M11 via immunization with a subcutaneous administration of PEGylated ovalbumin (PEG-OVA) were successfully generated. The generated IgMs showed efficient reactivity to mPEG 2000 conjugated to 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine (DSPE), PEGylated liposome (SL) and PEG-OVA. It appears that HIK-M09 recognizes ethoxy (OCH 2 CH 2 ) repeat units along with a terminal motif of PEG, while HIK-M11 recognizes only ethoxy repeat units of PEG. Such unique properties allow HIK-M09 to bind with dense PEG. In addition, their impact on the in vivo clearance of the PEGylated products was investigated. It was found that the generated ant-PEG IgMs induced a clearance of SL as they were intravenously administered with SL. Interestingly, the HIK-M11, generated by PEG-OVA, induced the clearance of both SL and PEG-OVA, while the HIK-M09, generated by SL, induced the clearance of SL only. We here revealed that the presence of serum anti-PEG IgM and the subsequent binding of anti-PEG IgM to the PEGylated products are not necessarily related to the enhanced clearance of the products. It appears that subsequent complement activation following anti-PEG IgM binding is the most important step in dictating the in vivo fate of PEGylated products. This study may have implications for the design, development and clinical application of PEGylated products and therapeutics. - Highlights: • Two monoclonal anti-PEG Ig

Generation, characterization and in vivo biological activity of two distinct monoclonal anti-PEG IgMs

Energy Technology Data Exchange (ETDEWEB)

Hashimoto, Yosuke; Shimizu, Taro; Mima, Yu [Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima, 1-78-1, Sho-machi, Tokushima 770-8505 (Japan); Abu Lila, Amr S. [Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima, 1-78-1, Sho-machi, Tokushima 770-8505 (Japan); Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Zagazig University, Sharqia Governorate, Zagazig 44519 (Egypt); Ishida, Tatsuhiro, E-mail: ishida@tokushima-u.ac.jp [Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima, 1-78-1, Sho-machi, Tokushima 770-8505 (Japan); Kiwada, Hiroshi [Department of Pharmacokinetics and Biopharmaceutics, Subdivision of Biopharmaceutical Sciences, Institute of Health Biosciences, The University of Tokushima, 1-78-1, Sho-machi, Tokushima 770-8505 (Japan)

2014-05-15

PEGylation, the attachment of polyethylene glycol (PEG) to nanocarriers and proteins, is a widely accepted approach to improving the in vivo efficacy of the non-PEGylated products. However, both PEGylated liposomes and PEGylated proteins reportedly trigger the production of specific antibodies, mainly IgM, against the PEG moiety, which possibly leads to a reduction in safety and therapeutic efficacy of the PEGylated products. In the present study, two monoclonal anti-PEG IgMs — HIK-M09 via immunization with an intravenous injection of PEGylated liposomes (SLs) and HIK-M11 via immunization with a subcutaneous administration of PEGylated ovalbumin (PEG-OVA) were successfully generated. The generated IgMs showed efficient reactivity to mPEG{sub 2000} conjugated to 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine (DSPE), PEGylated liposome (SL) and PEG-OVA. It appears that HIK-M09 recognizes ethoxy (OCH{sub 2}CH{sub 2}) repeat units along with a terminal motif of PEG, while HIK-M11 recognizes only ethoxy repeat units of PEG. Such unique properties allow HIK-M09 to bind with dense PEG. In addition, their impact on the in vivo clearance of the PEGylated products was investigated. It was found that the generated ant-PEG IgMs induced a clearance of SL as they were intravenously administered with SL. Interestingly, the HIK-M11, generated by PEG-OVA, induced the clearance of both SL and PEG-OVA, while the HIK-M09, generated by SL, induced the clearance of SL only. We here revealed that the presence of serum anti-PEG IgM and the subsequent binding of anti-PEG IgM to the PEGylated products are not necessarily related to the enhanced clearance of the products. It appears that subsequent complement activation following anti-PEG IgM binding is the most important step in dictating the in vivo fate of PEGylated products. This study may have implications for the design, development and clinical application of PEGylated products and therapeutics. - Highlights: • Two monoclonal

MINOR HUMAN-ANTIBODY RESPONSE TO A MOUSE AND CHIMERIC MONOCLONAL-ANTIBODY AFTER A SINGLE IV INFUSION IN OVARIAN-CARCINOMA PATIENTS - A COMPARISON OF 5 ASSAYS

NARCIS (Netherlands)

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

Detecting fish parvalbumin with commercial mouse monoclonal anti-frog parvalbumin IgG.

Science.gov (United States)

Chen, Lingyun; Hefle, Sue L; Taylor, Steve L; Swoboda, Ines; Goodman, Richard E

2006-07-26

Parvalbumin is a calcium-binding muscle protein that is highly conserved across fish species and amphibians. It is the major cross-reactive allergen associated with both fish and frog allergy. We used two-dimensional electrophoretic and immunoblotting techniques to investigate the utility of a commercial monoclonal anti-frog parvalbumin IgG for detecting parvalbumin present in some commonly consumed fish species. The 2D electrophoresis and immunoblots revealed species-specific differences in proteins that appear to represent various numbers of isoforms of parvalbumin in carp (5), catfish (3), cod (1) and tilapia (2). No parvalbumin was detected in yellowfin tuna. Based on minor differences in relative intensities of protein staining and immunodetection, parvalbumin isoforms may have slight differences in the epitope region recognized by the anti-frog parvalbumin antibody. These results suggest that the frog anti-parvalbumin antibody can be used as a valuable tool to detect parvalbumins from the fish tested in this study, except yellowfin tuna.

Rheumatoid factor interference in immunogenicity assays for human monoclonal antibody therapeutics.

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Tatarewicz, Suzanna; Miller, Jill M; Swanson, Steven J; Moxness, Michael S

2010-05-31

Rheumatoid factors (RFs) are endogenous human antibodies that bind to human gamma globulins. RFs demonstrate preferential binding to aggregated gamma globulins and are involved in the clearing mechanism of immune complexes. Immunoassays designed to measure human anti-human antibodies (HAHA) after administration of monoclonal antibody therapeutics are thus vulnerable to interference from RFs. When using a sensitive electrochemiluminescent (ECL) bridging immunoassay, samples from subjects with rheumatoid arthritis demonstrated much higher baseline reactivity than healthy subjects. Interference was found to be dependent on the aggregation state of the therapeutic antibody that had been conjugated with the detection reagent (ruthenium). Size exclusion high performance liquid chromatography (SE-HPLC) demonstrated that of the total integrated peaks, as little as 0.55% high molecular weight aggregates (>600kDa) were sufficient to cause increased reactivity. Stability studies of the ruthenium and biotin conjugated therapeutic antibody indicated that storage time, temperature and buffer formulation were critical in maintaining the integrity of the reagents. Through careful SE-HPLC monitoring we were able to choose appropriate storage and buffer conditions which led to a reduction in the false reactivity rate in therapeutic-naïve serum from a rheumatoid arthritis population.

At least two Fc Neu5Gc residues of monoclonal antibodies are required for binding to anti-Neu5Gc antibody

OpenAIRE

Yu, Chuanfei; Gao, Kai; Zhu, Lei; Wang, Wenbo; Wang, Lan; Zhang, Feng; Liu, Chunyu; Li, Meng; Wormald, Mark R.; Rudd, Pauline M.; Wang, Junzhi

2016-01-01

Two non-human glycan epitopes, galactose-Į-1,3-galactose (Į-gal) and Neu5Gc-Į-2-6-galactose (Neu5Gc) have been shown to be antigenic when attached to Fab oligosaccharides of monoclonal antibodies (mAbs) , while Į-gal attached to Fc glycans were not. However, the antigenicity of Neu5Gc on the Fc glycans remains unclear in the context that most mAbs carry only Fc glycans. After studying two clinical mAbs carrying significant amounts of Fc Neu5Gc, we show that their binding activity with anti-Ne...

Interaction between EGFR and EphA2

DEFF Research Database (Denmark)

Larsen, Alice Bjerregaard

2010-01-01

Enhanced or altered epidermal growth factor receptor (EGFR) activity has been reported in many human cancers and several molecular targeting therapies has been developed. However, despite intense research, therapies targeting EGFR have shown conflicting results in clinical studies, indicating...... the involvement of other important molecular players. Several different EGFR mutations have been reported in cancer, one of which is the cancer specific type III EGFR deletion mutant (EGFRvIII, de2-7EGFR, ΔEGFR). In a global search for EGFR and EGFRvIII regulated genes we identified the receptor tyrosine kinase...... (RTK) EphA2. EphA2 belongs to the large Eph-receptor family, which has mainly been associated with neuronal development. More recently, expression of several Eph-receptors has been detected in many different cancer types. Elevated EphA2 expression has been reported in a broad range of human cancer...

Interaction between EGFR and EphA2

DEFF Research Database (Denmark)

Larsen, Alice Bjerregaard

2010-01-01

Enhanced or altered epidermal growth factor receptor (EGFR) activity has been reported in many human cancers and several molecular targeting therapies has been developed. However, despite intense research, therapies targeting EGFR have shown conflicting results in clinical studies, indicating...... the involvement of other important molecular players. Several different EGFR mutations have been reported in cancer, one of which is the cancer specific type III EGFR deletion mutant (EGFRvIII, de2-7EGFR, ¿EGFR). In a global search for EGFR and EGFRvIII regulated genes we identified the receptor tyrosine kinase...... (RTK) EphA2. EphA2 belongs to the large Eph-receptor family, which has mainly been associated with neuronal development. More recently, expression of several Eph-receptors has been detected in many different cancer types. Elevated EphA2 expression has been reported in a broad range of human cancer...

Immunoglobulin gene usage in the human anti-pathogen response.

Science.gov (United States)

Newkirk, M M; Rioux, J D

1995-09-01

The human antibody response to foreign pathogens is generated to a relatively small number of target surface proteins and carbohydrates that nonetheless have an extensive array of epitopes. The study of human monoclonal antibodies to different pathogens shows that there are a diversity of mechanisms used to generate a sufficient repertoire of antibodies to combat the invading pathogens. Although many different immunoglobulin gene elements are used to construct the anti-pathogen response, some elements are used more often than would be expected if all elements were used randomly. For example, the immune response to Haemophilus influenzae polysaccharide appears to be quite narrow, being restricted primarily to a specific heavy-chain gene, 3-15, and a lambda light-chain family II member, 4A. In contrast, for the immune response to cytomegalovirus proteins, a wider group of gene elements is needed. It is also surprising that despite an investigator bias for IgG- rather than IgM-secreting immortal B cells (because of their high affinity and neutralizing abilities), 26% of light chains and 13% of heavy chains showed a very low level of somatic mutation, equivalent to an IgM molecule that has not undergone affinity maturation. Although some highly mutated IgG molecules are present in the anti-pathogen response, most of the monoclonal antibodies specific for viruses or bacteria have a level of somatic hypermutation similar to that of the adult IgM repertoire. A number of studies have shown that there are similarities in the antibody responses to pathogens and to self (autoantibodies).(ABSTRACT TRUNCATED AT 250 WORDS)

Human pharmacokinetics, biodistribution and dosimetry of the kit of monoclonal antibody IOR EGF/R3 labelled with 99m Tc

International Nuclear Information System (INIS)

Torres, L.A.; Ramos, M.; Perera, A.; Hernandez, A.; Iznaga, M.E. N.

1998-01-01

The aim of this work was to assess the human pharmacokinetics, biodistribution and dosimetry of the 99m Tc-labeled MAb ior egf/r3. Five patients were included in the biodistribution and dosimetric studies and three in the pharmacokinetic analysis. Multiple blood and urine samples we recollected and sequential anterior and posterior whole-body scintigraphies u pto 24 hr post-injection were performed to all patients . The internal radiation dosimetry was estimated from gamma camera imaging data using the methods developed by the Medical Internal radiation dosimetry (MIRD)committee. Raw data were computed from operations between gamma graphic images and regions of interest (ROI) using the Bio-Dose software and time-activity curves were calculated in order to determine the residence times of the source organs. The Pharmacokinetics and Biodistribution results showed that this compound have a bio exponential plasmatic and blood clearance with a rapid biodistribution phase of 9.1 ± 8.4 min and 12.2±4.4 min, respectively, and a slower elimination phase of 6.6 ± 1.6 hr and 10.8 ± 6.8 hr. respectively. The urinary and hepatobiliary excretion showed 4.7 ± 0.4 % and 9.9 ± 1.8 % of the total administered dose,eliminated by these ways. Liver was the target organ of this product and had an uptake peak at 1 hr post-injection (61.2%) and a great retention of the MAb(T 1/2 eff = 5.3 hr, T 1/2 Biol. = 45.0 hr). The dosimetric results showed that liver, gallbladder and spleen received the higher absorbed. The effective dose and the effective equivalent dose were 1,2E-01 mSv/MBq and 9,2E-02 mSv/MBq respectively. These results allow to see the i or egf/r3 kit in a safe and controlled way. (Author)

Cloning of the first human anti-JCPyV/VP1 neutralizing monoclonal antibody: epitope definition and implications in risk stratification of patients under natalizumab therapy.

Science.gov (United States)

Diotti, Roberta Antonia; Mancini, Nicasio; Clementi, Nicola; Sautto, Giuseppe; Moreno, Guisella Janett; Criscuolo, Elena; Cappelletti, Francesca; Man, Petr; Forest, Eric; Remy, Louise; Giannecchini, Simone; Clementi, Massimo; Burioni, Roberto

2014-08-01

JC virus (JCPyV) has gained novel clinical importance as cause of progressive multifocal leukoencephalopathy (PML), a rare demyelinating disease recently associated to immunomodulatory drugs, such as natalizumab used in multiple sclerosis (MS) cases. Little is known about the mechanisms leading to PML, and this makes the need of PML risk stratification among natalizumab-treated patients very compelling. Clinical and laboratory-based risk-stratification markers have been proposed, one of these is represented by the JCPyV-seropositive status, which includes about 54% of MS patients. We recently proposed to investigate the possible protective role of neutralizing humoral immune response in preventing JCPyV reactivation. In this proof-of-concept study, by cloning the first human monoclonal antibody (GRE1) directed against a neutralizing epitope on JCPyV/VP1, we optimized a robust anti-JCPyV neutralization assay. This allowed us to evaluate the neutralizing activity in JCPyV-positive sera from MS patients, demonstrating the lack of correlation between the level of anti-JCPyV antibody and anti-JCPyV neutralizing activity. Relevant consequences may derive from future clinical studies induced by these findings; indeed the study of the serum anti-JCPyV neutralizing activity could allow not only a better risk stratification of the patients during natalizumab treatment, but also a better understanding of the pathophysiological mechanisms leading to PML, highlighting the contribution of peripheral versus central nervous system JCPyV reactivation. Noteworthy, the availability of GRE1 could allow the design of novel immunoprophylactic strategies during the immunomodulatory treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

Minor human antibody response to a mouse and chimeric monoclonal antibody after a single i.v. infusion in ovarian carcinoma patients: a comparison of five assays

NARCIS (Netherlands)

Buist, M. R.; Kenemans, P.; van Kamp, G. J.; Haisma, H. J.

1995-01-01

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).

Novel flow cytometric analysis of the progress and route of internalization of a monoclonal anti-carcinoembryonic antigen (CEA) antibody.

Science.gov (United States)

Ford, C H; Tsaltas, G C; Osborne, P A; Addetia, K

1996-03-01

A flow cytometric method of studying the internalization of a monoclonal antibody (Mab) directed against carcinoembryonic antigen (CEA) has been compared with Western blotting, using three human colonic cancer cell lines which express varying amounts of the target antigen. Cell samples incubated for increasing time intervals with fluoresceinated or unlabelled Mab were analyzed using flow cytometry or polyacrylamide gel electrophoresis and Western blotting. SDS/PAGE analysis of cytosolic and membrane components of solubilized cells from the cell lines provided evidence of non-degraded internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 min. Internalized anti-CEA was detected in the case of high CEA expressing cell lines (LS174T, SKCO1). Very similar results were obtained with an anti-fluorescein flow cytometric assay. Given that these two methods consistently provided comparable results, use of flow cytometry for the detection of internalized antibody is suggested as a rapid alternative to most currently used methods for assessing antibody internalization. The question of the endocytic route followed by CEA-anti-CEA complexes was addressed by using hypertonic medium to block clathrin mediated endocytosis.

Effects of Monoclonal Antibody Cetuximab on Proliferation of Non-small Cell Lung Cancer Cell lines

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Zhen CHEN

2010-08-01

Full Text Available Background and objective The epidermal growth factor receptor (EGFR monoclonal antibody cetuximab has been used widely in non-small cell lung cancer patients. The aim of this study is to explore the effect of lung cancer cells (A549, H460, H1299, SPC-A-1 which were treated by cetuximab in vitro. Methods We studied the effects of increasing concentrations of cetuximab (1 nmol/L-625 nmol/L in four human lung cancer cell lines (A549, SPC-A-1, H460, H1229. CCK8 measured the inhibition of cell proliferation in each group. A549, SPC-A-1 were marked by PI and the statuses of apoptosis were observed. Western blot were used to detect the proliferation-related signaling protein and apoptosis-related protein in A549. Results The treatment with cetuximab resulted in the effect on cell proliferation and apoptosis in a time- and dosedependent manner. The expression of activated key enzymes (p-AKT, p-EGFR, p-MAPK in EGFR signaling transduction pathway were down-regulated more obviously. Conclusion Cetuximab is an effective targeted drug in the treatment of lung cancer cell lines, tissues, most likely to contribute to the inhibition of key enzymes in EGFR signaling transduction pathway.

Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice

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Leili Aghebati

2013-02-01

Full Text Available Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells.

Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice

Science.gov (United States)

Sineh sepehr, Koushan; Baradaran, Behzad; Majidi, Jafar; Abdolalizadeh, Jalal; Aghebati, leili; Zare Shahneh, Fatemeh

2013-01-01

Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs) with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells. PMID:24312821

H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

Science.gov (United States)

Itai, Shunsuke; Fujii, Yuki; Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Takahashi, Maki; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-08-01

Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H 2 Mab-77 (IgG 1 , kappa) was selected. Finally, immunohistochemical analyses with H 2 Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H 2 Mab-77 to detect HER2 in pathological analyses of breast cancers.

In vivo activity of a mixture of two human monoclonal antibodies (anti-HBs) in a chronic hepatitis B virus carrier chimpanzee

NARCIS (Netherlands)

R. Heijtink; W. Paulij; P.A.C. van Bergen (Patrick); M.H. van Roosmalen (Mark); D. Rohm; B. Eichentopf (Bertram); E. Muchmore; A.D.M.E. Osterhaus (Albert); R.A. de Man (Robert)

1999-01-01

textabstractA 35-year-old female hepatitis B virus carrier chimpanzee was infused with one dose of a mixture of human monoclonal antibodies 9H9 and 4-7B (antibodies against hepatitis B virus surface antigen; HBsAg). Blood samples were taken before and up to 3 weeks

Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

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M Jedi-Tehrani

2005-10-01

Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

A camelid nanobody against EGFR was easily obtained through refolding of inclusion body expressed in Escherichia coli.

Science.gov (United States)

Xu, Li; Song, Xiaoyu; Jia, Lingyun

2017-11-01

Using anti-EGFR (epidermal growth factor receptor) nanobody is a good choice for diagnoses and therapeutics for high EGFR expression diseases. In the present study, the percentage composition of anti-EGFR nanobody attained 25% of the total cell protein expressed in Escherichia coli BL21 (DE3). However, almost all nanobodies were expressed as inclusion bodies. To acquire active nanobodies, a series of dilution refolding procedures were optimized after inclusion bodies were dissolved into 6 M urea and purified with immobilized metal affinity chromatography. The results showed the refolding rate of the anti-EGFR nanobodies attained to 73%, and about 100 mg nanobodies were refolded from 1 L cells under the conditions that the initial nanobody concentration was 0.3 mg/mL, the dilution speed was 2.5 mL/Min, the dilution buffer was Tris-HCl at pH 8.0, the additives were 0.2 M Arg, 5 mM reduced glutathione (GSH), and 1 mM oxidized glutathione (GSSG). Then the activity of the refolded nanobodies was confirmed. The results showed that the refolded anti-EGFR nanobodies, in a dose-dependent manner, bounded to the tumor cell surface of A431 and MCF-7 and significantly inhibited the proliferation of A431 caused by the epidermal growth factor. Our study provides a facile method to rapidly, efficiently, and massively prepare anti-EGFR antibodies and promotes anti-EGFR-based recognition in cancer diagnoses and therapeutics. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Generation and Characterization of Novel Human IRAS Monoclonal Antibodies

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Bo Wang

2009-01-01

Full Text Available Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS, has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10–120aa through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10–120aa. Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

Phase I trial of anti-GD2 monoclonal antibody hu3F8 plus GM-CSF: Impact of body weight, immunogenicity and anti-GD2 response on pharmacokinetics and survival.

Science.gov (United States)

Cheung, Irene Y; Kushner, Brian H; Modak, Shakeel; Basu, Ellen M; Roberts, Stephen S; Cheung, Nai-Kong V

2017-01-01

Fifty-seven stage 4 patients with refractory/relapsed neuroblastoma were enrolled in a phase I trial (Clinicaltrials.gov NCT01757626) using humanized anti-GD2 monoclonal antibody hu3F8 in combination with granulocyte-macrophage colony-stimulating factor. The influence of body weight and human anti-human antibody (HAHA) on the pharmacokinetics (PK) of hu3F8, and the effect of de novo anti-GD2 response on patient outcome were explored. Serum samples before hu3F8 infusion, and serially up to day 12 during treatment cycle #1, and at 5 min after each hu3F8 infusion for all subsequent cycles were collected. PK was analyzed using non-compartmental modeling. Immunogenicity was assayed by HAHA response, and vaccination effect by induced host anti-GD2 response measured periodically until disease progression or last followup. Progression-free and overall survival was estimated by the Kaplan-Meier method. Despite dosing being based on body weight, smaller patients had consistently lower area-under-the-curve and faster clearance over the 15 dose levels (0.9 to 9.6 mg/kg per treatment cycle) in this trial. Positive HAHA, defined by the upper limit of normal, when measured within 10 days from the last hu3F8 dose received, was associated with significantly lower serum hu3F8. Despite prior sensitization to other anti-GD2 antibody, e.g. mouse 3F8 or ch14.18, 75% of the patients never developed HAHA response even after getting more treatment cycles. Hu3F8 induced a de novo anti-GD2 response in patients, which was prognostic of progression-free survival. We conclude that hu3F8 had low immunogenicity. During treatment, positive HAHA and low body weight affected PK adversely, whereas induced anti-GD2 response was an outcome predictor.

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

International Nuclear Information System (INIS)

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

2014-01-01

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

New monoclonal antibody to human apolipoprotein J

Czech Academy of Sciences Publication Activity Database

Čapková, Jana; Geussová, Gizela; Pěknicová, Jana

2002-01-01

Roč. 2002, č. 48 (2002), s. 40-42 ISSN 0015-5500 R&D Projects: GA ČR GV524/96/K162 Grant - others:NFDK-MAOB(XE) 1985-NFDK-MAOB Institutional research plan: CEZ:AV0Z5052915 Keywords : apo J * human spermatoza * monoclonal antibody Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.615, year: 2002

Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer

International Nuclear Information System (INIS)

Jörißen, Hannah; Klockenbring, Torsten; Bektas, Nuran; Dahl, Edgar; Hartmann, Arndt; Haaf, Anette ten; Di Fiore, Stefano; Kiefer, Hans; Thess, Andreas; Barth, Stefan

2009-01-01

RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3 expression in normal and cancerous human

Individual and combining effects of anti-RANKL monoclonal antibody and teriparatide in ovariectomized mice

Directory of Open Access Journals (Sweden)

Naoto Tokuyama

2015-06-01

Full Text Available We examined the individual and combined effects of teriparatide and anti-RANKL (receptor activator of nuclear factor κB ligand monoclonal antibody in ovariectomized mice. Three-month-old female C57BL/6 mice were ovariectomized (OVX or sham operated. Four weeks after OVX, they were assigned to 3 different groups to receive anti-RANKL monoclonal antibody (Ab alone (5 mg/kg single injection at 4 weeks after OVX, Ab group, teriparatide alone (80 μg/kg daily injection for 4 weeks from 4 weeks after OVX, PTH group, or mAb plus teriparatide (Ab + PTH group. Mice were sacrificed 8 weeks after OVX. Bone mineral density (BMD was measured at the femur and lumbar spine. Hind limbs were subjected to histological and histomorphometric analysis. Serum osteocalcin and CTX-I levels were measured to investigate the bone turnover. Compared with Ab group, Ab + PTH group showed a significant increase in BMD at distal femur and femoral shaft. Cortical bone volume was significantly increased in PTH and Ab + PTH groups compared with Ab group. Bone turnover in Ab + PTH group was suppressed to the same degree as in Ab group. The number of TRAP-positive multinucleated cells was markedly reduced in Ab and Ab + PTH groups. These results suggest that combined treatment of teriparatide with anti-RANKL antibody has additive effects on BMD in OVX mice compared with individual treatment.

The Role of Anti-Drug Antibodies in the Pharmacokinetics, Disposition, Target Engagement, and Efficacy of a GITR Agonist Monoclonal Antibody in Mice.

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Brunn, Nicholas D; Mauze, Smita; Gu, Danling; Wiswell, Derek; Ueda, Roanna; Hodges, Douglas; Beebe, Amy M; Zhang, Shuli; Escandón, Enrique

2016-03-01

Administration of biologics to enhance T-cell function is part of a rapidly growing field of cancer immunotherapy demonstrated by the unprecedented clinical success of several immunoregulatory receptor targeting antibodies. While these biologic agents confer significant anti-tumor activity through targeted immune response modulation, they can also elicit broad immune responses potentially including the production of anti-drug antibodies (ADAs). DTA-1, an agonist monoclonal antibody against GITR, is a highly effective anti-tumor treatment in preclinical models. We demonstrate that repeated dosing with murinized DTA-1 (mDTA-1) generates ADAs with corresponding reductions in drug exposure and engagement of GITR on circulating CD3(+) CD4(+) T cells, due to rapid hepatic drug uptake and catabolism. Mice implanted with tumors after induction of preexisting mDTA-1 ADA show no anti-tumor efficacy when given 3 mg/kg mDTA-1, an efficacious dose in naive mice. Nonetheless, increasing mDTA-1 treatment to 30 mg/kg in ADA-positive mice restores mDTA-1 exposure and GITR engagement on circulating CD3(+) CD4(+) T cells, thereby partially restoring anti-tumor efficacy. Formation of anti-mDTA-1 antibodies and changes in drug exposure and disposition does not occur in GITR(-/-) mice, consistent with a role for GITR agonism in humoral immunity. Finally, the administration of muDX400, a murinized monoclonal antibody against the checkpoint inhibitor PD-1, dosed alone or combined with mDTA-1 did not result in reduced muDX400 exposure, nor did it change the nature of the anti-mDTA-1 response. This indicates that anti-GITR immunogenicity may not necessarily impact the pharmacology of coadministered monoclonal antibodies, supporting combination immunomodulatory strategies. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

3-Dimensional culture systems for anti-cancer compound profiling and high-throughput screening reveal increases in EGFR inhibitor-mediated cytotoxicity compared to monolayer culture systems.

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Howes, Amy L; Richardson, Robyn D; Finlay, Darren; Vuori, Kristiina

2014-01-01

3-dimensional (3D) culture models have the potential to bridge the gap between monolayer cell culture and in vivo studies. To benefit anti-cancer drug discovery from 3D models, new techniques are needed that enable their use in high-throughput (HT) screening amenable formats. We have established miniaturized 3D culture methods robust enough for automated HT screens. We have applied these methods to evaluate the sensitivity of normal and tumorigenic breast epithelial cell lines against a panel of oncology drugs when cultured as monolayers (2D) and spheroids (3D). We have identified two classes of compounds that exhibit preferential cytotoxicity against cancer cells over normal cells when cultured as 3D spheroids: microtubule-targeting agents and epidermal growth factor receptor (EGFR) inhibitors. Further improving upon our 3D model, superior differentiation of EC50 values in the proof-of-concept screens was obtained by co-culturing the breast cancer cells with normal human fibroblasts and endothelial cells. Further, the selective sensitivity of the cancer cells towards chemotherapeutics was observed in 3D co-culture conditions, rather than as 2D co-culture monolayers, highlighting the importance of 3D cultures. Finally, we examined the putative mechanisms that drive the differing potency displayed by EGFR inhibitors. In summary, our studies establish robust 3D culture models of human cells for HT assessment of tumor cell-selective agents. This methodology is anticipated to provide a useful tool for the study of biological differences within 2D and 3D culture conditions in HT format, and an important platform for novel anti-cancer drug discovery.

Wild-type EGFR Is Stabilized by Direct Interaction with HSP90 in Cancer Cells and Tumors

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Aarif Ahsan

2012-08-01

Full Text Available The epidermal growth factor receptor (EGFR has been targeted for inhibition using tyrosine kinase inhibitors and monoclonal antibodies, with improvement in outcome in subsets of patients with head and neck, lung, and colorectal carcinomas. We have previously found that EGFR stability plays a key role in cell survival after chemotherapy and radiotherapy. Heat shock protein 90 (HSP90 is known to stabilize mutant EGFR and ErbB2, but its role in cancers with wild-type (WT WT-EGFR is unclear. In this report, we demonstrate that fully mature, membrane-bound WT-EGFR interacts with HSP90 independent of ErbB2. Further, the HSP90 inhibitors geldanamycin (GA and AT13387 cause a decrease in WT-EGFR in cultured head and neck cancer cells. This decrease results from a significantly reduced half-life of WT-EGFR. WT-EGFR was also lost in head and neck xenograft specimens after treatment with AT13387 under conditions that inhibited tumor growth and prolonged survival of the mice. Our findings demonstrate that WT-EGFR is a client protein of HSP90 and that their interaction is critical for maintaining both the stability of the receptor as well as the growth of EGFR-dependent cancers. Furthermore, these findings support the search for specific agents that disrupt HSP90's ability to act as an EGFR chaperone.

Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

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Andrabi Raiees

2012-09-01

Full Text Available Abstract Background Analysis of human monoclonal antibodies (mAbs developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3 is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5 binding and presence of epitopes recognized by broadly neutralizing antibodies. Methods Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females within the age range of 20–57 years (median = 33 years were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. Results We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL, suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. Conclusions Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope

Lipopolysaccharide induces VCAM-1 expression and neutrophil adhesion to human tracheal smooth muscle cells: Involvement of Src/EGFR/PI3-K/Akt pathway

International Nuclear Information System (INIS)

Lin, W.-N.; Luo, S.-F.; Wu, C.-B.; Lin, C.-C.; Yang, C.-M.

2008-01-01

In our previous study, LPS has been shown to induce vascular cell adhesion molecule-1(VCAM-1) expression through MAPKs and NF-κB in human tracheal smooth muscle cells (HTSMCs). In addition to these pathways, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3K) have been shown to be implicated in the expression of several inflammatory target proteins. Here, we reported that LPS-induced up-regulation of VCAM-1 enhanced the adhesion of neutrophils onto HTSMC monolayer, which was inhibited by LY294002 and wortmannin. LPS stimulated phosphorylation of protein tyrosine kinases including Src, PYK2, and EGFR, which were further confirmed using specific anti-phospho-Src, PYK2, or EGFR Ab, respectively, revealed by Western blotting. LPS-stimulated Src, PYK2, EGFR, and Akt phosphorylation and VCAM-1 expression were attenuated by the inhibitors of Src (PP1), EGFR (AG1478), PI3-K (LY294002 and wortmannin), and Akt (SH-5), respectively, or transfection with siRNAs of Src or Akt and shRNA of p110. LPS-induced VCAM-1 expression was also blocked by pretreatment with curcumin (a p300 inhibitor) or transfection with p300 siRNA. LPS-stimulated Akt activation translocated into nucleus and associated with p300 and VCAM-1 promoter region was further confirmed by immunofluorescence, immunoprecipitation, and chromatin immunoprecipitation assays. This association of Akt and p300 to VCAM-1 promoter was inhibited by pretreatment with PP1, AG1478, wortmannin, and SH-5. LPS-induced p300 activation enhanced VCAM-1 promoter activity and VCAM-1 mRNA expression. These results suggested that in HTSMCs, Akt phosphorylation mediated through transactivation of Src/PYK2/EGFR promoted the transcriptional p300 activity and eventually led to VCAM-1 expression induced by LPS

Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system.

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Hur, Byung-ung; Yoon, Jae-bong; Liu, Li-Kun; Cha, Sang-hoon

2010-11-30

Specific antibodies that possess a subnanomolar affinity are very difficult to obtain from human naïve immunoglobulin repertoires without the use of lengthy affinity optimization procedures. Here, we designed a hierarchical phage-displayed antibody library system to generate an enormous diversity of combinatorial Fab fragments (6×10(17)) and attempted to isolate high-affinity Fabs against the human epidermal growth factor receptor (EGFR). A primary antibody library, designated HuDVFab-8L, comprising 4.5×10(9) human naïve heavy chains and eight unspecified human naïve light chains was selected against the EGFR-Fc protein by biopanning, and four anti-EGFR Fab clones were isolated. Because one of the Fab clones, denoted EG-L2-11, recognized a native EGFR expressed on A431 cells, the heavy chain of the Fab was shuffled with a human naïve light chain repertoire with a diversity of 1.4×10(8) and selected a second time against the EGFR-Fc protein again. One EG-L2-11 variant, denoted EG-19-11, recognized an EGFR epitope that was almost the same as that bound by cetuximab and had a K(D) of approximately 540 pM for soluble EGFR, which is about 7-fold higher than that of the FabC225 derived from cetuximab. This variant was also internalized by A431 cells, likely via receptor-mediated endocytosis, and it efficiently inhibited EGF-mediated tyrosine phosphorylation of the EGFR. These results demonstrate that the use of our hierarchical antibody library system is advantageous in generating fully human antibodies especially with a therapeutic purpose. Copyright © 2010 Elsevier B.V. All rights reserved.

Human pharmacokinetics, biodistribution and dosimetry of the kit of monoclonal antibody IOR EGF/R3 labelled with {sup 99m} Tc

Energy Technology Data Exchange (ETDEWEB)

Torres, L.A.; Ramos, M.; Perera, A.; Hernandez, A.; Iznaga, M.E. N. [Solano, Ivette Alvarez, Jose L. Rodriguez. Centro de InvestigacionesClinicas. 34 no.4501 e/45 y 47 Kohly, Playa, C. Habana (Cuba)

1998-12-31

The aim of this work was to assess the human pharmacokinetics, biodistribution and dosimetry of the {sup 99m} Tc-labeled MAb ior egf/r3. Five patients were included in the biodistribution and dosimetric studies and three in the pharmacokinetic analysis. Multiple blood and urine samples we recollected and sequential anterior and posterior whole-body scintigraphies u pto 24 hr post-injection were performed to all patients . The internal radiation dosimetry was estimated from gamma camera imaging data using the methods developed by the Medical Internal radiation dosimetry (MIRD)committee. Raw data were computed from operations between gamma graphic images and regions of interest (ROI) using the Bio-Dose software and time-activity curves were calculated in order to determine the residence times of the source organs. The Pharmacokinetics and Biodistribution results showed that this compound have a bio exponential plasmatic and blood clearance with a rapid biodistribution phase of 9.1 {+-} 8.4 min and 12.2{+-}4.4 min, respectively, and a slower elimination phase of 6.6 {+-} 1.6 hr and 10.8 {+-} 6.8 hr. respectively. The urinary and hepatobiliary excretion showed 4.7 {+-} 0.4 % and 9.9 {+-} 1.8 % of the total administered dose,eliminated by these ways. Liver was the target organ of this product and had an uptake peak at 1 hr post-injection (61.2%) and a great retention of the MAb(T 1/2 eff = 5.3 hr, T 1/2 Biol. = 45.0 hr). The dosimetric results showed that liver, gallbladder and spleen received the higher absorbed. The effective dose and the effective equivalent dose were 1,2E-01 mSv/MBq and 9,2E-02 mSv/MBq respectively. These results allow to see the i or egf/r3 kit in a safe and controlled way. (Author)

Imaging Potential Evaluation of Fab Derived from the Anti-EGFRvIII Monoclonal Antibody 4G1.

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Jing, Shen; He, Yujia; He, Yanqiong; Wang, Liang; Jia, Jianhua; Shan, Xiaomin; Liu, Shuang; Tang, Min; Peng, Zhiping; Liu, Xujie

2018-05-31

As one of the most crucial epidermal growth factor receptor (EGFR) variants, EGFRvIII can be detected in various tumors but rarely in normal tissues, making it an ideal target for prognosis, diagnosis or immune therapy. The recently developed anti-EGFRvIII monoclonal antibody (mAb), 4G1, has been validated as a promising molecular probe to detect EGFRvIII expression in tumors by single-photon emission computed tomography/computed tomography imaging. To overcome shortcomings associated with the whole antibody, including long-term retention, circulation and enhanced permeability and retention effects, the Fab fragment of 4G1 (Fab-4G1) was generated, labeled with 131 I and evaluated in vitro and in vivo to test its potential application in molecular imaging. Whole mAb 4G1 was first digested by immobilized ficin and then purified through a protein A column to generate the Fab fragment, Fab-4G1. Next, SDS-PAGE, Western blot, indirect fluorescence assay, flow cytometry and enzyme-linked immunosorbent assay were performed to verify molecular weight, specificity and affinity of Fab-4G1. Finally, biodistribution planar gamma imaging was performed by injection of 131 I-labeled Fab-4G1 into xenografted EGFRvIII-overexpressed tumors in nude mice. Parallel studies were also performed with intact 4G1. The molecular weight of Fab was determined to be 35-40 kDa by SDS-PAGE. In vitro tests confirmed both intact 4G1 and Fab-4G1 specifically bound EGFRvIII but not wild-type EGFR, and Fab-4G1 showed decreased affinity. Compared to 131 I-4G1, biodistribution studies showed lower tumor uptake of 131 I-Fab-4G1 at all time points, but much faster elimination in all normal organs. As for planar gamma imaging, 131 I-Fab-4G1 and 31 I-4G1 showed similar imaging effect at 2 h after injection of tracer, while 131 I-Fab-4G1 was eliminated more quickly with time, suggesting radiolabeled Fab-4G1 could be potentially used for imaging of EGFRvIII-positive tumors at early time points. Radiolabeled

Chromogenic in situ hybridization to detect EGFR gene copy number in cell blocks from fine-needle aspirates of non small cell lung carcinomas and lung metastases from colo-rectal cancer

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Terrenato Irene

2010-09-01

Full Text Available Abstract Background Several studies demonstrated that epidermal growth factor receptor (EGFR gene copy number (GCN correlates to the response to tyrosine kinase inhibitors in non small cell lung cancer (NSCLC and to anti-EGFR monoclonal antibodies (MoAbs in metastatic colorectal cancer (CRC. In the presence of lung nodules, cytology is often the only possible diagnostic approach. Chromogenic in situ hybridization (CISH is an alternative technique to fluorescence in situ hybridization (FISH, but its feasibility in detecting EGFR GCN in cell blocks from fine-needle aspiration cytology (FNAC of lung nodules has not yet been established. Methods We evaluated the feasibility of CISH on 33 FNAC from 20 primary NSCLC (5 squamous carcinomas, 8 large cell carcinomas and 7 adenocarcinomas and 13 lung metastases from CRC. Results Of the 33 FNAC analyzed by CISH, 27 (82% presented a balanced increase in EGFR gene and chromosome 7 number: 10 cases (30% showed a low polysomy, 15 (45% a high polysomy and 2 (6% NSCLC were amplified. No significant differences between NSCLC and CRC lung metastases were found in relation to disomic or polysomic status. In addition, no correlation between EGFR GCN and EGFR immunohistochemical overexpression was found. Furthermore, we compared CISH results with those obtained by FISH on the same samples and we found 97% overall agreement between the two assays (k = 0.78, p Conclusions Our study shows that CISH is a valid method to detect EGFR GCN in cell blocks from FNAC of primary NSCLC or metastatic CRC to the lung.

Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting

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Sander A. A. Kooijmans

2016-03-01

Full Text Available Background: Extracellular vesicles (EVs are attractive candidate drug delivery systems due to their ability to functionally transport biological cargo to recipient cells. However, the apparent lack of target cell specificity of exogenously administered EVs limits their therapeutic applicability. In this study, we propose a novel method to equip EVs with targeting properties, in order to improve their interaction with tumour cells. Methods: EV producing cells were transfected with vectors encoding for anti-epidermal growth factor receptor (EGFR nanobodies, which served as targeting ligands for tumour cells, fused to glycosylphosphatidylinositol (GPI anchor signal peptides derived from decay-accelerating factor (DAF. EVs were isolated using ultrafiltration/size-exclusion liquid chromatography and characterized using western blotting, Nanoparticle Tracking Analysis, and electron microscopy. EV–tumour cell interactions were analyzed under static conditions using flow cytometry and under flow conditions using a live-cell fluorescence microscopy-coupled perfusion system. Results: V analysis showed that GPI-linked nanobodies were successfully displayed on EV surfaces and were highly enriched in EVs compared with parent cells. Display of GPI-linked nanobodies on EVs did not alter general EV characteristics (i.e. morphology, size distribution and protein marker expression, but greatly improved EV binding to tumour cells dependent on EGFR density under static conditions. Moreover, nanobody-displaying EVs showed a significantly improved cell association to EGFR-expressing tumour cells under flow conditions. Conclusions: We show that nanobodies can be anchored on the surface of EVs via GPI, which alters their cell targeting behaviour. Furthermore, this study highlights GPI-anchoring as a new tool in the EV toolbox, which may be applied for EV display of a variety of proteins, such as antibodies, reporter proteins and signaling molecules.

Monoclonal antibodies to human chorionic gonadotropin and their application to two-site sandwich radioimmunoassay

International Nuclear Information System (INIS)

Mizuchi, A.; Iio, M.; Miyachi, Y.

1984-01-01

Monoclonal antibodies were prepared against human chorionic gonadotropin (HCG). One monoclonal antibody recognized a conformational determinant expressed only on native HCG molecule and another monoclonal antibody had the specificity for the epitopes located on the β-subunit of HCG. Monoclonal antibodies reacting with different antigenic determinants on the HCG molecule were used to develop a simplified 2-site sandwich radioimmunoassay in which one monoclonal antibody was immobilized and another labeled with 125 iodine. This assay was highly specific for HCG and there was no cross-reactivity with α,β-subunit of HCG, luteinizing hormone and follicle stimulating hormone. (Auth.)

Characterization of a lipopolysaccharide mutant of Leptospira derived by growth in the presence of an anti-lipopolysaccharide monoclonal antibody

NARCIS (Netherlands)

Zapata, Sonia; Trueba, Gabriel; Bulach, Dieter M.; Boucher, David; Adler, Ben; Hartskeerl, Rudy

2010-01-01

A lipopolysaccharide mutant of Leptospira interrogans (LaiMut) was obtained by growth in the presence of an agglutinating monoclonal antibody (mAb) against lipopolysaccharide. Agglutination reactions with anti-lipopolysaccharide mAbs and polyclonal antibodies showed that LaiMut had lost some

Sensitive chain specific radioimmunoassay for human immunoglobulins using monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Sikora, K; Alderson, T St.J.; Ellis, J [Ludwig Institute for Cancer Research, Cambridge (UK)

1983-02-25

A sensitive radioimmunoassay is described for human immunoglobulins. This solid-phase assay uses commercially available monoclonal antibodies and is specific for different Ig chain types. Levels of less than 20 ng/ml Ig are detectable. The assay is suitable for the analysis of human hybridoma supernatants.

Brief report: Afatinib and cetuximab in four patients with EGFR exon 20 insertion positive advanced non-small-cell lung cancer.

Science.gov (United States)

van Veggel, Bianca; de Langen, Adrianus J; Hashemi, Sayed; Monkhorst, Kim; Heideman, Daniëlle A M; Thunnissen, Erik; Smit, Egbert F

2018-04-24

Epidermal growth factor receptor (EGFR) exon 20 insertions comprise 4-9% of EGFR mutated non-small-cell lung cancer (NSCLC). Despite being an oncogenic driver, they are associated with primary resistance to EGFR tyrosine kinase inhibitors (TKIs). We hypothesized that dual EGFR blockade with afatinib, an irreversible EGFR TKI, and cetuximab, a monoclonal antibody against EGFR, could induce tumor responses. Four patients with EGFR exon 20 insertion positive NSCLC were treated with afatinib 40 mg once daily and cetuximab 250-500 mg/m 2 every two weeks. All patients had stage IV adenocarcinoma of the lung harboring an EGFR exon 20 insertion mutation. Previous lines of treatment consisted of platinum doublet chemotherapy (n=4) and EGFR TKI (n=2). Three out of four patients showed a partial response according to RECIST 1.1. Median progression-free survival was 5.4 months (95% confidence interval 0.0 - 14.2 months; range 2.7 - 17.6 months). Toxicity was manageable with appropriate skin management and dose reduction being required in two patients. Dual EGFR blockade with afatinib and cetuximab may induce tumor responses in patients with EGFR exon 20 insertion positive NSCLC. Copyright © 2018. Published by Elsevier Inc.

Porcine humoral immune responses to multiple injections of murine monoclonal antibodies

DEFF Research Database (Denmark)

Lohse, Louise; Nielsen, Jens; Kamstrup, Søren

2005-01-01

In humans and cattle, multiple injections of murine monoclonal antibodies (m-mAbs) induce anti-mouse antibody responses. The objectives of the present. study were to investigate whether a similar response could be seen when pigs were subjected to m-mAb therapy, and to study the kinetics of such a...

Comparative tumour localization properties of radiolabelled monoclonal antibody preparations of defined immunoreactivities

International Nuclear Information System (INIS)

Pimm, M.V.; Baldwin, R.W.

1987-01-01

The immunoreactive fraction of an anti-CEA monoclonal antibody preparation has been progressively decreased by the addition of increasing proportions of impurity in the form of immunologically inert mouse immunoglobulin. Following radioiodination, the immunoreactive fractions of the preparations were determined and their localization in a human tumour xenograft in nude mice was assessed. There was a progressive decline in tumour localization, from tumour to blood ratios of 2:1 with unadulterated antibody to 0.6:1 with preparations only 15% with respect to the initial antibody. These findings demonstrate that the immunoreactive fraction of monoclonal antibody preparations is a major limiting factor in tumour localization and this has implications for experimental and clinical applications of monoclonal antibodies. (orig.)

Anti-influenza M2e antibody

Science.gov (United States)

Bradbury, Andrew M [Santa Fe, NM

2011-12-20

Humanized recombinant and monoclonal antibodies specific for the ectodomain of the influenza virus M2 ion channel protein are disclosed. The antibodies of the invention have anti-viral activity and may be useful as anti-viral therapeutics and/or prophylactic/vaccine agents for inhibiting influenza virus replication and for treating individuals infected with influenza.

Generation of chimeric bispecific G250/anti-CD3 monoclonal antibody, a tool to combat renal cell carcinoma

NARCIS (Netherlands)

Luiten, R. M.; Coney, L. R.; Fleuren, G. J.; Warnaar, S. O.; Litvinov, S. V.

1996-01-01

The monoclonal antibody (MAb) G250 binds to a tumour-associated antigen, expressed in renal cell carcinoma (RCC), which has been demonstrated to be a suitable target for antibody-mediated immunotherapy. A bispecific antibody having both G250 and anti-CD3 specificity can cross-link G250

Treatment of metastatic colorectal cancer: focus on panitumumab

International Nuclear Information System (INIS)

Tay, Rebecca Y; Wong, Rachel; Hawkes, Eliza A

2015-01-01

Targeted agents are an important therapeutic option in the treatment of metastatic colorectal cancer (mCRC). Panitumumab is a recombinant, fully humanized, immunoglobulin G2 monoclonal antibody that targets the epidermal growth factor receptor (EGFR) with efficacy in mCRC as monotherapy and in combination with chemotherapy. Kirsten rat sarcoma (KRAS) mutation status has emerged as an important biomarker to predict response to anti-EGFR therapy. Optimal timing for panitumumab use in the mCRC treatment algorithm has not been established. This review discusses the mechanism of action, predictive biomarkers, and role of panitumumab in the treatment of mCRC

Nicotine enhances proliferation, migration, and radioresistance of human malignant glioma cells through EGFR activation

International Nuclear Information System (INIS)

Khalil, A.A.; Jameson, M.J.; Broaddus, W.C.; Lin, P.S.; Chung, T.D.

2013-01-01

It has been suggested that continued tobacco use during radiation therapy contributes to maintenance of neoplastic growth despite treatment with radiation. Nicotine is a cigarette component that is an established risk factor for many diseases, neoplastic and otherwise. The hypothesis of this work is that nicotine promotes the proliferation, migration, and radioresistance of human malignant glioma cells. The effect of nicotine on cellular proliferation, migration, signaling, and radiation sensitivity were evaluated for malignant glioma U87 and GBM12 cells by use of the AlamarBlue, scratch healing, and clonogenic survival assays. Signal transduction was assessed by immunoblotting for activated EGFR, extracellular regulated kinase (ERK), and AKT. At concentrations comparable with those found in chronic smokers, nicotine induced malignant glioma cell migration, growth, colony formation, and radioresistance. Nicotine increased phosphorylation of EGFR tyr992 , AKT ser473 , and ERK. These molecular effects were reduced by pharmacological inhibitors of EGFR, PI3K, and MEK. It was therefore concluded that nicotine stimulates the malignant behavior of glioma cells in vitro by activation of the EGFR and downstream AKT and ERK pathways. (author)

Individualized therapies in colorectal cancer: KRAS as a marker for response to EGFR-targeted therapy

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Li Kuiyuan

2009-04-01

Full Text Available Abstract Individualized therapies that are tailored to a patient's genetic composition will be of tremendous value for treatment of cancer. Recently, Kirsten ras (KRAS status has emerged as a predictor of response to epidermal growth factor receptor (EGFR targeted therapies. In this article, we will discuss targeted therapies for colorectal cancers (CRC based on EGFR signaling pathway and review published data about the potential usefulness of KRAS as a biological marker for response to these therapies. Results from relevant studies published since 2005 and unpublished results presented at national meetings were retrieved and summarized. These studies reflected response (or lack of response to EGFR-targeted therapies in patients with metastatic CRC as a function of KRAS status. It has become clear that patients with colorectal cancer whose tumor has an activating mutation in KRAS do not respond to monoclonal antibody therapies targeting EGFR. It should now become a standard practice that any patients being considered for EGFR targeted therapies have their tumors tested for KRAS status and only those with wild-type KRAS being offered such therapies.

Identification of the zinc finger 216 (ZNF216) in human carcinoma cells: a potential regulator of EGFR activity

Science.gov (United States)

Mincione, Gabriella; Di Marcantonio, Maria Carmela; Tarantelli, Chiara; Savino, Luca; Ponti, Donatella; Marchisio, Marco; Lanuti, Paola; Sancilio, Silvia; Calogero, Antonella; Di Pietro, Roberta; Muraro, Raffaella

2016-01-01

Epidermal Growth Factor Receptor (EGFR), a member of the ErbB family of receptor tyrosine kinase (RTK) proteins, is aberrantly expressed or deregulated in tumors and plays pivotal roles in cancer onset and metastatic progression. ZNF216 gene has been identified as one of Immediate Early Genes (IEGs) induced by RTKs. Overexpression of ZNF216 protein sensitizes 293 cell line to TNF-α induced apoptosis. However, ZNF216 overexpression has been reported in medulloblastomas and metastatic nasopharyngeal carcinomas. Thus, the role of this protein is still not clearly understood. In this study, the inverse correlation between EGFR and ZNF216 expression was confirmed in various human cancer cell lines differently expressing EGFR. EGF treatment of NIH3T3 cells overexpressing both EGFR and ZNF216 (NIH3T3-EGFR/ZNF216), induced a long lasting activation of EGFR in the cytosolic fraction and an accumulation of phosphorylated EGFR (pEGFR) more in the nuclear than in the cytosolic fraction compared to NIH3T3-EGFR cells. Moreover, EGF was able to stimulate an increased expression of ZNF216 in the cytosolic compartment and its nuclear translocation in a time-dependent manner in NIH3T3-EGFR/ZNF216. A similar trend was observed in A431 cells endogenously expressing the EGFR and transfected with Znf216. The increased levels of pEGFR and ZNF216 in the nuclear fraction of NIH3T3-EGFR/ZNF216 cells were paralleled by increased levels of phospho-MAPK and phospho-Akt. Surprisingly, EGF treatment of NIH3T3-EGFR/ZNF216 cells induced a significant increase of apoptosis thus indicating that ZNF216 could sensitize cells to EGF-induced apoptosis and suggesting that it may be involved in the regulation and effects of EGFR signaling. PMID:27732953

Anti-EGFR Antibody Efficiently and Specifically Inhibits Human TSC2−/− Smooth Muscle Cell Proliferation. Possible Treatment Options for TSC and LAM

Science.gov (United States)

Lesma, Elena; Grande, Vera; Ancona, Silvia; Carelli, Stephana; Di Giulio, Anna Maria; Gorio, Alfredo

2008-01-01

Background Tuberous sclerosis complex (TSC), a tumor syndrome caused by mutations in TSC1 or TSC2 genes, is characterized by the development of hamartomas. We previously isolated, from an angiomyolipoma of a TSC2 patient, a homogenous population of smooth muscle-like cells (TSC2−/− ASM cells) that have a mutation in the TSC2 gene as well as TSC2 loss of heterozygosity (LOH) and consequently, do not produce the TSC2 gene product, tuberin. TSC2−/− ASM cell proliferation is EGF-dependent. Methods and Findings Effects of EGF on proliferation of TSC2−/− ASM cells and TSC2−/− ASM cells transfected with TSC2 gene were determined. In contrast to TSC2−/− ASM cells, growth of TSC2-transfected cells was not dependent on EGF. Moreover, phosphorylation of Akt, PTEN, Erk and S6 was significantly decreased. EGF is a proliferative factor of TSC2−/− ASM cells. Exposure of TSC2−/− ASM cells to anti-EGFR antibodies significantly inhibited their proliferation, reverted reactivity to HMB45 antibody, a marker of TSC2−/− cell phenotype, and inhibited constitutive phosphorylation of S6 and ERK. Exposure of TSC2−/− ASM cells to rapamycin reduced the proliferation rate, but only when added at plating time. Although rapamycin efficiently inhibited S6 phosphorylation, it was less efficient than anti-EGFR antibody in reverting HMB45 reactivity and blocking ERK phosphorylation. In TSC2−/− ASM cells specific PI3K inhibitors (e.g. LY294002, wortmannin) and Akt1 siRNA had little effect on S6 and ERK phosphorylation. Following TSC2-gene transfection, Akt inhibitor sensitivity was observed. Conclusion Our results show that an EGF independent pathway is more important than that involving IGF-I for growth and survival of TSC−/− ASM cells, and such EGF-dependency is the result of the lack of tuberin. PMID:18958173

Role of EGFR transactivation in preventing apoptosis in Pseudomonas aeruginosa-infected human corneal epithelial cells.

Science.gov (United States)

Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X

2004-08-01

To determine the role of epidermal growth factor (EGF) receptor (EGFR)-mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa-infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase-mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis. Bacterial infection of HCECs induces

Role of EGFR Transactivation in Preventing Apoptosis in Pseudomonas aeruginosa–Infected Human Corneal Epithelial Cells

Science.gov (United States)

Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X.

2009-01-01

PURPOSE To determine the role of epidermal growth factor (EGF) receptor (EGFR)–mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). METHODS Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. RESULTS P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa–infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase–mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis

Monoclonal antibodies and cancer

International Nuclear Information System (INIS)

Haisma, H.J.

1987-01-01

The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111 In, 67 Ga and 131 I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

Kinetics of EGFR expression during fractionated irradiation varies between different human squamous cell carcinoma lines in nude mice

International Nuclear Information System (INIS)

Eicheler, Wolfgang; Krause, Mechthild; Hessel, Franziska; Zips, Daniel; Baumann, Michael

2005-01-01

Background and purpose: Preclinical and clinical data indicate that high pretherapeutic EGFR expression is associated with poor local tumour control, possibly caused by a high repopulation rate of clonogenic cells during radiotherapy in these tumours. Previous data reported from our laboratory showed a correlation between EGFR expression and acceleration of repopulation in poorly differentiated FaDu human squamous cell carcinoma (SCC) during fractionated irradiation. To test whether this is a general phenomenon, two further SCC were investigated in the present study. Patients and methods: GL and UT-SCC-14, two moderately well differentiated and keratinising hSCC, were grown as xenografts in nude mice. Functional data on the repopulation kinetics during fractionated irradiation for these tumour models have been previously determined. The expression of EGFR during fractionation was analysed by immunohistochemistry. Endpoints were the membrane-staining score and the proportion of EGFR-positive cells (EGFR labelling index). Results: Different kinetics of EGFR expression during fractionated RT were found. In UT-SCC-14, EGFR staining score and labelling index increased significantly during radiotherapy. In GL SCC, the EGFR expression was unchanged. Both tumours are characterized by a small but significant repopulation rate during radiotherapy. Conclusions: The expression of EGFR may change significantly during fractionated irradiation. No clear correlation between EGFR expression and the repopulation kinetics of clonogenic tumour cells during fractionated irradiation was found. The changes in EGFR expression during irradiation warrant further investigation on their prognostic implications and on their importance for therapeutic interventions

Therapeutic effect of anti-feline TNF-alpha monoclonal antibody for feline infectious peritonitis.

Science.gov (United States)

Doki, Tomoyoshi; Takano, Tomomi; Kawagoe, Kohei; Kito, Akihiko; Hohdatsu, Tsutomu

2016-02-01

Feline infectious peritonitis virus (FIPV) replication in macrophages/monocytes induced tumor necrosis factor (TNF)-alpha production, and that the TNF-alpha produced was involved in aggravating the pathology of FIP. We previously reported the preparation of a feline TNF-alpha (fTNF-alpha)-neutralizing mouse monoclonal antibody (anti-fTNF-alpha mAb). This anti-fTNF-alpha mAb 2-4 was confirmed to inhibit the following fTNF-alpha-induced conditions in vitro. In the present study, we investigated whether mAb 2-4 improved the FIP symptoms and survival rate of experimentally FIPV-inoculated SPF cats. Progression to FIP was prevented in 2 out of 3 cats treated with mAb 2-4, whereas all 3 cats developed FIP in the placebo control group. Plasma alpha1-glycoprotein and vascular endothelial growth factor levels were improved by the administration of mAb 2-4, and the peripheral lymphocyte count also recovered. These results strongly suggested that the anti-fTNF-alpha antibody is effective for the treatment of FIP. Copyright © 2015 Elsevier Ltd. All rights reserved.

Vorinostat and metformin sensitize EGFR-TKI resistant NSCLC cells via BIM-dependent apoptosis induction.

Science.gov (United States)

Chen, Hengyi; Wang, Yubo; Lin, Caiyu; Lu, Conghua; Han, Rui; Jiao, Lin; Li, Li; He, Yong

2017-11-07

There is a close relationship between low expression of BIM and resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI). Vorinostat is a pan-histone deacetylase inhibitor (HDACi) that augments BIM expression in various types of tumor cells, however, this effect is attenuated by the high expression of anti-apoptotic proteins in EGFR-TKI resistant non-small cell lung cancer (NSCLC) cells. Vorinostat in combination with metformin - a compound that can inhibit anti-apoptotic proteins expression, might cooperate to activate apoptotic signaling and overcome EGFR-TKI resistance. This study aimed to investigate the cooperative effect and evaluate possible molecular mechanisms. The results showed that vorinostat combined with gefitinib augmented BIM expression and increased the sensitivity of EGFR-TKI resistant NSCLC cells to gefitinib, adding metformin simultaneously could obviously inhibit the expression of anti-apoptotic proteins, and further increased expression levels of BIM and BAX, and as a result, further improved the sensitivity of gefitinib both on the NSCLC cells with intrinsic and acquired resistance to EGFR-TKI. In addition, autophagy induced by gefitinib and vorinostat could be significantly suppressed by metformin, which might also contribute to enhance apoptosis and improve sensitivity of gefitinib. These results suggested that the combination of vorinostat and metformin might represent a novel strategy to overcome EGFR-TKI resistance associated with BIM-dependent apoptosis in larger heterogeneous populations.

IgM but not IgG monoclonal anti-Nocardia brasiliensis antibodies confer protection against experimental actinomycetoma in BALB/c mice.

Science.gov (United States)

Gonzalez-Suarez, Maria L; Salinas-Carmona, Mario C; Pérez-Rivera, Isabel

2009-10-01

Nocardia brasiliensis is a facultative intracellular microorganism that produces a human chronic infection known as actinomycetoma. Human and mouse anti-N. brasiliensis antibody response identify P24, P26 and P61 immunodominant antigens. In this work, we generated immunoglobulin M (IgM) and IgG monoclonal antibodies (mAbs) specific to immunodominant P61 antigen. The monoclonal IgM (NbM1) and IgG2a (NbG1) antibodies were assessed for their in vitro bactericidal activity, in vivo protective effect and ability to block catalase activity. These mAbs specifically recognized P61, but they did not inhibit its enzyme activity. The in vitro bactericidal effect of NbG1 was higher than the killing ability of the IgM mAb. In vivo experiments with a murine model of experimental infection with N. brasiliensis injected into rear footpads was used to test the effect of NbM1 and NbG1. The negative untreated group developed a chronic actinomycetoma within 4 weeks. IgM mAbs conferred protection to BALB/c mice infected with N. brasiliensis. IgG mAb lacked this protective effect. IgM mAb showed a dose-response correlation between antibody concentration and lesion size. These results demonstrate that humoral immune response mediated by antigen-specific IgM antibody protects against an intracellular bacterial infection.

Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

Science.gov (United States)

Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

1992-09-01

The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

Different classes of EGFR inhibitors may have different potential to improve local tumour control after fractionated irradiation: a study on C225 in FaDu hSCC

International Nuclear Information System (INIS)

Krause, M.; Schuetze, C.; Petersen, C.; Pimentel, N.; Hessel, F.; Harstrick, A.; Baumann, M.

2005-01-01

Background and purpose: Previous experiments reported from this laboratory have shown that simultaneous application of the selective epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitor BIBX1382BS during fractionated irradiation significantly prolonged growth delay of FaDu human squamous cell carcinoma but did not improve local tumour control. The present study investigates the effect of the EGFR monoclonal antibody (mAb) C225 on local tumour control of FaDu tumours after combined treatment with single dose and fractionated irradiation to address whether different classes of EGFR inhibitors have different potential to improve the outcome of radiotherapy in the same tumour model. Material and methods: In unirradiated tumours, C225 was given either once or 4 times i.p. to the nude mice. Irradiation experiments were performed with graded single doses under clamp hypoxic conditions or with 30 fractions in 6 weeks with graded total doses under ambient blood flow. C225 was given 6 h before or 6 h before and 2, 5 and 7 days after single dose irradiation. During fractionated irradiation C225 was given once per week. Experimental endpoints were tumour growth delay and local tumour control 120 after end of irradiation. Results: C225 treatment resulted in prolongation of tumour growth delay after drug treatment alone as well as after single dose and fractionated irradiation. TCD 50 values were reduced from 56.3 Gy [95% CI 50; 62 Gy] after single dose irradiation alone to 46.0 Gy [41;51] (enhancement ratio [ER]=1.22, P 50 ) was 73.0 Gy [64; 82] in control tumours and 63.1 Gy [57; 69] after simultaneous C225 treatment, corresponding to an ER of 1.2 (P=0.01). Conclusion: Treatment of FaDu hSCC with the anti-EGFR mAb C225 resulted in a significant prolongation of tumour growth delay after single dose and fractionated irradiation. In contrast to previous results on the EGFR-TK inhibitor BIBX1382BS, this prolongation of growth delay translated into a slight but

C-kit-targeted imaging of gastrointestinal stromal tumor using radiolabeled anti-c-kit monoclonal antibody in a mouse tumor model

International Nuclear Information System (INIS)

Sogawa, Chizuru; Tsuji, Atsushi B.; Sudo, Hitomi; Sugyo, Aya; Yoshida, Chisato; Odaka, Kenichi; Uehara, Tomoya; Arano, Yasushi; Koizumi, Mitsuru; Saga, Tsuneo

2010-01-01

Introduction: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor arising from the gastrointestinal tract and highly expresses mutated c-kit. We aimed to develop a specific and sensitive method for detecting GISTs using radiolabeled anti-c-kit monoclonal antibody. Methods: A mutated c-kit-expressing cell clone was established by transfecting an expressing vector of mutated c-kit gene into HEK293 human embryonic kidney cells. The tumors were developed by inoculating c-kit-expressing cells into nude mice. 125 I- and 111 In-labeled anti-c-kit antibodies (12A8 and 41A11) were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays, and in vivo by biodistribution and imaging studies in tumor-bearing mice. Results: Both 125 I- and 111 In-labeled antibodies showed specific binding with c-kit-expressing cells with high affinity (dissociation constants = 2.2-7.1x10 9 M -1 ). Internalization assay showed that 125 I-labeled antibodies were rapidly internalized and dehalogenated, with the release of 125 I from the cells, resulting in reduction of cell-associated radioactivity with time. In contrast, 111 In-labeled antibody was internalized but did not result in the reduced radioactivity associated with tumor cells. Reflecting this phenomenon, the in vivo tumor uptake of 125 I-labeled antibody was low on Day 1, further decreasing with time, while tumor uptake of 111 In-labeled antibody was high on Day 1, further increasing with time. The xenografted tumor was clearly visualized by scintigraphy after injection of 111 In-labeled antibody. Conclusion: The anti-c-kit monoclonal antibody labeled with a metal radionuclide would be promising for c-kit-targeted imaging of GISTs.

Radioimmunoimaging of human colon carcinoma grafted into nudemice using 131I-labeled monoclonal anticea antibody and its F(ab')2 fragments

International Nuclear Information System (INIS)

Liu Guangda

1988-01-01

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

Generation and Characterization of Anti-CD34 Monoclonal Antibodies that React with Hematopoietic Stem Cells

Science.gov (United States)

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Movassaghpour, Aliakbar; Abdolalizadeh, Jalal

2014-01-01

CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells (UCB) in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies (mAb) can be a useful tool for isolation and purification of human hematopoietic stem cells (HSCs). PMID:24611141

Characterization of anti-P monoclonal antibodies directed against the ribosomal protein-RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice.

Science.gov (United States)

Sato, H; Onozuka, M; Hagiya, A; Hoshino, S; Narita, I; Uchiumi, T

2015-02-01

Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus. © 2014 British Society for Immunology.

Characterization of anti-P monoclonal antibodies directed against the ribosomal protein–RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice

Science.gov (United States)

Sato, H; Onozuka, M; Hagiya, A; Hoshino, S; Narita, I; Uchiumi, T

2015-01-01

Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus. PMID:25255895

Phase I/II clinical and pharmacokinetic study evaluating a fully human monoclonal antibody against EGFr (HuMax-EGFr) in patients with advanced squamous cell carcinoma of the head and neck

DEFF Research Database (Denmark)

Bastholt, Lars; Specht, Lena; Jensen, Kenneth

2007-01-01

PURPOSE: To assess safety, tolerability, pharmacokinetics and clinical activity of HuMax-EGFr in patients with SCCHN. PATIENTS AND METHODS: Twenty-eight patients with SCCHN were enrolled. The study comprised a single-dose escalation part for assessment of safety issues followed by a repeat dose e...

Using anti-VEGF monoclonal antibody and magnetic nanoparticles

International Nuclear Information System (INIS)

Chen Jing; Wuhua; Hang Deyan; Xie Changsheng

2004-01-01

Objective: To study the biodistribution of 131 I-anti-vascular endothelial growth factor (VEGF) monoclonal antibody (Sc-7269)-dextran magnetic nanoparticles (DMN) in nude mice bearing human liver cancer where an external magnetic field was focused on, and to evaluate its therapeutic effects and safety. Methods: Eighteen nude mice bearing human liver cancer where an external magnetic field was focused on, were used for the bio-distribution study after intratumoral injection (n=9) or intravenous injection (n=9) of 131 I-Sc-7269-DMN. Another 25 tumor-bearing nude mice were divided into five groups, four groups of them were treated with 74 MBq/ml 131 I-Sc-7269-DMN, 131 I-Sc-7269, 131 I-DMN and 131 I by a single intratumoral injection, respectively. And an external magnetic field was bound to the tumor of the nude mice that were injected 131 I-Sc-7269-DMN or 131 I-DMN. For control study, the remaining one group was injected with physiological saline. Tumor growth delay (TGD) and tumor inhibition rate were observed as antitumor effects. Peripheral white cell counts and the loss of body weight were tested as indicators of systemic toxicity. Results: The retention percentages of radioactivity (%ID/g) in tumors after intratumoral injection were 104.06, 101.58 and 100.96%ID/g at 4, 24 and 48 h, respectively, while in the case of intravenous injection, the %ID/g values were lower (85.33, 89.67 and 90.00%ID/g, respectively, P 131 I-Sc-7269-DMN [ (13.3 ± 3.3) d] was the longest, and tumor inhibition rate (89.0%)was the highest compared with that in other groups (P 131 I-Sc-7269-DMN-treated mice as monitored by the decrease in peripheral white cell counts and the loss of body weight. Conclusions: The radioimmunotherapy with intratumoral injection of 131 I-Sc-7269-DMN may be safe and efficient for the treatment of liver cancer. Furthermore, the radioimmunotherapy using DMN as a carrier system may be a highly potential approach in targeted treatment of other kinds of tumors

Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

Science.gov (United States)

Barriuso, Begoña; Antolín, Pilar; Arias, F Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

2016-06-10

Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

EGFR gene overexpression retained in an invasive xenograft model by solid orthotopic transplantation of human glioblastoma multiforme into nude mice.

Science.gov (United States)

Yi, Diao; Hua, Tian Xin; Lin, Huang Yan

2011-03-01

Orthotopic xenograft animal model from human glioblastoma multiforme (GBM) cell lines often do not recapitulate an extremely important aspect of invasive growth and epidermal growth factor receptor (EGFR) gene overexpression of human GBM. We developed an orthotopic xenograft model by solid transplantation of human GBM into the brain of nude mouse. The orthotopic xenografts sharing the same histopathological features with their original human GBMs were highly invasive and retained the overexpression of EGFR gene. The murine orthotopic GBM models constitute a valuable in vivo system for preclinical studies to test novel therapies for human GBM.

A sandwich immunoassay for human prolyl 4-hydroxylase using monoclonal antibody

International Nuclear Information System (INIS)

Yoshida, Shinichi

1986-01-01

Monoclonal antibody was used in a sandwich enzyme immunoassay and in a radioimmunoassay for human serum immunoreactive prolyl 4-hydroxylase. The enzyme immunoassay utilized a monoclonal antibody as a solid phase and horseradish peroxidase-labeled rabbit antibody to human prolyl 4-hydroxylase as a conjugate. Sensitivity was 0.1 ng of enzyme per tube. With a conjugate purified by an enzyme-bound affinity column, sensitivity was increased to 0.01 ng per tube, and linearity was obtained between 0.01 to 30 ng per tube. The radioimmunoassay used a 125 I-labeled rabbit antibody (IgG) as the conjugate. Sensitivity of this technique was 0.4 ng of enzyme per tube. (Auth.)

Current status of cancer immunodetection with radiolabeled human monoclonal antibodies.

Science.gov (United States)

De Jager, R; Abdel-Nabi, H; Serafini, A; Pecking, A; Klein, J L; Hanna, M G

1993-04-01

The use of radiolabeled murine monoclonal antibodies (MoAbs) for cancer immunodetection has been limited by the development of human antimouse antibodies (HAMA). Human monoclonal antibodies do not elicit a significant human antihuman (HAHA) response. The generation and production of human monoclonal antibodies met with technical difficulties that resulted in delaying their clinical testing. Human monoclonal antibodies of all isotypes have been obtained. Most were immunoglobulin (Ig) M directed against intracellular antigens. Two antibodies, 16.88 (IgM) and 88BV59 (IgG3k), recognize different epitopes on a tumor-associated antigen, CTA 16.88, homologous to cytokeratins 8, 18, and 19. CTA 16.88 is expressed by most epithelial-derived tumors including carcinomas of the colon, pancreas, breast, ovary, and lung. The in vivo targeting by these antibodies is related to their localization in nonnecrotic areas of tumors. Repeated administration of 16.88 over 5 weeks to a cumulative dose of 1,000 mg did not elicit a HAHA response. Two of 53 patients developed a low titer of HAHA 1 to 3 months after a single administration of 88BV59. Planar imaging of colorectal cancer with Iodine-131 (131I)-16.88 was positive in two studies in 9 of 12 and 16 of 20 patients preselected by immunohistochemistry. Tumors less than 2 cm in diameter are usually not detected. The lack of immunogenicity and long tumor residence time (average = 17 days) makes 16.88 a good candidate for therapy. Radioimmunlymphoscintigraphy with indium-111 (111In)-LiLo-16.88 administered by an intramammary route was used in the presurgical staging of primary breast cancer. The negative predictive value of lymph node metastases for tumors less than 3 cm was 90.5%. Planar and single photon emission computed tomography imaging of colorectal carcinoma with technetium-99m (99mTc) 88BV59 was compared with computed tomography (CT) scan in 36 surgical patients. The antibody scan was more sensitive than the CT scan in detecting

Expression of blood group I and i active carbohydrate sequences on cultured human and animal cell lines assessed by radioimmunoassays with monoclonal cold agglutinins

International Nuclear Information System (INIS)

Childs, R.A.; Kapadia, A.; Feizi, T.

1980-01-01

Human monoclonal anti-I and anti-i, reactive with known carbohydrate sequences, have been used as reagents to quantitate (by radioimmunoassay) and visualize (by immunofluorescence) the expression of the various blood group I and i antigenic determinants in a variety of cultured cell lines commonly used in laboratory investigations. It has been shown that the antigens they recognize are widely distributed on the surface of human and animal cell lines, expressed in varying amounts in different cell lines and on individual cells within a given cell line. In two cell lines, a transformation-associated increase in the expression of I antigen was observed. Because of their precise specificity for defined carbohydrate chain domains, these autoantibodies have become valuable reagents in biological chemistry. (orig.) [de

Acute neurological worsening after Rituximab treatment in patients with anti-MAG neuropathy.

Science.gov (United States)

Sala, Emilie; Robert-Varvat, Florence; Paul, Stéphane; Camdessanché, Jean-Philippe; Antoine, Jean-Christophe

2014-10-15

Patients with peripheral neuropathy and anti-MAG monoclonal IgM may respond to Rituximab, a humanized monoclonal anti-CD20 antibody. We report on three patients with peripheral neuropathy and anti-MAG monoclonal IgM who deteriorated under Rituximab and reviewed seven previously published cases. Worsening was acute and severe, and occurred during the treatment period. All the patients improved after deterioration but at final evaluation only one was improved comparatively to baseline, five were worsened and four were stabilized. Deterioration was not clearly associated with an increase of the anti-MAG antibody titer. Two patients received Rituximab prior or after the course which induced worsening without adverse reaction. Although rare, acute worsening of the neuropathy can occur after Rituximab. The deterioration is however reversible within some weeks to several months. Copyright © 2014 Elsevier B.V. All rights reserved.

Oxidized LDL-induced angiogenesis involves sphingosine 1-phosphate: prevention by anti-S1P antibody.

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Camaré, Caroline; Trayssac, Magali; Garmy-Susini, Barbara; Mucher, Elodie; Sabbadini, Roger; Salvayre, Robert; Negre-Salvayre, Anne

2015-01-01

Neovascularization occurring in atherosclerotic lesions may promote plaque expansion, intraplaque haemorrhage and rupture. Oxidized LDL (oxLDL) are atherogenic, but their angiogenic effect is controversial; both angiogenic and anti-angiogenic effects have been reported. The angiogenic mechanism of oxLDL is partly understood, but the role of the angiogenic sphingolipid, sphingosine 1-phosphate (S1P), in this process is not known. Thus, we investigated whether S1P is involved in the oxLDL-induced angiogenesis and whether an anti-S1P monoclonal antibody can prevent this effect. Angiogenesis was assessed by capillary tube formation by human microvascular endothelial cells (HMEC-1) cultured on Matrigel and in vivo by the Matrigel plug assay in C57BL/6 mice. Human oxLDL exhibited a biphasic angiogenic effect on HMEC-1; low concentrations were angiogenic, higher concentrations were cytotoxic. The angiogenic response to oxLDL was blocked by the sphingosine kinase (SPHK) inhibitor, dimethylsphingosine, by SPHK1-siRNA and by an anti-S1P monoclonal antibody. Moreover, inhibition of oxLDL uptake and subsequent redox signalling by anti-CD36 and anti-LOX-1 receptor antibodies and by N-acetylcysteine, respectively, blocked SPHK1 activation and tube formation. In vivo, in the Matrigel plug assay, low concentrations of human oxLDL or murine oxVLDL also triggered angiogenesis, which was prevented by i.p. injection of the anti-S1P antibody. These data highlight the role of S1P in angiogenesis induced by oxLDL both in HMEC-1 cultured on Matrigel and in vivo in the Matrigel plug model in mice, and demonstrate that the anti-S1P antibody effectively blocks the angiogenic effect of oxLDL. © 2014 The British Pharmacological Society.

Therapeutic value of EGFR inhibition in CRC and NSCLC: 15 years of clinical evidence.

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Troiani, Teresa; Napolitano, Stefania; Della Corte, Carminia Maria; Martini, Giulia; Martinelli, Erika; Morgillo, Floriana; Ciardiello, Fortunato

2016-01-01

Epidermal growth factor receptor (EGFR) plays a key role in tumour evolution, proliferation and immune evasion, and is one of the most important targets for biological therapy, especially for non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC). In the past 15 years, several EGFR antagonists have been approved for the treatment of NSCLC and metastatic CRC (mCRC). To optimise the use of anti-EGFR agents in clinical practice, various clinical and molecular biomarkers have been investigated, thus moving their indication from unselected to selected populations. Nowadays, anti-EGFR drugs represent a gold-standard therapy for metastatic NSCLC harbouring EGFR activating mutation and for RAS wild-type mCRC. Their clinical efficacy is limited by the presence of intrinsic resistance or the onset of acquired resistance. In this review, we provide an overview of the antitumour activity of EGFR inhibitors in NSCLC and CRC and of mechanisms of resistance, focusing on the development of a personalised approach through 15 years of preclinical and clinical research.

Epidermal growth factor receptor inhibition by anti-CD147 therapy in cutaneous squamous cell carcinoma.

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Frederick, John W; Sweeny, Larissa; Hartman, Yolanda; Zhou, Tong; Rosenthal, Eben L

2016-02-01

Advanced cutaneous squamous cell carcinoma (SCC) is an uncommon and aggressive malignancy. As a result, there is limited understanding of its biology and pathogenesis. CD147 and epidermal growth factor receptor (EGFR) have been identified as oncologically important targets, but their relationship remains undefined in cutaneous SCC. Multiple cutaneous SCC cell lines (Colo-16, SRB-1, and SRB-12), were treated in vitro with a range of chimeric anti-CD147 monoclonal antibody (mAb) (0, 50, 100, and 200 µg/mL) or transfected with a small interfering RNA against CD147 (SiCD147). Cell proliferation, migration (scratch wound healing assay), and protein expression was then assessed. In vivo, Colo-16 flank xenografts were treated anti-CD147 mAb (150 µg i.p. triweekly). After treatment with anti-CD147 (200 µg/mL), there was a significant decrease in proliferation for all cell lines relative to controls (p CD147 (200 µg/mL) resulted in decreased cell migration for all cell lines, with an average of 43% reduction in closure compared to controls (p CD147 antibody therapy and siRNA mediated reduction in CD147 expression were both found to decrease protein expression of EGFR, which correlated with a reduction in downstream total and phosphorylated protein kinase B (pAKT). Tumor growth in vivo was reduced for both the anti-CD147 treatment group and the SiCD147 group relative to controls. Inhibition and downregulation of CD147 in cutaneous SCC resulted in suppression of the malignant phenotype in vitro and in vivo, which may be mediated in part by an alteration in EGFR expression. As a result, CD147 may serve as a potential therapeutic target for advanced cutaneous SCC. © 2014 Wiley Periodicals, Inc.

Frequent EGFR Positivity and Overexpression in High-Grade Areas of Human MPNSTs

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Séverine Tabone-Eglinger

2008-01-01

Full Text Available Malignant peripheral nerve sheath tumours (MPNSTs are highly malignant and resistant. Transformation might implicate up regulation of epidermal growth factor receptor (EGFR. Fifty-two MPNST samples were studied for EGFR, Ki-67, p53, and survivin expression by immunohistochemistry and for EGFR amplification by in situ hybridization. Results were correlated with clinical data. EGFR RNA was also quantified by RT-PCR in 20 other MPNSTs and 14 dermal neurofibromas. Half of the patients had a neurofibromatosis type 1 (NF1. EGFR expression, detected in 86% of MPNSTs, was more frequent in NF1 specimens and closely associated with high-grade and p53-positive areas. MPNSTs expressed more EGFR transcripts than neurofibromas. No amplification of EGFR locus was observed. NF1 status was the only prognostic factor in multivariate analysis, with median survivals of 18 and 43 months for patients with or without NF1. Finally, EGFR might become a new target for MPNSTs treatment, especially in NF1-associated MPNSTs.

Sequences of 12 monoclonal anti-dinitrophenyl spin-label antibodies for NMR studies

International Nuclear Information System (INIS)

Leahy, D.J.; Rule, G.S.; Whittaker, M.M.; McConnell, H.M.

1988-01-01

Eleven monoclonal antibodies specific for a spin-labeled dinitrophenyl hapten (DNP-SL) have been produces for use in NMR studies. They have been named AN01 and ANO3-AN12. The stability constants for the association of these antibodies with DNP-SL and related haptens were measured by fluorescence quenching. cDNA clones coding for the heavy and light chains of each antibody and of an additional anti-DNP-SL monoclonal antibody, ANO2, have been isolated. The nucleic acid sequence of the 5' end of each clone has been determined, and the amino acid sequence of the variable regions of each antibody has been deduced from the cDNA sequence. The sequences are relatively heterogeneous, but both the heavy and the light chains of ANO1 and ANO3 are derived from the same variable-region gene families as those of the ANO2 antibody. ANO7 has a heavy chain that is related to that of ANO2, and ANO9 has a related light chain. ANO5 and ANO6 are unrelated to ANO2 but share virtually identical heavy and light chains. Preliminary NMR difference spectra comparing related antibodies show that sequence-specific assignment of resonances is possible. Such spectra also provide a measure of structural relatedness

Nimotuzumab enhances temozolomide-induced growth suppression of glioma cells expressing mutant EGFR in vivo

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Nitta, Yusuke; Shimizu, Saki; Shishido-Hara, Yukiko; Suzuki, Kaori; Shiokawa, Yoshiaki; Nagane, Motoo

2016-01-01

A mutant form of epidermal growth factor receptor (EGFR), EGFRvIII, is common in glioblastoma (GBM) and confers enhanced tumorigenic activity and drug resistance. Nimotuzumab, an anti-EGFR antibody, has shown preclinical and clinical activity to GBM, but its specific activity against EGFRvIII has not been fully investigated. Human glioma U87MG or LNZ308 cells overexpressing either wild-type (wt) EGFR or EGFRvIII were treated with nimotuzumab, temozolomide, or both. Expression and phosphorylation status of molecules were determined by Western blot analysis. Methylation status of promoter region of O 6 -methylguanine-DNA methyltransferase (MGMT) was detected by methylation-specific PCR. Antitumor activity was tested using nude mice bearing either subcutaneous or intracerebral xenografts along with analyses of EGFR phosphorylation status, proliferation, apoptosis, and vessel density. Nimotuzumab treatment resulted in reduction of EGFRvIII tyrosine phosphorylation with a decrease in Akt phosphorylation that was greater than that of wtEGFR. Correspondingly, antitumor effects, growth suppression and survival elongation, were more significant in mice bearing either subcutaneous or intracerebral tumor expressing EGFRvIII than in those expressing wtEGFR. These effects were markedly increased when temozolomide was combined with nimotuzumab. The post-treatment recurrent brain tumors exhibited a decrease in expression of the mismatch repair (MMR) proteins, MSH6 and MLH1, but their methylated MGMT status did not changed. Nimotuzumab has in vivo antitumor activity against GBM, especially those expressing EGFRvIII, when combined with temozolomide. This could provide a basis for preselection of patients with GBM by EGFR status who might benefit from the nimotuzumab and temozolomide combination therapy

A sensitive chain specific radioimmunoassay for human immunoglobulins using monoclonal antibodies

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Sikora, K.; Alderson, T.St.J.; Ellis, J.

1983-01-01

A sensitive radioimmunoassay is described for human immunoglobulins. This solid-phase assay uses commercially available monoclonal antibodies and is specific for different Ig chain types. Levels of less than 20 ng/ml Ig are detectable. The assay is suitable for the analysis of human hybridoma supernatants. (Auth.)

The epidermal growth factor receptor (EGFr) as a target for in situ radiation therapy

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Vallis, K.A.; Reilly, R.M.

2003-01-01

In situ radiation therapy traditionally involves the use of a monoclonal antibody (mAb) directed against a specific tumor-associated antigen and labeled with α-particle emitter such as 131-I. An alternative strategy is to use a low molecular weight peptide rather than a mAb as the carrier molecule. Also, recent evidence shows that radioactive elements that emit Auger electrons may be useful for inducing receptor/cell-specific cytotoxicity. Auger electrons provide low energy emissions (<10-20 keV). Although they have a short range in tissue (a few mm), Auger electrons have a high rate of energy deposition that is comparable to high linear energy transfer radiation such as -particles. Human epidermal growth factor (hEGF) is a natural peptide ligand for EGFr, which is frequently overexpressed in breast cancer. EGF is rapidly internalized and translocated to the cell nucleus following binding to EGFr. We are developing a strategy of EGF conjugated to an Auger electron-emitting radionuclide, 111-In, as a treatment for EGFr-overexpressing breast cancers. This strategy has several advantages over the mAb approach, as EGF is an endogenous peptide and should not be immunogenic. Also, its small molecular size should facilitate extravasation and tumor penetration. We have shown that 111In-hEGF is highly and selectively radiotoxic to MDA-MB-468 human breast cancer cells overexpressing EGFr but was not radiotoxic to MCF-7 breast cancer cells with a 100-fold lower level of EGFr expression. We have also demonstrated that 111-In-hEGF was greater than 80-fold more potent on a molar concentration basis at inhibiting the growth of MDA-MB-468 breast cancer cells than paclitaxel (IC50 70 pM vs. 6 nM respectively) and greater than 400-fold more potent than doxorubicin (IC50 20 nM). We have evaluated the therapeutic efficacy of 111-In-hEGF in athymic mice implanted subcutaneously with MDA-MB-468 breast cancer xenografts. Tumour growth was strongly inhibited following administration of

Decreased repopulation as well as increased reoxygenation contribute to the improvement in local control after targeting of the EGFR by C225 during fractionated irradiation

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Krause, Mechthild; Ostermann, Gernot; Petersen, Cordula; Yaromina, Ala; Hessel, Franziska; Harstrick, Andreas; Kogel, Albert J van der; Thames, Howard D; Baumann, Michael

2005-01-01

Background and purpose: Inhibition of repopulation and enhanced reoxygenation has been suggested to contribute to improvement of local tumour control after fractionated irradiation combined with inhibitors of the epidermal growth factor receptor (EGFR). The present study addresses this hypothesis in FaDu human squamous cell carcinoma. For this tumour model marked repopulation and incomplete reoxygenation during fractionated irradiation has previously been demonstrated. Furthermore, the anti-EGFR monoclonal antibody C225 has been shown to significantly improve the results of fractionated irradiation in this tumour. Materials and methods: FaDu tumours in nude mice were irradiated with 18 fractions in 18 days (18f/18d) or 18 fractions in 36 days (18f/36d). Three Gy fractions were given either under ambient or under clamp hypoxic conditions. C225 or carrier was applied four times during the course of treatment. Fractionated irradiations were followed by graded top-up doses to obtain complete dose-response curves for local tumour control. Tumour control dose 50% (TCD 50 ) was determined at day 120 after end of treatment. Results: Significant repopulation and reoxygenation occurred during fractionated irradiation of FaDu tumours (P-values between 0.028 and 50 for 18f/36d under ambient conditions (P=0.04). Bootstrap analysis revealed decreased repopulation and increased reoxygenation after application of C225 (P=0.06 for the combined effect). This was further corroborated by a significant effect of C225 on the 'repopulated' dose under ambient conditions which is influenced by both, reoxygenation and repopulation (P=0.012). Conclusions: Our study provides evidence that both decreased repopulation as well as increased reoxygenation contribute to the improvement of local control after targeting of EGFR by C225 during fractionated irradiation of FaDu tumours

Monoclonal antibodies reveal multiple forms of expression of human microsomal epoxide hydrolase

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Duan, Hongying; Takagi, Akira [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kayano, Hidekazu [Department of Pathology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Koyama, Isamu [Department of Digestive and General Surgery, Saitama International Medical Center, Faculty of Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

2012-04-01

In a previous study, we developed five kinds of monoclonal antibodies against different portions of human mEH: three, anti-N-terminal; one, anti-C-terminal; one, anti-conformational epitope. Using them, we stained the intact and the permeabilized human cells of various kinds and performed flow cytometric analysis. Primary hepatocytes and peripheral blood mononuclear cells (PBMC) showed remarkable differences. On the surface, hepatocytes exhibited 4 out of 5 epitopes whereas PBMC did not show any of the epitopes. mEH was detected inside both cell types, but the most prominent expression was observed for the conformational epitope in the hepatocytes and the two N-terminal epitopes in PBMC. These differences were also observed between hepatocyte-derived cell lines and mononuclear cell-derived cell lines. In addition, among each group, there were several differences which may be related to the cultivation, the degree of differentiation, or the original cell subsets. We also noted that two glioblastoma cell lines reveal marked expression of the conformational epitope on the surface which seemed to correlate with the brain tumor-associated antigen reported elsewhere. Several cell lines also underwent selective permeabilization before flow cytometric analysis, and we noticed that the topological orientation of mEH on the ER membrane in those cells was in accordance with the previous report. However, the orientation on the cell surface was inconsistent with the report and had a great variation between the cells. These findings show the multiple mode of expression of mEH which may be possibly related to the multiple roles that mEH plays in different cells. -- Highlights: ► We examine expression of five mEH epitopes in human cells. ► Remarkable differences exist between hepatocytes and PBMC. ► mEH expression in cell lines differs depending on several factors. ► Some glioblastoma cell lines reveal marked surface expression of mEH. ► Topology of mEH on the cell

Anti-tumor activity of high-dose EGFR tyrosine kinase inhibitor and sequential docetaxel in wild type EGFR non-small cell lung cancer cell nude mouse xenografts

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Tang, Ning; Zhang, Qianqian; Fang, Shu; Han, Xiao; Wang, Zhehai

2016-01-01

Treatment of non-small-cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (EGFR) is still a challenge. This study explored antitumor activity of high-dose icotinib (an EGFR tyrosine kinase inhibitor) plus sequential docetaxel against wild-type EGFR NSCLC cells-generated nude mouse xenografts. Nude mice were subcutaneously injected with wild-type EGFR NSCLC A549 cells and divided into different groups for 3-week treatment. Tumor xenograft volumes were monitored and record...

Pyrosequencing, a method approved to detect the two major EGFR mutations for anti EGFR therapy in NSCLC

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Richard Marie-Jeanne

2011-05-01

Full Text Available Abstract Background Epidermal Growth Factor Receptor (EGFR mutations, especially in-frame deletions in exon 19 (ΔLRE and a point mutation in exon 21 (L858R predict gefitinib sensitivity in patients with non-small cell lung cancer. Several methods are currently described for their detection but the gold standard for tissue samples remains direct DNA sequencing, which requires samples containing at least 50% of tumor cells. Methods We designed a pyrosequencing assay based on nested PCR for the characterization of theses mutations on formalin-fixed and paraffin-embedded tumor tissue. Results This method is highly specific and permits precise characterization of all the exon 19 deletions. Its sensitivity is higher than that of "BigDye terminator" sequencing and enabled detection of 3 additional mutations in the 58 NSCLC tested. The concordance between the two methods was very good (97.4%. In the prospective analysis of 213 samples, 7 (3.3% samples were not analyzed and EGFR mutations were detected in 18 (8.7% patients. However, we observed a deficit of mutation detection when the samples were very poor in tumor cells. Conclusions pyrosequencing is then a highly accurate method for detecting ΔLRE and L858R EGFR mutations in patients with NSCLC when the samples contain at least 20% of tumor cells.

EGFR gene amplification is relatively common and associates with outcome in intestinal adenocarcinoma of the stomach, gastro-oesophageal junction and distal oesophagus

International Nuclear Information System (INIS)

Birkman, Eva-Maria; Ålgars, Annika; Lintunen, Minnamaija; Ristamäki, Raija; Sundström, Jari; Carpén, Olli

2016-01-01

Approximately 50 % of gastric adenocarcinomas belong to a molecular subgroup characterised by chromosomal instability and a strong association with the intestinal histological subtype. This subgroup typically contains alterations in the receptor tyrosine kinase–RAS pathway, for example EGFR or HER2 gene amplifications leading to protein overexpression. In clinical practice, HER2 overexpressing metastatic gastric cancer is known to respond to treatment with anti-HER2 antibodies. By contrast, anti-EGFR antibodies have not been able to provide survival benefit in clinical trials, which, however, have not included patient selection based on the histological subtype or EGFR gene copy number analysis of the tumours. To examine the role of EGFR as a potential biomarker, we studied the prevalence, clinicopathological associations as well as prognostic role of EGFR and HER2 expression and gene amplification in intestinal adenocarcinomas of the stomach, gastro-oesophageal junction and distal oesophagus. Tissue samples from 220 patients were analysed with EGFR and HER2 immunohistochemistry. Those samples with moderate/strong staining intensity were further analysed with silver in situ hybridization to quantify gene copy numbers. The results were associated with clinical patient characteristics and survival. Moderate/strong EGFR protein expression was found in 72/220 (32.7 %) and EGFR gene amplification in 31/220 (14.1 %) of the tumours, while moderate/strong HER2 protein expression was detected in 31/220 (14.1 %) and HER2 gene amplification in 29/220 (13.2 %) of the tumours. EGFR and HER2 genes were co-amplified in eight tumours (3.6 %). EGFR gene amplification was more common in tumours of distal oesophagus/gastro-oesophageal junction/cardia than in those of gastric corpus (p = 0.013). It was associated with shortened time to cancer recurrence (p = 0.026) and cancer specific survival (p = 0.033). EGFR gene amplification is relatively common in intestinal adenocarcinomas

Development of a complete human anti-human transferrin receptor C antibody as a novel marker of oral dysplasia and oral cancer

International Nuclear Information System (INIS)

Nagai, Kentaro; Nakahata, Shingo; Shimosaki, Shunsuke; Tamura, Tomohiro; Kondo, Yuudai; Baba, Takashi; Taki, Tomohiko; Taniwaki, Masafumi; Kurosawa, Gene; Sudo, Yukio; Okada, Seiji; Sakoda, Sumio; Morishita, Kazuhiro

2014-01-01

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. Up to 20% of oral dysplasia cases have been suggested to undergo malignant transformation to OSCC; however, there are no methods to predict OSCC development. In this study, to identify the genes associated with oral dysplasia progression, we performed genomic copy number analyses of genomic DNA samples isolated from primary oral dysplasia and OSCC via the microdissection method and found elevated expression of transferrin receptor C (TfR1/TFRC) with genomic amplification in oral dysplasia and OSCC. The expression rate of TFRC in OSCC was significantly higher than that in dysplasia, suggesting that OSCC disease progression might be related to TFRC expression. Additionally, we investigated the in vitro and in vivo impacts of a newly established anti-human TFRC monoclonal antibody, which was isolated from a human cDNA library using the phage-display method, on cell proliferation and survival. The anti-TFRC antibody blocked the interaction between transferrin and TFRC and consequently inhibited iron uptake, leading to the iron deprivation-mediated suppression of cell growth and induction of apoptosis. Moreover, we demonstrated that the anti-TFRC antibody efficiently inhibited tumor growth in a murine xenograft OSCC model. Therefore, we suggest our developed complete human anti-human TFRC antibody as a useful, novel treatment for oral dysplasia and OSCC

Characterization of a Broadly Reactive Anti-CD40 Agonistic Monoclonal Antibody for Potential Use as an Adjuvant.

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Cameron Martin

Full Text Available Lack of safe and effective adjuvants is a major hindrance to the development of efficacious vaccines. Signaling via CD40 pathway leads to enhanced antigen processing and presentation, nitric oxide expression, pro-inflammatory cytokine expression by antigen presenting cells, and stimulation of B-cells to undergo somatic hypermutation, immunoglobulin class switching, and proliferation. Agonistic anti-CD40 antibodies have shown promising adjuvant qualities in human and mouse vaccine studies. An anti-CD40 monoclonal antibody (mAb, designated 2E4E4, was identified and shown to have strong agonistic effects on primary cells from multiple livestock species. The mAb recognize swine, bovine, caprine, and ovine CD40, and evoked 25-fold or greater proliferation of peripheral blood mononuclear cells (PBMCs from these species relative to cells incubated with an isotype control (p<0.001. In addition, the mAb induced significant nitric oxide (p<0.0001 release by bovine macrophages. Furthermore, the mAb upregulated the expression of MHC-II by PBMCs, and stimulated significant (p<0.0001 IL-1α, IL6, IL-8, and TNF-α expression by PBMCs. These results suggest that the mAb 2E4E4 can target and stimulate cells from multiple livestock species and thus, it is a potential candidate for adjuvant development. This is the first study to report an anti-swine CD40 agonistic mAb that is also broadly reactive against multiple species.

Anti-Human Endoglin (hCD105 Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

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Begoña Barriuso

2016-06-01

Full Text Available Endoglin (CD105 is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio propionate (SPDP. The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

Appraisal of radioimmunotherapy with 131I anti-alpha fetoprotein monoclonal antibody in patients with primary liver cancer

International Nuclear Information System (INIS)

Huang Yaozhang

1992-01-01

Mixed anti-alpha-fetoprotein monoclonal antibodies (AFPMcAb) labeled with 131 I were used in the treatment of 23 patients of moderate to advanced primary liver cancer. In 16 cases treated with 24 doses, the survival periods were 18-605 days with a mean of 135 days. Two patients with moderately advanced liver cancer had a mean survival period of 465 days. According to our experience, the larger dose of 131 I and of anti-AFPMcAb, the longer the survival period and the better the therapeutic results were observed. The relationship between the ratio of cancer/liver radioactivity and the survival period remains to be elucidated

Efficacy of Wnt-1 monoclonal antibody in sarcoma cells

International Nuclear Information System (INIS)

Mikami, Iwao; Koizumi, Kiyoshi; Jablons, David M; You, Liang; He, Biao; Xu, Zhidong; Batra, Sonny; Lee, Amie Y; Mazieres, Julien; Reguart, Noemi; Uematsu, Kazutsugu

2005-01-01

Sarcomas are one of the most refractory diseases among malignant tumors. More effective therapies based on an increased understanding of the molecular biology of sarcomas are needed as current forms of therapy remain inadequate. Recently, it has been reported that Wnt-1/β-catenin signaling inhibits apoptosis in several cancers. In this study, we investigated the efficacy of a monoclonal anti-Wnt-1 antibody in sarcoma cells. We treated cell lines A-204, SJSA-1, and fresh primary cultures of lung metastasis of sarcoma with a monoclonal anti-Wnt-1 antibody. Wnt-1 siRNA treatment was carried out in A-204. We assessed cell death using Crystal Violet staining. Apoptosis induction was estimated by flow cytometry analysis (Annexin V and PI staining). Cell signaling changes were determined by western blotting analysis. We detected Wnt-1 expression in all tissue samples and cell lines. Significant apoptosis induction was found in monoclonal anti-Wnt-1 antibody treated cells compared to control monoclonal antibody treated cells (p < 0.02). Similarly, we observed increased apoptosis in Wnt-1 siRNA treated cells. Blockade of Wnt-1 signaling in both experiments was confirmed by analyzing intracellular levels of Dishevelled-3 and of cytosolic β-catenin. Furthermore, the monoclonal anti-Wnt-1 antibody also induced cell death in fresh primary cultures of metastatic sarcoma in which Wnt-1 signaling was active. Our results indicate that Wnt-1 blockade by either monoclonal antibody or siRNA induces cell death in sarcoma cells. These data suggest that Wnt-1 may be a novel therapeutic target for the treatment of a subset of sarcoma cells in which Wnt-1/β-catenin signaling is active

Passive vaccination with a human monoclonal antibody: generation of antibodies and studies for efficacy in Bacillus anthracis infections.

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vor dem Esche, Ulrich; Huber, Maria; Zgaga-Griesz, Andrea; Grunow, Roland; Beyer, Wolfgang; Hahn, Ulrike; Bessler, Wolfgang G

2011-07-01

A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121-150 or 451-470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination. Copyright © 2010 Elsevier GmbH. All rights reserved.

Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2018-04-01

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L 1 Mab-13 (IgG 1 , kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L 1 Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L 1 Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L 1 Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.

Lemongrass (Cymbopogon flexuosus) essential oil demonstrated anti-inflammatory effect in pre-inflamed human dermal fibroblasts.

Science.gov (United States)

Han, Xuesheng; Parker, Tory L

2017-06-01

Lemongrass ( Cymbopogon flexuosus ) essential oil (LEO), which has citral as its main component, has exhibited anti-inflammatory effect in both animal and human cells. In this study, we evaluated the anti-inflammatory activity of a commercially available LEO in pre-inflamed human dermal fibroblasts. We first studied the impact of LEO on 17 protein biomarkers that are critically associated with inflammation and tissue remodeling. LEO significantly inhibited production of the inflammatory biomarkers vascular cell adhesion molecule 1 (VCAM-1), interferon gamma-induced protein 10 (IP-10), interferon-inducible T-cell alpha chemoattractant (I-TAC), and monokine induced by gamma interferon (MIG); decreased levels of the tissue remodeling biomarkers collagen-I and III, epidermal growth factor receptor (EGFR), and plasminogen activator inhibitor (PAI-1); and inhibited the immunomodulatory biomarker macrophage colony-stimulating factor (M-CSF). Furthermore, we studied the impact of LEO on genome-wide gene expression profiles. LEO significantly modulated global gene expression and robustly impacted signaling pathways, many of which are critical for inflammation and tissue remodeling processes. This study provides the first evidence of the anti-inflammatory activity of LEO in human skin cells and indicates that it is a good therapeutic candidate for treating inflammatory conditions of the skin.

Prognostic and predictive values of EGFR overexpression and EGFR copy number alteration in HER2-positive breast cancer.

Science.gov (United States)

Lee, H J; Seo, A N; Kim, E J; Jang, M H; Kim, Y J; Kim, J H; Kim, S-W; Ryu, H S; Park, I A; Im, S-A; Gong, G; Jung, K H; Kim, H J; Park, S Y

2015-01-06

Epidermal growth factor receptor (EGFR) is overexpressed in a subset of human epidermal growth factor receptor 2 (HER2)-positive breast cancers, and coexpression of HER2 and EGFR has been reported to be associated with poor clinical outcome. Moreover, interaction between HER2 and EGFR has been suggested to be a possible basis for trastuzumab resistance. We analysed the clinical significance of EGFR overexpression and EGFR gene copy number alterations in 242 HER2-positive primary breast cancers. In addition, we examined the correlations between EGFR overexpression, trastuzumab response and clinical outcome in 447 primary, and 112 metastatic HER2-positive breast cancer patients treated by trastuzumab. Of the 242 primary cases, the level of EGFR overexpression was 2+ in 12.7% and 3+ in 11.8%. High EGFR gene copy number was detected in 10.3%. Epidermal growth factor receptor overexpression was associated with hormone receptor negativity and high Ki-67 proliferation index. In survival analyses, EGFR overexpression, but not high EGFR copy number, was associated with poor disease-free survival in all patients, and in the subgroup not receiving adjuvant trastuzumab. In 447 HER2-positive primary breast cancer patients treated with adjuvant trastuzumab, EGFR overexpression was also an independent poor prognostic factor. However, EGFR overexpression was not associated with trastuzumab response, progression-free survival or overall survival in the metastatic setting. Epidermal growth factor receptor overexpression, but not high EGFR copy number, is a poor prognostic factor in HER2-positive primary breast cancer. Epidermal growth factor receptor overexpression is a predictive factor for trastuzumab response in HER2-positive primary breast cancer, but not in metastatic breast cancer.

CCR 20th Anniversary commentary: in search of cetuximab's first indication-combination therapy with irinotecan in colorectal cancer.

Science.gov (United States)

Hicklin, Daniel J

2015-04-01

The research article by Prewett and colleagues, published in the May 1, 2002, issue of Clinical Cancer Research, provided important translational data that extended earlier preclinical and clinical studies with the human-murine chimeric anti-EGFR monoclonal antibody C225. Subsequent clinical trials with C225 led to the demonstration of its efficacy in combination with irinotecan and regulatory approval for the treatment of metastatic colorectal cancer. ©2015 American Association for Cancer Research.

Radioimmunotherapy of Nude Mice Bearing Human Colon Carcinoma with I-131 Labeled Anti-carcinoembryonic Antigen

International Nuclear Information System (INIS)

Kim, Byung Tae; Lee, Kyung Han; Kim, Sang Eun; Choi, Yong; Chi, Dae Yoon; Chung, June Key; Lee, Myung Chul; Koh, Chang Soon; Chung, Hong Keun

1995-01-01

This study was designed to evaluate the effects of various factors on the therapeutic effect of the I-l3l labeled anti-carcinoembryonic antigen monoclonal antibody(anti-CEA antibody). Tetrazolium-based colorimetric assay (MTT) was used to compare in vitro cytotoxicity of 3 Korean colon cancer cell lines (SNU-C2A, SNU-C4, SNU-C5) for selection of proper 2 cell lines in this study. The changes of the size of tumor which was xenografted to nude mice (balb/c nu/nu) were compared in 4 groups (group treated I-131 labeled anti-CEA antibody, group treated with non-radiolabeled anti-CEA antibody, group treated with I-131. labeled anti-human chorionic gonadotropin monoclonal antibody (anti-hCG antibody) as nonspecific antibody, and group injected with normal saline as a control). Immunohistochemical staining and in vivo autoradiography were performed after excision of the xenografted tumor. The results were as below mentioned. The in vitro cytotoxic effect of I-131 labeled anti-CEA antibody is most prominent in SNU-C5 cell line between 3 cancer cell lines. The changes of xenografted tumor size in both SNU-C4 and SNU-C5 cell tumors at the thirteenth day after injection of the antibodies were smallest in the group treated with I-131 labeled anti-CEA antibody (SNU-C4/SNU-C5; 324/342%) comparing with other groups, group treated with anti-CEA antibody (622/660%), group treated with I-131 anti-hCG antibody (538/546%), and control group(1030/724%) (p<0.02 in SNU-C4 and p<0.1in SNU-C5 at the 13th day after injection of antibodies). On the thirteenth day after injection of the antibodies nude mice were sacreficed to count the radiouptake of tumor and to check the changes of tumor size. Correlations between radiouptake and change of tumor size were calculated in each groups and significant negative correlation was only obtained in the group treated with I-131 anti-CEA antibody (p<0.05). There were no correlations between antigenic expression of carcinoembryonic antigen and

Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo.

Science.gov (United States)

Chang, De-Kuan; Moniz, Raymond J; Xu, Zhongyao; Sun, Jiusong; Signoretti, Sabina; Zhu, Quan; Marasco, Wayne A

2015-06-11

Carbonic anhydrase (CA) IX is a surface-expressed protein that is upregulated by the hypoxia inducible factor (HIF) and represents a prototypic tumor-associated antigen that is overexpressed on renal cell carcinoma (RCC). Therapeutic approaches targeting CAIX have focused on the development of CAIX inhibitors and specific immunotherapies including monoclonal antibodies (mAbs). However, current in vivo mouse models used to characterize the anti-tumor properties of fully human anti-CAIX mAbs have significant limitations since the role of human effector cells in tumor cell killing in vivo is not directly evaluated. The role of human anti-CAIX mAbs on CAIX(+) RCC tumor cell killing by immunocytes or complement was tested in vitro by antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP) as well as on CAIX(+) RCC cellular motility, wound healing, migration and proliferation. The in vivo therapeutic activity mediated by anti-CAIX mAbs was determined by using a novel orthotopic RCC xenograft humanized animal model and analyzed by histology and FACS staining. Our studies demonstrate the capacity of human anti-CAIX mAbs that inhibit CA enzymatic activity to result in immune-mediated killing of RCC, including nature killer (NK) cell-mediated ADCC, CDC, and macrophage-mediated ADCP. The killing activity correlated positively with the level of CAIX expression on RCC tumor cell lines. In addition, Fc engineering of anti-CAIX mAbs was shown to enhance the ADCC activity against RCC. We also demonstrate that these anti-CAIX mAbs inhibit migration of RCC cells in vitro. Finally, through the implementation of a novel orthotopic RCC model utilizing allogeneic human peripheral blood mononuclear cells in NOD/SCID/IL2Rγ(-/-) mice, we show that anti-CAIX mAbs are capable of mediating human immune response in vivo including tumor infiltration of NK cells and activation of T cells, resulting in

Molecular and Therapeutic Characterization of Anti-ectodysplasin A Receptor (EDAR) Agonist Monoclonal Antibodies*

Science.gov (United States)

Kowalczyk, Christine; Dunkel, Nathalie; Willen, Laure; Casal, Margret L.; Mauldin, Elizabeth A.; Gaide, Olivier; Tardivel, Aubry; Badic, Giovanna; Etter, Anne-Lise; Favre, Manuel; Jefferson, Douglas M.; Headon, Denis J.; Demotz, Stéphane; Schneider, Pascal

2011-01-01

The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC50 of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments. PMID:21730053

Detection of auto-anti-idiotypic antibodies to Lol p I (rye I) IgE antibodies in human sera by the use of murine idiotypes: levels in atopic and non-atopic subjects and effects of immunotherapy.

Science.gov (United States)

Hébert, J; Bernier, D; Mourad, W

1990-06-01

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.

Fully-human Monoclonal Antibodies Against Human EphrinB2 and EphB4 | NCI Technology Transfer Center | TTC

Science.gov (United States)

The National Cancer Institute's Cancer and Inflammation Program is seeking statements of capability or interest from parties interested in licensing fully-human monoclonal antibodies against human EphrinB2 and EphB4.

Binding Affinity, Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200

DEFF Research Database (Denmark)

Lindegren, Sture; Andrade, Luciana N S; Bäck, Tom

2015-01-01

The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual...

A human monoclonal antibody with neutralizing activity against highly divergent influenza subtypes.

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Nicola Clementi

Full Text Available The interest in broad-range anti-influenza A monoclonal antibodies (mAbs has recently been strengthened by the identification of anti-hemagglutinin (HA mAbs endowed with heterosubtypic neutralizing activity to be used in the design of "universal" prophylactic or therapeutic tools. However, the majority of the single mAbs described to date do not bind and neutralize viral isolates belonging to highly divergent subtypes clustering into the two different HA-based influenza phylogenetic groups: the group 1 including, among others, subtypes H1, H2, H5 and H9 and the group 2 including, among others, H3 subtype. Here, we describe a human mAb, named PN-SIA28, capable of binding and neutralizing all tested isolates belonging to phylogenetic group 1, including H1N1, H2N2, H5N1 and H9N2 subtypes and several isolates belonging to group 2, including H3N2 isolates from the first period of the 1968 pandemic. Therefore, PN-SIA28 is capable of neutralizing isolates belonging to subtypes responsible of all the reported pandemics, as well as other subtypes with pandemic potential. The region recognized by PN-SIA28 has been identified on the stem region of HA and includes residues highly conserved among the different influenza subtypes. A deep characterization of PN-SIA28 features may represent a useful help in the improvement of available anti-influenza therapeutic strategies and can provide new tools for the development of universal vaccinal strategies.

Biodistribution and pharmacokinetics of monoclonal antibody T1h and variant anti-CD6 murine 10D12 in healthy animals and in experimental arthritis model

International Nuclear Information System (INIS)

León, M; Hernández, I; Aldana, L; Ayra, F; Castro, Y; Leyva, R; García, L; Pérez, S.; Casaco, A.

2016-01-01

Biodistribution and pharmacokinetic of two radio labeled monoclonal antibodies was performed with the help of imaging techniques. Isotopic labeling was carried out by means of standardized methods. Pharmacokinetic evaluation was performed using the population approach and sparse data design. Introduction: Targeted therapy with monoclonal antibodies (MAb) is an efficient option for the treatment of rheumatoid arthritis. Th1 is a MAb anti human CD6 developed for the treatment of autoimmune disease and 10D12 is its counterpart anti murine CD6 developed as a pharmacological tool to get deep into the response mechanisms in animals models of rheumatoid arthritis.To investigate the behavior of both antibodies in the assay system, molecules were labeled with 125I to evaluate pharmacokinetic in healthy animals and with 99mTc to evaluate the antibody uptake in inflamed area of induced arthritis. Materials and methods: Antibodies were supplied by the Center of Molecular immunology. Iodination was performed by the iodogen method and technetium labeling was carried out directly by Schwarz method. Female C57BL6 from CENPALAB were used for experiments. Biodistribution and pharmacokinetic was performed by a sparse data design using the population approach. Uptake in region of inflammation was quantified by gammagraphy at the same time points of blood sampling. A compartmental model was build to quantify uptake kinetic. Pharmacokinetic profiles were analyzed using MONOLIX software version 4.2. Results: Minor pharmacokinetic differences were found between monoclonal antibodies labeled with 125I and 99mTc. As a humanized antibody, T1h shows a faster clearance than 10D12 and a biodistribution pattern reflecting preference for excretion mechanisms. The arthritis accumulation was not consistent with a targeted mediated uptake. On the other hand, radio labeled 10D12 shows an accumulation profile in arthritis with two peaks of maximum concentration representing an initial transit to

A novel monoclonal anti-CD81 antibody produced by genetic immunization efficiently inhibits Hepatitis C virus cell-cell transmission.

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Isabel Fofana

Full Text Available Hepatitis C virus (HCV infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies.Using genetic immunization, we produced four monoclonal antibodies (mAbs against the HCV host entry factor CD81. The effects of antibodies on inhibition of HCV infection and dissemination were analyzed in HCV permissive human liver cell lines.The anti-CD81 mAbs efficiently inhibited infection by HCV of different genotypes as well as a HCV escape variant selected during liver transplantation and re-infecting the liver graft. Kinetic studies indicated that anti-CD81 mAbs target a post-binding step during HCV entry. In addition to inhibiting cell-free HCV infection, one antibody was also able to block neutralizing antibody-resistant HCV cell-cell transmission and viral dissemination without displaying any detectable toxicity.A novel anti-CD81 mAb generated by genetic immunization efficiently blocks HCV spread and dissemination. This antibody will be useful to further unravel the role of virus-host interactions during HCV entry and cell-cell transmission. Furthermore, this antibody may be of interest for the development of antivirals for prevention and treatment of HCV infection.

Monoclonal Antibody Fragments for Targeting Therapeutics to Growth Plate Cartilage | NCI Technology Transfer Center | TTC

Science.gov (United States)

Researchers at The Eunice Kennedy Shriver National Institute on Child Health and Human Development (NICHD) have discovered monoclonal antibodies that bind to matrilin-3, a protein specifically expressed in cartilage tissue, that could be used for treating or inhibiting growth plate disorders, such as a skeletal dysplasia or short stature. The monoclonal antibodies can also be used to target therapeutic agents, such as anti-arthritis agents, to cartilage tissue. NICHD seeks statements of capability or interest from parties interested in collaborative research to co-develop, evaluate, or commercialize treatment of skeletal disorders using targeting antibodies.

A pathological study on overexpression of c-erbB-2, EGFR proteins in human radiation skin ulcer

International Nuclear Information System (INIS)

Zhao Po; Yang Zhixiang; Wang Dewen; Li Guomin; Gao Yabin

1996-01-01

We performed an immunohistochemical study in 25 cases of human radiation skin ulcer by using c-erbB-2, Epidermal growth factor receptor (EGFR), protein antibodies and repairing method of antigen with microwave oven. The results show that the positive rate for overexpression of c-erbB-2 oncoprotein was 92.0% (23/25), and EGFR was 73.7%(14/19). The positive position for overexpression of c-crbB-2 was mainly observed on cell membrane of squamous epithelial cells and cell plasma of fibroblasts, endotheliocytes, and leiomyocytes in media and fibrocytes in adventitia of the arterioles in mesenchyme. It is suggested that the overexpression of c-erbB-2 and EGFR proteins might be related to cancer transformation and poor healing in radiation skin ulcers

Antibody-dependent cellular cytotoxicity (ADCC) activity of a novel anti-PD-L1 antibody avelumab (MSB0010718C) on human tumor cells

OpenAIRE

Boyerinas, Benjamin; Jochems, Caroline; Fantini, Massimo; Heery, Christopher R.; Gulley, James L.; Tsang, Kwong Yok; Schlom, Jeffrey

2015-01-01

Several anti-PD1/PD-L1 monoclonal antibodies (MAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these MAbs is to inhibit PD1 on immune cells interacting with PD-L1 on tumor cells. These MAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective MAb-mediated cancer therapies. A fully human anti-PD-L...

Assessment of Epidermal Growth Factor Receptor (EGFR expression in human meningioma

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Perry Arie

2010-05-01

Full Text Available Abstract Purpose This study explores whether meningioma expresses epidermal growth factor receptor (EGFR and determines if there is a correlation between the WHO grade of this tumor and the degree of EGFR expression. Methods Following institutional review board approval, 113 meningioma specimens from 89 patients were chosen. Of these, 85 were used for final analysis. After a blinded review, immunohistochemical stains for EGFR were performed. Staining intensity (SI was scored on a scale 0-3 (from no staining to strong staining. Staining percentage of immunoreactive cells (SP was scored 1-5 (from the least to the maximum percent of the specimen staining. Immunohistochemical score (IHS was calculated as the product of SI and SP. Results Eighty-five samples of meningioma were classified in accordance with World Health Organization (WHO criteria: benign 57/85 (67%, atypical 23/85 (27%, and malignant 5/85 (6%. The majority of samples demonstrated a moderate SI for EGFR. IHS for EGFR demonstrated a significant association between SI and histopathologic subtype. Also, there was a correlation between the SP and histopathologic subtype (p = 0.029. A significant association was determined when the benign and the atypical samples were compared to the malignant with respect to the SP (p = 0.009. While there was a range of the IHS for the benign and the atypical histologic subtypes, malignant tumors exhibited the lowest score and were statistically different from the benign and the atypical specimens (p Conclusions To our knowledge, this represents the largest series of meningioma samples analyzed for EGFR expression reported in the literature. EGFR expression is greatest in benign meningiomas and may serve a potential target for therapeutic intervention with selective EGFR inhibitors.

[AntiEGFRnano inhibites proliferation and migration of estrogen-dependent Ishikawa cells of human endometrial cancer cell line].

Science.gov (United States)

Diao, Zhen-yu; Lu, Wu-guang; Cao, Peng; Hu, Yun-long; Zhou, Xing; Xue, Ping-ping; Shen, Li; Sun, Hai-xiang

2012-10-01

Nanobody is a kind of antibody from camel, which misses light chain. Nanobody has the same antigen binding specificity and affinity as mAb. Moreover, because of its small molecular weight, high stability and easy preparation, nanobody has great value of biomedical applications. In this study, we successfully prepared highly pure antiEGFR nanobody in E.coli using genetic engineering techniques. Cell proliferation assay (CCK-8 assay) and migration experiments (cell scratch test and Transwell assay) indicated that the recombinant antiEGFRnano can significantly inhibit the proliferation and migration of endometrial cancer cells. These results provide a new way of thinking and methods for EGFR-targeted therapy of endometrial cancer.

Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells.

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Keisuke Tabara

Full Text Available Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs. However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2 or BIBW2992 (pan-TKI of EGFR family proteins. Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.

Preparation of human cardiac anti-myosin: a review

International Nuclear Information System (INIS)

Okada, H.; Souza, I.T.T.

1990-01-01

The present communication is a review of the physicochemical characterization and immunological properties of myosin isolated from the cardiac muscle, the production of monoclonal antibody anti-myosin, the radiolabeling of this antibody and its applications as radiopharmaceuticals to imaging myocardial infarcts. The classical example of radioimmunologic diagnosis of non malignant tissues is the detection of myocardial infarction by radiolabeled antibodies to myosin. (author)

EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR AND HUMAN PAPILLOMAVIRUS (HPV L1 CAPSID PROTEIN IN CERVICAL SQUAMOUS INTRAEPITHELIAL LESIONS

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Balan Raluca

2010-09-01

Full Text Available We analyzed the immunohistochemical pattern of epidermal growth factor receptor (EGFR in cervical squamous intraepithelial lesions (SILs in correlation with L1 HPV capsid protein, in order to determine the relationship between EGFR expression and the infection status of human papillomavirus (HPV. The study included 40 cases, 24 LSIL (low grade SIL (CIN1, cervical intraepithelial neoplasia and 16 HSIL (high grade SIL (6 cases of CIN2 and 10 cases of CIN3. The immunoexpression of L1 HPV protein was assessed on conventional cervico-vaginal smears and EGFR was immunohistochemically evaluated on the corresponding cervical biopsies. The HPV L1 capsid protein was expressed in 45.83% of LSIL and 25% of HSIL. EGFR was overexpressed in 62,4% of HSIL (58,4% CIN2 and 41,6% CIN3 and 37,6% LSIL. The immunoexpression of L1 HPV has clinical application in the progression assessment of the cervical precancerous lesions without a correlation to the grade of the cervical SIL. EGFR is expressed by all proliferating squamous epithelial cells, thus corresponding with the grade of SIL. The evaluation of EGFR status, correlated with L1 HPV protein expression, can provide useful data of progression risk of cervical squamous intraepithelial lesions

Chimeric Monoclonal Antibody Cetuximab Targeting Epidermal Growth Factor-Receptor in Advanced Non-Melanoma Skin Cancer.

Science.gov (United States)

Wollina, Uwe; Tchernev, Georgi; Lotti, Torello

2018-01-25

Non-melanoma skin cancer (NMSC) is the most common malignancy in humans. Targeted therapy with monoclonal antibody cetuximab is an option in case of advanced tumor or metastasis. We present and update of the use of cetuximab in NMSC searching PUBMED 2011-2017. The monoclonal antibody cetuximab against epidermal growth factor receptor (EGFR) has been investigated for its use in NMSC during the years 2011 to 2017 by a PUBMED research using the following items: "Non-melanoma skin cancer AND cetuximab," "cutaneous squamous cell carcinoma AND cetuximab," and "basal cell carcinoma AND cetuximab", and "cetuximab AND skin toxicity". Available data were analyzed including case reports. Current evidence of cetuximab efficacy in NMSC was mainly obtained in cutaneous SCC and to a lesser extend in BCC. Response rates vary for neoadjuvant, adjuvant, mono- and combined therapy with cetuximab. Management of cutaneous toxicities is necessary. Guidelines are available. Cetuximab is an option for recurrent or advanced NMSC of the skin. It seems to be justified particularly in very high-risk tumors. There is a need for phase III trials.

[Comparative Study for Anti-Hepatitis B Surface Antigen Titers Based on Two Measurement Methods: Using Monoclonal Antibodies Isolated from Hepatitis B Vaccinated Recipients].

Science.gov (United States)

Oone, Kumiko; Kani, Satomi; Oohashi, Minoru; Shinkai, Noboru; Inoue, Takako; Wakimoto, Yukio; Tanaka, Yasuhito

2015-08-01

As anti-hepatitis B surface antigen (anti-HBs) titers vary depending on the measurement methods, we compared two different methods to measure anti-HBs titers in sera and HBs monoclonal antibodies. The sera from 182 HB virus-resolved patients who were negative for HBsAg but positive for antiHB core protein (HBc) and/or anti-HBs were obtained. The measurement of anti-HBs was compared using either Lumipulse G1200 or Architect i2000SR. Six different monoclonal antibody (mAbs) clones isolated from healthy individuals inoculated with hepatitis B vaccine Bimmugen (genotype C) were used. A statistically significant correlation in anti-HBs titers was found between the two methods tested (Y = 0.951X + 100.7, R = 0.813, p Lumipulse and 12 (6.6%) were opposite results. Measuring 2 mAbs with HBV neutralizing activity, the titers of the 116 antibody (1.0 μg/mL) were comparable (689.3 mIU/mL by Lumipulse and 440.7 mIU/mL by Architect), whereas those of the 478 antibody (1.0 μg/mL) were much lower by Architect than by Lumipulse (42.6 vs. 818.6 mIU/mL, respectively). Of four other mAbs without HBV neutralizing activity, equal titers were observed for one; two mAbs had less anti-HB titers by Architect; and one was below the cut-off index (Lumipulse, and the potential ability to detect the 478 antibody with neutralizing activity is low, indicating that Architect might underestimate anti-HBs titers. Future studies should standardize the anti-HBs titer measurement system.

[Anti-EGFR Antibody Combination Chemotherapy Was Effective against Locally Advanced Ascending Colon Cancer as Well as a Recurrent Lesion - A Case Report].

Science.gov (United States)

Yamada, Yasufumi; Yokomizo, Hajime; Yano, Yuki; Okayama, Sachiyo; Satake, Masaya; Ida, Arika; Usui, Takebumi; Yamaguchi, Kentaro; Shiozawa, Shunichi; Yoshimatsu, Kazuhiko; Shimakawa, Takeshi; Katsube, Takao; Naritaka, Yoshihiko; Kato, Hiroyuki

2017-10-01

Here we report a case in which a locally advanced ascending colon cancer was successfully treated with anti-EGFR immunotherapy combined with chemotherapy and curative resection, and recurrent cancer was treated with the same chemotherapy. A 71-year-old man was diagnosed with ascending colon cancer in our department. No distant metastasis was observed, but curative resection was considered impossible because of extensive local cancer invasion. Because a genetic analysis revealed the presence of the wild-type KRAS gene, 6 courses of mFOLFOX6 plus cetuximab were administered. A cPR was obtained and curative resection was performed. The final diagnosis was ypT3N1M0, ypStage III a colon cancer, and chemotherapy improved the cancer stage to Grade 1b. Six courses of FOLFOX6 were then administered, followed by observation. After 2 years 6 months, a tumor of approximately 5 cm in size was noted in the right buttock using surveillance CT and was diagnosed as recurrent colon cancer. We considered further curative resection difficult and therefore 6 courses of mFOLFOX6 plus panitumumab were administered, a cPR was obtained, and right hip tumor extirpation surgery was performed. These results suggest that chemotherapy combined with anti-EGFR antibody immunotherapy is effective in treating recurrent colon cancer.

Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets

NARCIS (Netherlands)

ter Meulen, Jan; Bakker, Alexander B. H.; van den Brink, Edward N.; Weverling, Gerrit J.; Martina, Byron E. E.; Haagmans, Bart L.; Kuiken, Thijs; de Kruif, John; Preiser, Wolfgang; Spaan, Willy; Gelderblom, Hans R.; Goudsmit, Jaap; Osterhaus, Albert D. M. E.

2004-01-01

SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal

Phosphorylated EGFR expression may predict outcome of EGFR-TKIs therapy for the advanced NSCLC patients with wild-type EGFR

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Wang Fen

2012-08-01

Full Text Available Abstract Background EGFR mutation is a strong predictive factor of EGFR-TKIs therapy. However, at least 10% of patients with EGFR wild-type are responsive to TKIs, suggesting that other determinants of outcome besides EGFR mutation might exist. We hypothesized that activation of phosphorylated EGFR could be a potential predictive biomarker to EGFR-TKIs treatment among patients in wild-type EGFR. Method Total of 205 stage IIIb and IV NSCLC patients, tissue samples of whom were available for molecular analysis, were enrolled in this study. The phosphorylation of EGFR at tyrosine 1068 (pTyr1068 and 1173 (pTyr1173 were assessed by immunohistochemistry, and EGFR mutations were detected by denaturing high performance liquid chromatograph (DHPLC. Results Among 205 patients assessable for EGFR mutation and phosphorylation analysis, 92 (44.9% were EGFR mutant and 165 patients (57.6% had pTyr1173 expression. Superior progression-free survival (PFS was seen after EGFR-TKIs therapy in patients with pTyr1068 expression compared to pTyr1068 negative ones (median PFS 7.0 months vs. 1.2 months, P P = 0.016. In subgroup of patients with wild-type EGFR, pTyr1068 expression positive ones had a significantly prolonged PFS (4.2 months vs.1.2 months P  Conclusion pTyr1068 may be a predictive biomarker for screening the population for clinical response to EGFR-TKIs treatment; especially for patients with wild-type EGFR.

Differential role of EGF and BFGF in human GBM-TIC proliferation: relationship to EGFR-tyrosine kinase inhibitor sensibility.

Science.gov (United States)

Bajetto, A; Porcile, C; Pattarozzi, A; Scotti, L; Aceto, A; Daga, A; Barbieri, F; Florio, T

2013-01-01

Glioblastoma multiforme (GBM) is among the most devastating human tumors being rapidly fatal despite aggressive surgery, radiation and chemotherapies. It is characterized by extensive dissemination of tumor cells within the brain that hinders complete surgical resection. GBM tumor initiating-cells (TICs) are a rare subpopulation of cells responsible for tumor development, growth, invasiveness and recurrence after chemotherapy. TICs from human GBM can be selected in vitro using the same conditions permissive for the growth of normal neural cells, of which share some features including marker expression, self-renewal capacity, long-term proliferation, and ability to differentiate into neuronal and glial cells. EGFR overexpression and its constitutive activation is one of the most important signaling alteration identified in GBM, and its pharmacological targeting represents an attractive therapeutic goal. We previously demonstrated that human GBM TICs have different sensitivity to the EGFR kinase inhibitors erlotinib and gefitinib, depending on the differential modulation of downstream signaling cascades. In this work we investigated the mechanisms of resistance to erlotinib in two human GBM TIC cultures, analyzing EGF and bFGF individual contribution to proliferation, clonogenicity, and migration. We demonstrated the presence of a small cell subpopulation whose proliferation is supported by EGF and a larger one mainly dependent on bFGF. Thus, insensitivity to EGFR kinase inhibitors as far as TIC proliferation results from a predominant FGFR activation that hides the inhibitory effects induced on EGFR signaling. Conversely, EGF and bFGF induced cell migration with similar efficacy. In addition, unlike neural stem/progenitors cells, the removal of chondroitin sulphate proteoglycans from cell surface was unable to discern EGF- and bFGF-dependent subpopulations in GBM TICs.

Immobilization of Murine Anti-BMP-2 Monoclonal Antibody on Various Biomaterials for Bone Tissue Engineering

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Sahar Ansari

2014-01-01

Full Text Available Biomaterials are widely used as scaffolds for tissue engineering. We have developed a strategy for bone tissue engineering that entails application of immobilized anti-BMP-2 monoclonal antibodies (mAbs to capture endogenous BMPs in vivo and promote antibody-mediated osseous regeneration (AMOR. The purpose of the current study was to compare the efficacy of immobilization of a specific murine anti-BMP-2 mAb on three different types of biomaterials and to evaluate their suitability as scaffolds for AMOR. Anti-BMP-2 mAb or isotype control mAb was immobilized on titanium (Ti microbeads, alginate hydrogel, and ACS. The treated biomaterials were surgically implanted in rat critical-sized calvarial defects. After 8 weeks, de novo bone formation was assessed using micro-CT and histomorphometric analyses. Results showed de novo bone regeneration with all three scaffolds with immobilized anti-BMP-2 mAb, but not isotype control mAb. Ti microbeads showed the highest volume of bone regeneration, followed by ACS. Alginate showed the lowest volume of bone. Localization of BMP-2, -4, and -7 antigens was detected on all 3 scaffolds with immobilized anti-BMP-2 mAb implanted in calvarial defects. Altogether, these data suggested a potential mechanism for bone regeneration through entrapment of endogenous BMP-2, -4, and -7 proteins leading to bone formation using different types of scaffolds via AMOR.

Experimental radioimmunotherapy of a xenografted human glioma using [sup 131]I-labeled monoclonal antibody to epidermal growth factor receptor

Energy Technology Data Exchange (ETDEWEB)

Takahashi, Hiroshi; Nakazawa, Shozo [Nippon Medical School, Tokyo (Japan); Herlyn, D

1993-09-01

[sup 131]I-labeled F (ab')[sub 2] fragments of murine monoclonal antibodies (MAb) 425 specific to the epidermal growth factor receptor expressed on human gliomas were used in experimental human malignant glioma immunotherapy. Two injections of 150 [mu]Ci [sup 131]I-labeled 425 F(ab')[sub 2] achieved growth inhibition of U-87MG human malignant glioma xenografts in nude mice. This radiolabeled specific MAb F(ab')[sub 2] was significantly superior to radiolabeled fragments of an anti-hepatitis virus control MAb A5C3 in influencing tumor growth. However, similar treatment of established human malignant glioma xenografts did not inhibit progressive tumor growth significantly. No clear tumor inhibition was produced by unlabeled MAb 425F(ab')[sub 2]. These studies suggest that [sup 131]I-labeled MAbs have a significant antitumor effect where unmodified antibody is ineffective. Multiple doses of antibody may achieve an increase in labeled MAb concentration in tumors. (author).

Human cerebrospinal fluid monoclonal N-methyl-D-aspartate receptor autoantibodies are sufficient for encephalitis pathogenesis.

Science.gov (United States)

Kreye, Jakob; Wenke, Nina K; Chayka, Mariya; Leubner, Jonas; Murugan, Rajagopal; Maier, Nikolaus; Jurek, Betty; Ly, Lam-Thanh; Brandl, Doreen; Rost, Benjamin R; Stumpf, Alexander; Schulz, Paulina; Radbruch, Helena; Hauser, Anja E; Pache, Florence; Meisel, Andreas; Harms, Lutz; Paul, Friedemann; Dirnagl, Ulrich; Garner, Craig; Schmitz, Dietmar; Wardemann, Hedda; Prüss, Harald

2016-10-01

SEE ZEKERIDOU AND LENNON DOI101093/AWW213 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a recently discovered autoimmune syndrome associated with psychosis, dyskinesias, and seizures. Little is known about the cerebrospinal fluid autoantibody repertoire. Antibodies against the NR1 subunit of the NMDAR are thought to be pathogenic; however, direct proof is lacking as previous experiments could not distinguish the contribution of further anti-neuronal antibodies. Using single cell cloning of full-length immunoglobulin heavy and light chain genes, we generated a panel of recombinant monoclonal NR1 antibodies from cerebrospinal fluid memory B cells and antibody secreting cells of NMDAR encephalitis patients. Cells typically carried somatically mutated immunoglobulin genes and had undergone class-switching to immunoglobulin G, clonally expanded cells carried identical somatic hypermutation patterns. A fraction of NR1 antibodies were non-mutated, thus resembling 'naturally occurring antibodies' and indicating that tolerance induction against NMDAR was incomplete and somatic hypermutation not essential for functional antibodies. However, only a small percentage of cerebrospinal fluid-derived antibodies reacted against NR1. Instead, nearly all further antibodies bound specifically to diverse brain-expressed epitopes including neuronal surfaces, suggesting that a broad repertoire of antibody-secreting cells enrich in the central nervous system during encephalitis. Our functional data using primary hippocampal neurons indicate that human cerebrospinal fluid-derived monoclonal NR1 antibodies alone are sufficient to cause neuronal surface receptor downregulation and subsequent impairment of NMDAR-mediated currents, thus providing ultimate proof of antibody pathogenicity. The observed formation of immunological memory might be relevant for clinical relapses. © The Author (2016). Published by Oxford University Press on

Golimumab and certolizumab: The two new anti-tumor necrosis factor kids on the block

Directory of Open Access Journals (Sweden)

Mittal Mohit

2010-01-01

Full Text Available Anti-tumor necrosis factor (anti-TNF agents have revolutionized treatment of psoriasis and many other inflammatory diseases of autoimmune origin. They have considerable advantages over the existing immunomodulators. Anti-TNF agents are designed to target a very specific component of the immune-mediated inflammatory cascades. Thus, they have lower risks of systemic side-effects. In a brief period of 10 years, a growing number of biological therapies are entering the clinical arena while many more biologicals remain on the horizon. With time, the long-term side-effects and efficacies of these individual agents will become clearer and help to determine which ones are the most suitable for long-term care. Golimumab (a human monoclonal anti-TNF-α antibody and Certolizumab (a PEGylated Fab fragment of humanized monoclonal TNF-α antibody are the two latest additions to the anti-TNF regimen. Here, we are providing a brief description about these two drugs and their uses.

Transgenic nude mouse with green fluorescent protein expression-based human glioblastoma multiforme animal model with EGFR expression and invasiveness.

Science.gov (United States)

Tan, Guo-Wei; Lan, Fo-Lin; Gao, Jian-Guo; Jiang, Cai-Mou; Zhang, Yi; Huang, Xiao-Hong; Ma, Yue-Hong; Shao, He-Dui; He, Xue-Yang; Chen, Jin-Long; Long, Jian-Wu; Xiao, Hui-Sheng; Guo, Zhi-Tong; Diao, Yi

2012-08-01

Previously, we developed an orthotopic xenograft model of human glioblastoma multiforme (GBM) with high EGFR expression and invasiveness in Balb/c nu/nu nude mice. Now we also developed the same orthotopic xenograft model in transgenic nude mice with green fluorescent protein (GFP) expression. The present orthotopic xenografts labeled by phycoerythrin fluorescing red showed high EGFR expression profile, and invasive behavior under a bright green-red dual-color fluorescence background. A striking advantage in the present human GBM model is that the change of tumor growth can be observed visually instead of sacrificing animals in our further antitumor therapy studies.

Acrolein and thiol-reactive electrophiles suppress allergen-induced innate airway epithelial responses by inhibition of DUOX1 and EGFR.

Science.gov (United States)

Danyal, Karamatullah; de Jong, Willem; O'Brien, Edmund; Bauer, Robert A; Heppner, David E; Little, Andrew C; Hristova, Milena; Habibovic, Aida; van der Vliet, Albert

2016-11-01

Acrolein is a major thiol-reactive component of cigarette smoke (CS) that is thought to contribute to increased asthma incidence associated with smoking. Here, we explored the effects of acute acrolein exposure on innate airway responses to two common airborne allergens, house dust mite and Alternaria alternata, and observed that acrolein exposure of C57BL/6 mice (5 ppm, 4 h) dramatically inhibited innate airway responses to subsequent allergen challenge, demonstrated by attenuated release of the epithelial-derived cytokines IL-33, IL-25, and IL-1α. Acrolein and other anti-inflammatory thiol-reactive electrophiles, cinnamaldehyde, curcumin, and sulforaphane, similarly inhibited allergen-induced production of these cytokines from human or murine airway epithelial cells in vitro. Based on our previous observations indicating the importance of Ca 2+ -dependent signaling, activation of the NADPH oxidase DUOX1, and Src/EGFR-dependent signaling in allergen-induced epithelial secretion of these cytokines, we explored the impact of acrolein on these pathways. Acrolein and other thiol-reactive electrophiles were found to dramatically prevent allergen-induced activation of DUOX1 as well as EGFR, and acrolein was capable of inhibiting EGFR tyrosine kinase activity via modification of C797. Biotin-labeling strategies indicated increased cysteine modification and carbonylation of Src, EGFR, as well as DUOX1, in response to acrolein exposure in vitro and in vivo, suggesting that direct alkylation of these proteins on accessible cysteine residues may be responsible for their inhibition. Collectively, our findings indicate a novel anti-inflammatory mechanism of CS-derived acrolein and other thiol-reactive electrophiles, by directly inhibiting DUOX1- and EGFR-mediated airway epithelial responses to airborne allergens. Copyright © 2016 the American Physiological Society.

Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

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Malviya, G.; Dierckx, R.A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Conti, F. [Rheumatology Unit, I Faculty of Medicine and Surgery, Sapienza University of Rome (Italy); Chianelli, M. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Unit of Nuclear Medicine, Regina apostolorum Hospital, Albano, Rome (Italy); Scopinaro, F. [Nuclear Medicine Department, Sapienza University of Rome, St. Andrea Hospital, Rome (Italy); Signore, A. [Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen (Netherlands); Nuclear Medicine Department, Sapienza University of Rome, St. Andrea Hospital, Rome (Italy)

2010-02-15

The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-{alpha}, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with {sup 99m}Tc or {sup 111}In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform 'evidence-based biological therapy' of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for

Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

International Nuclear Information System (INIS)

Malviya, G.; Dierckx, R.A.; Conti, F.; Chianelli, M.; Scopinaro, F.; Signore, A.

2010-01-01

The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-α, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with 99m Tc or 111 In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform 'evidence-based biological therapy' of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for therapy decision-making and

Radiolabeled monoclonal antibodies for imaging and therapy: Potential, problems, and prospects: Scientific highlights

International Nuclear Information System (INIS)

Srivastava, S.C.; Buraggi, G.L.

1986-01-01

This meeting focused on areas of research on radiolabeled monoclonal antibodies. Topics covered included the production, purification, and fragmentation of monoclonal antibodies and immunochemistry of hybridomas; the production and the chemistry of radionuclides; the radiohalogenation and radiometal labeling techniques; the in-vivo pharmacokinetics of radiolabeled antibodies; the considerations of immunoreactivity of radiolabeled preparations; the instrumentation and imaging techniques as applied to radioimmunodetection; the radiation dosimetry in diagnostic and therapeutic use of labeled antibodies; the radioimmunoscintigraphy and radioimmunotherapy studies; and perspectives and directions for future research. Tutorial as well as scientific lectures describing the latest research data on the above topics were presented. Three workshop panels were convened on ''Methods for Determining Immunoreactivity of Radiolabeled Monoclonal Antibodies - Problems and Pitfalls,'' Radiobiological and Dosimetric Considerations for Immunotherapy with Labeled Antibodies,'' and ''The Human Anti-Mouse Antibody Response in Patients.''

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

Science.gov (United States)

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

2015-08-01

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

The biodistribution study of 99mTc labelled anti-CEA monoclonal antibody in tumor bearing nude mice

International Nuclear Information System (INIS)

Lou Zongxin

1992-01-01

The author report the optimal condition of 99m Tc labelling with anti-CEA monoclonal antibody using chelating of 99m Tc with dimethylformamide. The labelling rate of this method is 60%-80%, the radiochemical purity of labelling antibody over 90% and maintain its better immuno activity. The biodistribution of the tumor bearing nude mice demonstrates that as compared with the control group, 24 hours after the intraperitoneal injection the injected labelled antibody has its specific concentration in tumor tissue

Development and characterization of a TAPIR-like mouse monoclonal antibody to amyloid-beta.

Science.gov (United States)

Wang, Jun; Hara, Hideo; Makifuchi, Takao; Tabira, Takeshi

2008-06-01

Tissue amyloid plaque immuno-reactive (TAPIR) antibody was better related to the effect of immunotherapy in Alzheimer's disease (AD) than ELISA antibody. Here we used a hybridoma technique to develop a TAPIR-like anti-human amyloid-beta (Abeta) mouse monoclonal antibody. The obtained monoclonal antibody, 3.4A10, was an IgG2b isotype and recognized N-terminal portion of Abeta1-42 without binding denatured or native amyloid-beta protein precursor. It had higher affinity to Abeta1-42 than to Abeta1-40 by Biacore affinity analysis and stained preferably the peripheral part of senile plaques and recognized the plaque core less than 4G8. It inhibited the Abeta1-42 fibril formation as well as degraded pre-aggregated Abeta1-42 peptide in a thioflavin T fluorescence spectrophotometry assay. The in vivo studies showed that 3.4A10 treatment decreased amyloid burden compared to the control group and significantly reduced Abeta42 levels rather than Abeta40 levels in brain lysates as well as the Abeta*56 oligomer (12mer) in TBS fraction of the brain lysates. 3.4A10 entered brain and decorated some plaques, which is surrounded by more Iba1-positive microglia. 3.4A10 therapy did not induce lymphocytic infiltration and obvious increase in microhemorrhage. We conclude that 3.4A10 is a TAPIR-like anti-human amyloid monoclonal antibody, and has a potential of therapeutic application for AD.

The importance of immunohistochemical expression of EGFr in squamous cell carcinoma of the oral cavity treated with surgery and postoperative radiotherapy

International Nuclear Information System (INIS)

Smid, Ernst J.; Stoter, T. Rianne; Bloemena, Elisabeth; Lafleur, M. Vincent M.; Leemans, C. Rene; Waal, Isaac van der; Slotman, Ben J.; Langendijk, Johannes A.

2006-01-01

Purpose: The aim of this study was to investigate the prognostic significance of epidermal growth factor (EGFr) expression in oral cavity squamous cell carcinoma (OCSCC) treated with curative surgery and postoperative radiotherapy. Methods and Materials: This retrospective study included 165 OCSCC patients. The expression of EGFr was assessed on paraffin-embedded tissue of the primary tumor by immunohistochemistry using a monoclonal antibody directed against EGFr. Intensity of the EGFr expression was scored by two authors blinded for the clinical outcome. Results: In the univariate analysis, locoregional control at 3 years (LRC) in the EGFr-negative cases was 69% compared with 77% in the EGFr-positive cases (p 0.22). In the multivariate analysis for local control, a significant interaction was found between EGFr and overall treatment time of radiation (OTT). After stratification for EGFr expression, the OTT was of no importance in the EGFr-negative cases, whereas a significant difference in LRC was found in the EGFr-positive cases, in which the LRC after 3 years was 69% and 94% in case of an OTT of 0-42 days and >42 days, respectively (p = 0.009; hazard ratio = 3.42; 95% confidence interval, 1.28-8.96). No significant association was found between EGFr expression and overall survival. Conclusions: In the present study, no association was found between EGFr expression and outcome regarding locoregional control and overall survival. However, the results of the present study suggest that patients with squamous cell carcinoma of the oral cavity with high EGFr expression benefit more from a reduction of the overall treatment time of postoperative radiation than those with low EGFr expression

Characterization of sporozoite surface antigens of Plasmodium falciparum, using monoclonal antibodies. Part of a coordinated programme on the preparation of irradiated vaccines against some human diseases

International Nuclear Information System (INIS)

Groot, M.

1982-10-01

Sporozoites are considered as a source of potential vaccine. Characterization of their antigens is therefore important and can be achieved by monoclonal antibodies. The purpose of this project is to study the production of monoclonal antibodies against sporozoites of P. falciparum. Various infections of mosquitoes were carried out during the period 1981-1982 to obtain antigens for the production of hybridomas. Hybridomas were produced from mice immunized through the bites of infected mosquitoes and by intravenous inoculation. The anti-sporozoite activity of the hybridomas was tested by an immunofluorescent antibody test using P. falciparum sporozoites as antigens. Positive immunofluorescence was seen in hybridoma cell lines tested with P. falciparum, whereas negative results were obtained when the cell lines were cross-reacted with other human species (P. vivax) and with a rodent malaria parasite (P. berghei)

Targeted radiotherapy potentiates the cytotoxicity of a novel anti-human DR5 monoclonal antibody and the adenovirus encoding soluble TRAIL in prostate cancer

International Nuclear Information System (INIS)

Arafat, W.; Arafat, W.; Zhou, T.; Naoum, G.E.; Buchsbaum, D.J.

2015-01-01

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces a death signal following binding to death receptors (DR4, DR5). We have developed a novel anti-human DR-5 monoclonal antibody (TRA-8) and adenoviral encoding TRAIL (Ad/TRAIL). Herein, we are testing the combined effect of radiotherapy and TRA-8 or Ad TRAIL in prostate cancer cells. Human prostate cancer cell lines LnCap, PC-3 and DU145 were used in this study. Cells were treated either with TRA-8 alone or Ad/TRAIL, radiation alone, or a combination of each at different doses and intervals. Cell survival using the MTS assay and colony forming assay were used to determine radiosensitization. Immunohistochemistry was used to detect bax and bcl-2. Real-time PCR was performed on mRNA of treated prostate cancer cell lines. Finally, a murine model of subcutaneous prostate cancer was used to evaluate the in vivo effect. Cell survival assays detected by MTS assay showed that prostate cell lines treated with a combination of radiation and TRA-8 showed significantly lower survival than cells treated with either radiation or TRA-8 alone. Colony forming assay and cell proliferation assays showed increased killing after combination treatment with TRA-8 or Ad/TRAIL and radiation, than either single agent alone. Mechanistic studies showed that the killing effect was due to induction of apoptosis mostly by increased expression of bax in TRA-8 or Ad/TRAIL treated cells. Additionally, RT-PCR showed an increased copy number of bax in most cells treated with TRA-8 and radiation. It is concluded that radiation and TRA-8 or Ad/ TRAIL produced a synergistic effect in refractory prostrate cancer.

Generation of anti-idiotype antibodies for application in clinical immunotherapy laboratory analyses.

Science.gov (United States)

Liu, Zhanqi; Panousis, Con; Smyth, Fiona E; Murphy, Roger; Wirth, Veronika; Cartwright, Glenn; Johns, Terrance G; Scott, Andrew M

2003-08-01

The chimeric monoclonal antibody ch806 specifically targets the tumor-associated mutant epidermal growth factor receptor (de 2-7EGFR or EGFRVIII) and is currently under investigation for its potential use in cancer therapy. The humanised monoclonal antibody hu3S193 specifically targets the Lewis Y epithelial antigen and is currently in Phase I clinical trials in patients with advanced breast, colon, and ovarian carcinomas. To assist the clinical evaluation of ch806 and hu3S193, laboratory assays are required to monitor their serum pharmacokinetics and quantitate any immune responses to the antibodies. Mice immunized with ch806 or hu3S193 were used to generate hybridomas producing antibodies with specific binding to ch806 or hu3S193 and competitive for antigen binding. These anti-idiotype antibodies (designated Ludwig Melbourne Hybridomas, LMH) were investigated as reagents suitable for use as positive controls for HAHA or HACA analyses and for measuring hu3S193 or ch806 in human serum. Anti-idiotypes with the ability to concurrently bind two target antibody molecules were identified, which enabled the development of highly reproducible, sensitive, specific ELISA assays for determining serum concentrations of hu3S193 and ch806 with a 3 ng/mL limit of quantitation using LMH-3 and LMH-12, respectively. BIAcore analyses determined high apparent binding affinity for both idiotypes: LMH-3 binding immobilized hu3S193, Ka = 4.76 x 10(8) M(-1); LMH-12 binding immobilised ch806, Ka = 1.74 x 10(9) M(-1). Establishment of HAHA or HACA analysis of sera samples using BIAcore was possible using LMH-3 and LMH-12 as positive controls for quantitation of immune responses to hu3S193 or ch806 in patient sera. These anti-idiotypes could also be used to study the penetrance and binding of ch806 or hu3S193 to tumor cells through immunohistochemical analysis of tumor biopsies. The generation of anti-idiotype antibodies capable of concurrently binding a target antibody on each variable

Unique biological properties of catalytic domain directed human anti-CAIX antibodies discovered through phage-display technology.

Directory of Open Access Journals (Sweden)

Chen Xu

2010-03-01

Full Text Available Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC, but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule.

Human Monoclonal antibodies - A dual advantaged weapon to tackle cancer and viruses

Directory of Open Access Journals (Sweden)

Kurosawa G

2014-11-01

Full Text Available Human monoclonal antibodies (mAbs are powerful tools as pharmaceutical agents to tackle cancer and infectious diseases. Antibodies (Abs are present in blood at the concentration of 10 mg/ml and play a vital role in humoral immunity. Many therapeutic Abs have been reported since early 1980s. Human mAb technology was not available at that time and only the hybridoma technology for making mouse mAbs had been well established. In order to avoid various potential problems associated with use of mouse proteins, two different technologies to make human/mouse chimeric Ab as well as humanized Ab were developed crossing the various hurdles for almost twenty years and mAb based drugs such as rituximab, anti-CD20 Ab, and trastuzumab, anti-HER2 Ab, have been approved by the US Food and Drug Administration (FDA for treatment of non-Hodgkin's lymphoma and breast cancer in 1997 and 1998, respectively. These drugs are well recognized and accepted by clinicians for treatment of patients. The clinical outcome of the treatment with mAb has strongly encouraged the researchers to develop much more refined mAbs. In addition to chimeric Ab and humanized Ab, now human mAbs can be produced by two technologies. The first is transgenic mice that produce human Abs and the second is human Ab libraries using phage-display system. Until now, several hundreds of mAbs against several tens of antigens (Ags have been developed and subjected to clinical examinations. While many Abs have been approved as therapeutic agents against hematological malignancies, the successful mAbs against solid tumors are still limited. However, many researchers have suggested that developing potential mAbs agents should be possible and incurable cancers may become curable within another decade. Though it is hard to say explicitly that this prediction is correct, a passion for this development should be worth supporting to lead to a successful outcome which will lead to patient benefits. Our institute

Demonstration of two distinct antigenic determinants on hepatitis B e antigen by monoclonal antibodies

International Nuclear Information System (INIS)

Imai, M.; Nomura, M.; Gotanda, T.; Sano, T.; Tachibana, K.; Miyamoto, H.; Takahashi, K.; Toyama, S.; Miyakawa, Y.; Mayumi, M.

1982-01-01

Mice were immunized against hepatitis B e antigen (HBeAg) isolated from sera of asymptomatic carriers of hepatitis B virus. Their spleen cells were fused with mouse myeloma (NS-1) cells, and 5 clones of hybridoma cells secreting antibody against HBeAg (anti-HBe) were isolated. For the production of anti-HBe in large scale, cells were cultivated both in vitro and in the peritoneal cavity of ascitic mice. Although monoclonal antibodies produced by these clones showed a strong reactivity of anti-HBe in hemagglutination tests, individual monoclonal anti-HBe did not reveal any precipitin line in immunodiffusion. When 2 of the 5 monoclonal antibodies were mixed together, however, some combinations showed a precipitin line against HBeAg, whereas others did not. Utilizing solid-phase radioimmunoassay involving a number of combinations of monoclonal antibodies used for solid-phase and radiolabeling, the 5 antibodies were classified into 2 groups. Three of the anti-HBe antibodies were found to be directed to 1 determinant of HBeAg (determinant a); the remaining 2 to the other determinant (determinant b). Determinants a and b were detected on HBeAg in the serum, as well as on the polypeptide of 19,000 daltons (P19) derived from the nucleocapsid of hepatitis B virus. Monoclonal anti-HBe antibodies with different specificities may provide useful tools in delineating the antigenic structure of HBeAg and also in evaluating immune responses of the host directed to its subdeterminants

Evaluation of epidermal growth factor receptor (EGFR) by chromogenic in situ hybridization (CISH) and immunohistochemistry (IHC) in archival gliomas using bright-field microscopy.

Science.gov (United States)

Marquez, Abbey; Wu, Rina; Zhao, Jianxin; Tao, Jianhua; Shi, Zuorong

2004-03-01

Overexpression of EGFR secondary to EGFR gene amplification is a common feature in primary malignant gliomas. To correctly assess EGFR protein and gene level as possible prognostic and predictive markers in gliomas, straightforward assays, which can be used routinely in the pathology laboratory to evaluate EGFR status, becomes critical. EGFR gene amplification and chromosome 7 aneuploidy was detected in 34 formalin-fixed, paraffin-embedded benign and malignant gliomas by chromogenic in situ hybridization (CISH) using digoxigenin-labeled EGFR and biotin-labeled chromosome 7 centromeric probes. The results were evaluated by bright-field microscopy under a 40x objective lens. EGFR protein level was detected by immunohistochemistry (IHC) using monoclonal antibody 31G7. Five cases, 3 astrocytoma grade III (33%) and 2 glioblastoma multiforme (GBM) (33%), had EGFR amplification displayed as diaminobenzidine-stained multiple dots suggesting the pattern of double-minute chromosomes. Chromosome 7 polysomy was found in 68% gliomas, 100% GBM, 67% astrocytoma grade III, 42% astrocytoma grade II, 50% astrocytoma grade I, 100% ependymoma, and the 1 case of mixed glioma III. High expression of EGFR protein was present in 62% gliomas and displayed membrane and cytoplasmic staining. All tumors with EGFR gene amplification showed EGFR high expression. High expression of EGFR without gene amplification was observed in all grades of gliomas. Simultaneous detection of EGFR gene copies or chromosome 7 centromere signals along with tissue morphology allows us to compare CISH results easily with IHC results. Our results show that CISH is an objective, practical, and accurate assay to screen for EGFR gene status in gliomas.

A monoclonal antibody that specifically recognizes m6A nucleoside

OpenAIRE

Espuny, Ruth; Castro, Ana; Codony, Carles; Eritja Casadellà, Ramón; Bach-Elias, Montse

1998-01-01

A hybridoma against the nucleoside m6A has been obtained from mouse spleen. This hybridoma was named H65 and it secretes monoclonal antibodies anti-m6A. The competition assays showed that the monoclonal antibody was highly specific for m6A nucleoside.

Homology of ab1 and ab3 monoclonal antibodies that neutralize Semliki Forest virus

NARCIS (Netherlands)

Fernandez, IM; Bos, NA; Harmsen, M; Verheul, AFM; Snippe, H; Kraaijeveld, CA

2001-01-01

A noninternal image monoclonal antiidiotypic antibody (ab2 mAb), designated 1,13A321, that had proved its efficacy as vaccine against infection with Semliki Forest virus (SFV) in BALB/c mice, was used as immunogen to generate a panel of SFV-neutralizing monoclonal anti-anti-idiotypic antibodies (ab3

Induction of a systemic lupus erythematosus-like disease in mice by a common human anti-DNA idiotype

International Nuclear Information System (INIS)

Mendlovic, S.; Brocke, S.; Meshorer, A.; Mozes, E.; Shoenfeld, Y.; Bakimer, R.; Ben-Bassat, M.

1988-01-01

Systemic lupus erythematosus (SLE) is considered to be the quintessential autoimmune disease. It has not been possible to induce SLE in animal models by DNA immunization or by challenge with anti-DNA antibodies. The authors report a murine model of SLE-like disease induced by immunization of C3H.SW female mice with a common human monoclonal anti-DNA idiotype (16/6 idiotype). Following a booster injection with the 16/6 idiotype, high levels of murine anti-16/6 and anti-anti-16/6 antibodies (associated with anti-DNA activity) were detected in the sera of the immunized mice. Elevated titers of autoantibodies reacting with DNA, poly(I), poly(dT), ribonucleoprotein, autoantigens [Sm, SS-A (Ro), and SS-B (La)], and cardiolipin were noted. The serological findings were associated with increased erythrocyte sedimentation rate, leukopenia, proteinuria, immune complex deposition in the glomerular mesangium, and sclerosis of the glomeruli. The immune complexes in the kidneys were shown to contain the 16/6 idiotype. This experimental SLE-like model may be used to elucidate the mechanisms underlying SLE

A human/mouse chimeric monoclonal antibody against intercellular adhesion molecule-1 for tumor radioimmunoimaging

International Nuclear Information System (INIS)

Yamamura, Miyuki; Hinoda, Yuji; Sasaki, Shigeru; Tsujisaki, Masayuki; Imai, Kohzoh; Oriuchi, Noboru; Endo, Keigo.

1996-01-01

A mouse-human chimeric antibody for intercellular adhesion molecule-1 (ICAM-1) was established by using heavy chain loss mouse mutant hybridoma and human immunoglobulin expression vector. The HA58 hybridoma secreted anti-ICAM-1 monoclonal antibody (MoAb) (IgG1,κ). The gene of the mouse variable region of heavy chain was amplified and cloned by the polymerase chain reaction technique directly from the HA58 hybridoma RNA. The variable region of heavy chain was joined with an expression vector which contains human γ1 constant gene. The expression vector was transfected into heavy chain loss mutant cells HA58-7, which produced only murine immunoglobulin light chains. The resultant chimeric MoAb HA58, chHA58, retained full-binding reactivity to ICAM-1 compared with murine HA58 parental antibody. The chimeric MoAb chHA58 showed little antibody dependent cell-mediated cytotoxic activity against cultured tumor cells. Biodistribution studies with 99m Tc-labeled chHA58 in nude mice bearing human gastric carcinoma JRST cells, demonstrated that the tumor-blood ratio was 1.55 at 18 h after injection, when the tumors were clearly visible in gamma scintigraphy. These data suggest that chHA58 may be of practical use for radioimmunoimaging of a wide variety of tumors. (author)

Human monoclonal antibody 99mTc-88BV59: detection of colorectal cancer, recurrent or metastatic disease and immunogenicity assessment.

Science.gov (United States)

Krause, B J; Baum, R P; Staib-Sebler, E; Lorenz, M; Niesen, A; Hör, G

1997-01-01

This study presents immunoscintigraphic results in 24 patients suffering from primary colorectal cancer, recurrent or metastatic disease after the injection of 1197-1351 MBq technetium-99m labelled totally human monoclonal antibody 88BV59. Labelling efficacy of 99mTc-88BV59 ranged from 97% to 99%. Immunoscintigraphy was performed 18-20 h after injection. Scintigraphic findings were compared with those of computed tomography (CT). Patients underwent surgery in order to evaluate immunoscintigraphic findings histologically. Sera of the patients (before injection and 1 and 3 months post infusion) were analysed for the presence of human anti-human antibodies (HAHA). None of the patients showed a HAHA response as assessed by a solid-phase ELISA assay. The antibody scan detected about 25% more lesions than CT. In the detection of extrahepatic disease, the sensitivity of the antibody scan proved to be 68%, whereas the sensitivity of CT was 41%.

Mapping of cat albumin using monoclonal antibodies: identification of determinants common to cat and dog.

Science.gov (United States)

Boutin, Y; Hébert, J; Vrancken, E R; Mourad, W

1989-01-01

Cat and dog albumins from commercial extracts were used to produce monoclonal antibodies (MoAb). Anti-cat albumin MoAb recognized both cat and dog albumin equally, as did anti-dog albumin MoAb; this confirms cross-reactivity between cat and dog. The MoAb were separated into two groups according to their epitopic specificity; they recognized two overlapping epitopes of cat albumin. Furthermore, by competitive inhibition of radio-allergosorbent test (RAST), it was shown that one MoAb group inhibited significantly the binding of human IgE antibodies (from a pool of 13 patients allergic to both cats and dogs) to insolubilized cat or dog extracts. These observations suggest that murine anti-cat or anti-dog MoAb and human IgE antibodies recognize identical or closely related determinants on cat and dog albumin. Images Fig. 1 Fig. 2 PMID:2478325

Facilitation of syngeneic stem cell engraftment by anti-class I monoclonal antibody pretreatment of unirradiated recipients

International Nuclear Information System (INIS)

Voralia, M.; Semeluk, A.; Wegmann, T.G.

1987-01-01

We have established a murine model of syngeneic bone marrow transplantation based on the use of monoclonal antibody as the sole conditioning regimen in unirradiated recipients. Administration of a single injection of monoclonal antibody directed against major histocompatibility complex-encoded class I determinants facilitated permanent hemopoietic stem cell engraftment without any apparent side-effects. Whereas untreated hosts exhibited a maximal chimerism of 15% at donor cell doses of up to 12 X 10(7) bone marrow cells, pretreatment by 2 mg of anti-class I antibody one week prior to transplantation of 3 X 10(7) syngeneic bone marrow cells resulted in a mean donor representation of about 80%. The antibody can be given up to four weeks prior to transplantation, and the degree of donor engraftment observed is a function of the dose of antibody administered. The fact that specific antibody enhanced engraftment in two strain combinations indicates that antibody is the active agent in facilitating engraftment and that facilitation is not strain-restricted. Anti-class I antibodies of the IgG2a, but not IgG1, isotype are effective in promoting engraftment. Although the isotype requirement suggests a role for antibody-mediated cytotoxicity in promoting stem cell engraftment, the extensive time-frame of facilitation suggests that other effects of the antibody may also be involved. The model of syngeneic bone marrow transplantation we describe here will be useful in studying the mechanisms regulating stem cell engraftment and may have potential clinical application as an approach to autologous marrow transplantation

Immunoglobulin variable region sequences of two human monoclonal antibodies directed to an onco-developmental carbohydrate antigen, lactotetraosylceramide (LcOse4Cer).

Science.gov (United States)

Yago, K; Zenita, K; Ohwaki, I; Harada, R; Nozawa, S; Tsukazaki, K; Iwamori, M; Endo, N; Yasuda, N; Okuma, M

1993-11-01

A human monoclonal antibody, 11-50, was generated and was shown to recognize an onco-developmental carbohydrate antigen, LcOse4Cer. The isotype of this antibody was IgM, lambda, similar to the previously known human anti-LcOse4 antibodies, such as IgMWOO and HMST-1. We raised a murine anti-idiotypic antibody G3 (IgG1, kappa) against 11-50, and tested its reactivity towards the affinity purified human polyclonal anti-LcOse4 antibodies prepared from pooled human sera using a Gal beta 1-->3GlcNAc beta-immobilized column. The results indicated that at least a part of the human polyclonal anti-LcOse4 antibodies shared the G3 idiotype with 11-50. We further analyzed the sequence of variable regions of the two anti-LcOse4 antibodies, 11-50 and HMST-1. Sequence analysis of the heavy chain variable regions indicated that the VH regions of these two antibodies were highly homologous to each other (93.5% at the nucleic acid level), and these antibodies utilized the germline genes VH1.9III and hv3005f3 as the VH segments, which are closely related germline genes of the VHIII family. It was noted that these germline VH genes are frequently utilized in fetal B cells. The JH region of both antibodies was encoded by the JH4 gene. For the light chain, the V lambda segments of the two antibodies were 96.3% homologous to each other at the nucleic acid level. The V lambda segments of both antibodies showed the highest homology to the rearranged V lambda gene called V lambda II.DS among reported V lambda genes, while the exact germline V lambda genes encoding the two antibodies were not yet registered in available sequence databanks. The amino acid sequences of the J lambda segments of both antibodies were identical. These results indicate that the two human antibodies recognizing the onco-developmental carbohydrate antigen Lc4 are encoded by the same or very homologous germline genes.

Development and Characterization of a Humanized Anti-HER2 Antibody HuA21 with Potent Anti-Tumor Properties in Breast Cancer Cells

Directory of Open Access Journals (Sweden)

Ruilin Li

2016-04-01

Full Text Available Human epidermal growth factor receptor 2 (HER2 is one of the most studied tumor-associated antigens for cancer immunotherapy. An engineered anti-HER-2 chimeric A21 antibody (chA21 is a chimeric antibody targeted to subdomain I of the HER2 extracellular domain. Here, we report the anti-tumor activity of the novel engineered monoclonal antibody humanized chA21 (HuA21 that targets HER2 on the basis of chA21, and we describe the underlying mechanisms. Our results reveal that HuA21 markedly inhibits the proliferation and migration of HER2-overexpressing breast cancer cells and causes enhanced antibody-dependent cell-mediated cytotoxicity potency against HER2-overexpressing tumor cells. In particular, HuA21, but not trastuzumab (Tra, markedly suppresses growth and enhances the internalization of the antibody in Tra-resistant BT-474 breast cancer cells. These characteristics are highly associated with the intrinsic ability of HuA21 to down-regulate HER2 activation and inhibit the extracellular signal-regulated kinase 1/2 (ERK1/2 and protein kinase B (Akt signaling pathways. Furthermore, the combination of HuA21 with Tra synergistically enhances the anti-tumor effects in vitro and in vivo and inhibits HER2 activation and the ERK1/2 and Akt signaling pathways. Altogether, our results suggest that HuA21 may represent a unique anti-HER2 antibody with potential as a therapeutic candidate alone or in combination with other anti-HER2 reagents in cancer therapy.

Experimental study on 211At labelled monoclonal antibody 3H11 and its Fab fragment radioimmunotherapy for human gastric cancer xenografts in nude mice

International Nuclear Information System (INIS)

Jin Jiannan; Liu Ning; Zhang Shuyuan; Zhang Shiyuan; Luo Deyuan; Zhou Maolun

1996-01-01

Experimental radioimmunotherapy investigation of α-emitting radionuclide 211 At labelled anti-gastric cancer monoclonal antibody 3H11 and its Fab fragment for nude mice carrying human gastric cancer xenografts was conducted. Three i.p. injections of 14.8 or 22.2 kBq/g mouse were given, once every 5 days. The results showed that the growth of tumor xenografts was inhibited efficiently. The most evident therapy effect was observed at 15 days after treatment, and the tumor inhibition rates were 65% and 72%, respectively. No radiation injury of important organs was found

Purification of HBsAg produced by the human hepatoma cell line PLC/PRE/5 by affinity chromatography using monoclonal antibodies and application for ELISA diagnostic.

Science.gov (United States)

Merten, O W; Reiter, S; Scheirer, W; Katinger, H

1983-01-01

The human cell line PLC/PRF/5 (5) was used for the production of hepatitis B surface antigen subtype ad (HBsAg ad) and purified by affinity chromatography (AC) with monoclonal antibodies (mAb). mAb to HBsAg from mouse ascites have been purified by Protein A - AC prior coupling to AH-Sepharose 4B (Pharmacia). The combined procedure of ammonium-sulphate-precipitation of HBsAg from culture supernatants and immunosorbent-AC leads to approx. 700-fold purification. ELISA results using the mAb and the HBsAg for diagnostics of human serum, positive for anti-HBsAg-antibodies correlate with the RIA (AUSAB, Abbott).

Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of “preneoplastic antigen”-like molecules

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Duan, Hongying [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Yoshimura, Kazunori [Department of Physiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kobayashi, Nobuharu; Sugiyama, Kazuo [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Sawada, Jun-ichi; Saito, Yoshiro [Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

2012-04-01

Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases. -- Highlights: ► Monoclonal antibodies against different portions of mEH were developed. ► They discriminate between the membrane-bound and the linearized forms of mEH. ► We analyze the antigenic structure of the altered form of mEH in tumor cells. ► Preneoplastic antigen is a multimolecular complex of mEH with

Development of broad-spectrum human monoclonal antibodies for rabies post-exposure prophylaxis

International Nuclear Information System (INIS)

Benedictis, P. de; Minola, A.; Rota, E.; Aiello, R.; Zecchin, B.; Salomoni, A.; Foglierini, M.; Agatic, G.; Vanzetta, F.; Lavenir, R.; Lepelletier, A.; Bentley, E.; Weiss, R.; Cattoli, G.

2016-01-01

Full text: Currently available rabies post-exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad-spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non-RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20–RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post-vaccination antibody response. (author)

Nuclear medicine: Monoclonal antibodies

International Nuclear Information System (INIS)

Endo, K.; Sakahara, H.; Koizumi, M.; Kawamura, Y.; Torizuka, K.; Yokoyama, A.

1986-01-01

Antitumor monoclonal antibody was successfully labeled with Tc-99m by using dithiosemicarbazone (DTS) as a bifunctional chelating agent. In the first step, DTS was coupled to antibody without loss of immunoreactivity; the compound then efficiently formed a neutral 1:1 chelate with pentavalent or tetravalent Tc-99m. Imaging with Tc-99m-labeled monoclonal antibody to human osteosarcoma (OST-7) clearly displayed a small tumor in nude mice at 6 and 24 hours after intravenous administration. The tumor-to-blood ratio of the Tc-99m-labeled monoclonal antibody was higher than that of a radioiodinated antibody and similar to that of an In-111-labeled antibody. Thus, conjugation of DTS to monoclonal antibody followed by radiometalation is a simple and efficient method of preparing Tc-99m-labeled monoclonal antibody

Overview of the trastuzumab (Herceptin) anti-HER2 monoclonal antibody clinical program in HER2-overexpressing metastatic breast cancer. Herceptin Multinational Investigator Study Group.

Science.gov (United States)

Shak, S

1999-08-01

The recombinant humanized anti-HER2 monoclonal antibody trastuzumab (Herceptin; Genentech, San Francisco, CA) was evaluated in human clinical trials for treatment of women with metastatic breast cancer who have tumors that overexpress HER2. The trastuzumab clinical program consisted of a series of phase I, phase II, and phase III clinical trials. Clinical experience with this novel biologic has been obtained in more than 1,000 women with HER2-overexpressing metastatic breast cancer. Two pivotal trials were performed to evaluate trastuzumab efficacy and safety: (1) trastuzumab in combination with chemotherapy as first-line therapy and (2) trastuzumab as a single agent in second- and third-line chemotherapy. Preliminary results of the pivotal clinical trials that have been presented at national meetings are summarized below. The data suggest that trastuzumab will be an important new treatment option for women with HER2-overexpressing metastatic breast cancer.

The humanized anti-human AMHRII mAb 3C23K exerts an anti-tumor activity against human ovarian cancer through tumor-associated macrophages.