RESUMO
The Lewis Y (Ley) antigen is a blood group-related antigen that is expressed in a high proportion of epithelial cancers (including breast, colon, ovary, and lung cancer) and is an attractive target for monoclonal antibody-directed therapy. The murine monoclonal 3S193 (IgG3) was generated in BALB/c mice by immunization with Ley-expressing cells of the MCF-7 breast carcinoma cell-line. The murine 3S193 showed high specificity for Ley in ELISA tests with synthetic Ley and Ley-containing glycoproteins and glycolipids and also reacted strongly in rosetting assays and cytotoxic tests with Ley-expressing cells. We generated a humanized form of the murine 3S193 antibody by linking cDNA sequences encoding the variable region of murine 3S913 with frameworks of the human KOL heavy chain and REI K chain. The genes for the humanized 3S193 monoclonal antibody IgG1 were transfected into mouse myeloma NS0 cells and cloned for the establishment of high antibody-producing colonies. Humanized 3S193 antibody was subsequently produced through in vitro culture and under good manufacturing practice conditions using hollow-fiber bioreactors. The purified humanized 3S193 (hu3S193) was subsequently characterized and validated for use in preliminary immunotherapy investigations. hu3S193 reacted specifically with Ley antigen, with similar avidity to the murine form. hu3S193 demonstrated potent immune effector function, with higher antibody-dependent cell-mediated cytotoxicity than its murine counterpart and potent complement-dependent cytotoxicity (ED50, 1.0 microg/ml). The in vivo immunotherapeutic potential of hu3S193 was assessed in a human breast xenograft model using MCF-7, Ley-positive cells. Six i.v. doses of up to 1 mg of hu3S193 were administered to animals bearing established tumors (120-130 mm3) with no significant effect on tumor growth. In contrast, in an MCF-7 xenograft preventive model, a 1-mg hu3S193 dosage schedule was able to significantly slow tumor growth compared with placebo and isotype-matched control IgG1 antibody. hu3S193 has promise for immunotherapy of Ley-positive tumors and is currently entering Phase I clinical trials.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Neoplasias/terapia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Técnicas Biossensoriais , Neoplasias da Mama/terapia , Clonagem Molecular , DNA Complementar/metabolismo , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Humanos , Hibridomas/imunologia , Cinética , Neoplasias Mamárias Experimentais/terapia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Transplante de Neoplasias , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Chromosome counts were performed on 1,100 cells from 17 malignant breast carcinomas and on 168 cells of four normal tissue samples after amethopterin treatment and G-banding. Karyotypes were established from 216 cells of 11 tumor-derived cultures and from 47 cells of four nonmalignant tissue-derived cultures. Karyotypes of cells from nonmalignant samples showed a normal diploid chromosomal constitution with no consistent loss or gain of a specific chromosome. Structural chromosomal abnormalities were not observed. Tumor-derived cultures could be distinguished from normal cultures on the basis of a significantly increased incidence of numerical changes and structural chromosomal aberrations. In nine of 11 tumor-derived cultures, numerically normal cells were shown to be pseudodiploid, with frequencies ranging to 43% (mean, 13.2%) of the diploid cells. In agreement with previous reports, cytogenetic analyses showed predominantly diploid cells. Clonal numerical changes of chromosomes 17, 18, 20, and 21 could be detected in three tumor samples. Clonal structural abnormalities could be observed in two of 11 analyzed tumours. A t(6;12)(p21;p13) and an enlarged chromosome 7 (7q+) were found in a patient with invasive ductal carcinoma. An inversion of chromosome 7 [inv(7)(q11.2q32)] was observed in one case, also diagnosed as invasive ductal carcinoma. The significance of these findings in relation to clinical data is discussed.
Assuntos
Neoplasias da Mama/genética , Aberrações Cromossômicas , Adulto , Idoso , Feminino , Marcadores Genéticos , Humanos , Cariotipagem , Pessoa de Meia-Idade , PloidiasAssuntos
Neoplasias da Mama/análise , Mama/citologia , Receptores ErbB/análise , Queratinas/análise , Vimentina/análise , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Células Cultivadas/análise , Células Cultivadas/citologia , Meios de Cultura , Técnicas de Cultura/métodos , Citogenética , Células Epiteliais , Epitélio/análise , Humanos , Proteína Quinase C/metabolismo , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Células Tumorais CultivadasRESUMO
Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-2 (VEGFR-2/Flk1/KDR) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-D consists of a central receptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF-D. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-D. The anti-(VEGF-D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-D.
Assuntos
Anticorpos Monoclonais/imunologia , Fatores de Crescimento Endotelial/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Fatores de Crescimento/antagonistas & inibidores , Afinidade de Anticorpos , Especificidade de Anticorpos , Sequência de Bases , Divisão Celular/imunologia , Células Cultivadas , Primers do DNA , Fatores de Crescimento Endotelial/imunologia , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Mapeamento de Epitopos , Humanos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator D de Crescimento do Endotélio Vascular , Receptor 3 de Fatores de Crescimento do Endotélio VascularRESUMO
The kinetics, binding equilibria and stoichiometry of the interaction between epidermal growth factor and the soluble extracellular domain of the epidermal growth factor receptor (sEGFR), produced in CHO cells using a bioreactor, have been studied by three methods: analytical ultracentrifugation, biosensor analysis using surface plasmon resonance detection (BIAcore 2000) and fluorescence anisotropy. These studies were performed with an sEGFR preparation purified in the absence of detergent using a mild two step chromatographic procedure employing anion exchange and size exclusion HPLC. The fluorescence anisotropy and analytical ultracentrifugation data indicated a 1:1 molar binding ratio between EGF and the sEGFR. Analytical ultracentrifugation further indicated that the complex comprised 2EGF:2sEGFR, consistent with the model proposed recently by Lemmon et al. (1997). Global analysis of the BIAcore binding data showed that a simple Langmuirian interaction does not adequately describe the EGF:sEGFR interaction and that more complex interaction mechanisms are operative. Furthermore, analysis of solution binding data using either fluorescence anisotropy or the biosensor, to determine directly the concentration of free sEGFR in solution competition experiments, yielded Scatchard plots which were biphasic and Hill coefficients of less than unity. Taken together our data indicate that in solution there are two sEGFR populations; one which binds EGF with a KD of 2-20 nM and the other with a KD of 400-550 nM.