Construction of Anti-CD3 Human/Murine Chimeric Antibody Genes for Expression in Mammalian Cells / 中国肿瘤生物治疗杂志

For construction of anti-CD3 human/murine chimeric antibody genes, a selective first-strand cDNA synthesis from mRNA or RNA of murine McAb HIT3a was performed using murine Ig constant region primers, and then cDNA of heavy and light chain variable domains of murine immunoglobulin were amplified by PCR using a set of degenerated oligonucleotide primers. Using these cDNA fragments as probes, the L and H chain V region exons encoding the murine McAb anti-CD3 were isolated from the gene library of HIT3a DNA and inserted into mammalian expression vectors containing the human ? and yl constant region exons for construction of human/murine chimeric antibody genes.

Expression and Characterization of Human/Murine Chimeric Antibodies with Specificity for CD3 Antigen / 中国肿瘤生物治疗杂志

Exons encoding the variable regions of the light and heavy chain of the murine McAb HIT3a against CD3 antigen were isolated from HIT3a gene library and inserted into mammalian expression vectors containing the human K and y1 region exons. The chimeric genes were transfected into murine SP2/0 hybridoma cells by Lipofectin. Antibody levels of culture supernatants from transfectomas ranged 21~32 ?g/ml. The chimeric antibodies bound specifically to human T cells and competed effectivelly with the parental anti-CD3 murine McAb for binding to CD3 antigen on human T cells. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced with the chimeric antibodies as compared with anti-CD3 murine McAb. The mitogenic activity of human T cells can be suppressed and enhanced by the anti-CD3 chimeric antibodies. The cytotoxicity to tumor cells and proliferation of human T cells mediated by the anti-CD3 chimeric antibodies were much higher than IL-2 alone. Chimeric HIT3a antibody is a clinically relevant, genetically engineering antibody with potential use in treatment of graft-versus-host disease in tranplantation, autoimmune diseases and some tumors.

A group of monoclonal antibodies reactive with the human thymocyte differentiation antigens-Production and specificity analysis / 中国免疫学杂志

In a fusion of BABL/C murine spleen cells immunizated with human fetal thymocytes and P_3X_(3)Ag_(,3), myeloma cells, six monoclonal antibodies(McAb) were produced. They were termed HIT_1. HIT_2. HIT_3. HIT_4.HIT_(6-1) and HIT_(6-2), respectively. The specificity of these McAbs were analysed by indirect immunofluorescence technique and FACS.Results showed that they reacted with 80～90%thymocytes,but hardly with peripheral blood mononuclear cells and spleen cells in adults,and nonreactive with red blood cells, granulocytes and platelets, According to their reaction with the tonsil cells, we can divide these six McAbs into three groups: Groupl including HIT_1, HIT_2, and HIT_(?) McAbs reacted approximately with 1/3 tonsil cells; basically GroupⅡ including HIT_(6-1) and HIT_(6-2) McAbs gave negative reaction with tonsil cells; GroupⅢ McAb HIT_4 reacted with 15% tonsil cells, which suggested these were a heterogeneous group McAbs with different specificities. In comparision with OKT series of McAbs in thymus, peripheral blood and tonsil, HIT_(1-3) are similar to OKT_(10) and,HTT6-l and HIT_(6-2) are just like OKT_6,but HIT_4 seems to be a new McAb different frOm HIT_(1-3) and HIT_(6-1) HII_(6-2). The competitive binding assay showed that HIT_(6-1) and HIT_(6-2) labeled with FITC can be inhibited by unlabeled HIT_(6-1) and HIT_(6-2) each other and can also be blocked by OKT_6, suggesting further these antibodies recognized a same epitope on thymocytes. Cross reaction were also demonstrated on HIT_1, and HIT_2 but not on HIT_3, suggesting HIT_1 and HIT_2 recognized the same determinant and HIT_3 recognized another. So six antibodies are McAbs against T cell differentiation antigens.They are useful for research the differentiation of T cells and the classification of malignant lymphadenosis diseases.