ABSTRACT
Chimeric immunoglobulin genes were constructed by fusing murine variable region exons to human constant region exons. The ultimate goal was to produce an antibody capable of escaping surveillance by the human immune system while retaining the tumor specificity of a murine monoclonal. The murine variable regions were isolated from the functionally expressed kappa and gamma 1 immunoglobulin genes of the murine hybridoma cell line B6.2, the secreted monoclonal antibody of which reacts with a surface antigen from human breast, lung, and colon carcinomas. The kappa and gamma 1 chain fusion genes were co-introduced into non-antibody producing murine myeloma cells by electroporation. Transfectants that produced murine/human chimeric antibody were obtained at high frequency as indicated by immunoblots probed with an antisera specific for human immunoglobulin. Enzyme-linked immunoabsorbent assay analysis demonstrated that this chimeric antibody was secreted from the myeloma cells and retained the ability to bind selectively to membrane prepared from human tumor cells. The chimeric immunoglobulin was also shown by indirect fluorescence microscopy to bind to intact human carcinoma cells with specificity expected of B6.2. The ability of chimeric antibody to recognize human tumor-associated antigen makes feasible a novel approach to cancer immunotherapy.
Subject(s)
Antibodies, Monoclonal/genetics , Antibodies, Neoplasm/genetics , Antibody Specificity , Chimera , Genetic Engineering , Animals , Base Sequence , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/genetics , Mice , Mice, Inbred C57BL , TransfectionABSTRACT
cDNA clones carrying parts of murine fourth complement component (C4, serum substance protein) mRNA sequences have been identified by differential hybridization to mRNA from a high C4-producing strain, B10.WR, and a congeneic low C4 strain, B10.BR, followed by hybrid-selected translation and DNA sequence analysis. One clone, pMLC4/w7-2, encodes an open amino acid reading frame that includes four tandem arginine residues immediately preceding a sequence 85% homologous with the NH2-terminal sequence of the human C4 gamma-chain. The amino acid composition of the predicted sequence upstream of the tandem arginines matches quite closely with the composition of a similar sized peptide at the COOH terminus of the human C4 alpha chain. The latter result raises questions regarding the nature and extent of plasma-mediated postsynthetic processing of the C4 alpha-chain COOH terminus. The results also demonstrate that strain differences in plasma C4 levels (low C4 vs. high C4) reflect differences in steady-state levels of liver C4 mRNA in these strains.
