Secretion of a single-gene-encoded immunoglobulin from myeloma cells.
We describe construction of a single gene encoding a single-chain immunoglobulin-like molecule. This single-gene approach circumvents inefficiencies inherent in delivering two genes into a mammalian cell and in the assembly of a functional immunoglobulin molecule. It would also facilitate ex vivo transfection of cells for gene-therapy protocols. SP2/0 murine myeloma cells transfected with the single gene SG delta CLCH1 expressed a single-chain protein, SC delta CLCH1, comprising approximately 60 kDa of the anti-carcinoma monoclonal antibody (mAb) CC49. The single-chain protein consisted of the heavy- and light-chain variable (VH and VL) domains of the mAb covalently joined through a short linker peptide, while the carboxyl end of the VL domain was linked to the amino terminus of the human gamma 1 Fc region through the hinge region. The single-chain protein assembled into a dimeric molecule, termed SCA delta CLCH1, of approximately 120 kDa and was secreted into the tissue culture fluid. SDS/PAGE analysis of the secreted immunoglobulin purified by protein G affinity chromatography confirmed the size of the molecule. The native mAb CC49 and SCA delta CLCH1 of CC49 showed similar binding to the tumor-associated glycoprotein TAG-72, and the chimeric mAb CC49 and SCA delta CLCH1 showed similar cytotoxic activity. This single-gene construct approach provides a way of generating an immunoglobulin-like molecule which retains the specificity, binding properties, and cytolytic activity of the chimeric mAb CC49. The immunoglobulin-like molecule SCA delta CLCH1 is potentially a therapeutic and diagnostic reagent against a range of human carcinomas.
Shu
L
L
Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Qi
C F
CF
Schlom
J
J
Kashmiri
S V
SV
D000818:Animals; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D005784:Gene Amplification; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007136:Immunoglobulins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000235:genetics / Q000276:immunology*; D009838:Oligodeoxyribonucleotides; D014162:Transfection; D014407:Tumor Cells, Cultured
D007136:Immunoglobulins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides