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 SGDELTAC(L)C(H)1 expressed a single-chain protein, SCDELTAC(L)C(H)1, comprising almost-equal-to 60 kDa of the anti-carcinoma monoclonal antibody (mAb) CC49. The single-chain protein consisted of the heavy- and light-chain variable (V(H) and V(L)) domains of the mAb covalently joined through a short linker peptide, while the carboxyl end of the V(L) domain was linked to the amino terminus of the human gamma1 Fc region through the hinge region. The single-chain protein assembled into a dimeric molecule, termed SCADELTAC(L)C(H)1, of almost-equal-to 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 SCADELTAC(L)C(H)1 of CC49 showed similar binding to the tumor-associated glycoprotein TAG-72, and the chimeric mAb CC49 and SCADELTAC(L)C(H)1 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 SCADELTAC(L)C(H)1 is potentially a therapeutic and diagnostic reagent against a range of human carcinomas.