EXPRESSION AND SECRETION OF A RECOMBINANT RICIN IMMUNOTOXIN FROM MURINE MYELOMA CELLS

Expression plasmids carrying a humanized N901 immunoglobulin heavy cha in gene (hN901HC) fused to a gene encoding the native B chain of ricin toxin (RTB), hN901HC-RTB, or a sugar binding mutant of RTB, hN901HC-R TB Delta gly, were constructed. In each case, the fused gene construct ions were co-expressed in murine myeloma cells (Sp2/0) with the gene f or humanized N901 immunoglobulin light chain to produce the secreted r ecombinant products hN901-RTB and hN901-RTB Delta gly, respectively, W hen purified by affinity chromatography, both the hN901-RTB and hN901- RTB Delta gly products were found to have an apparent molecular mass o f M(r) = 210 000 and to be composed of two hN901 antibody heavy chains each fused to a full-length copy of RTB and two hN901 antibody light chains, In each of the recombinant fusions the hN901 antibody moiety r etained the full binding affinity and specificity for its cognate anti gen, CD56, Moreover, when mixtures of hN901-RTB and native ricin A cha in were incubated in the presence of the antigen-positive target cell line SW-2, antigen-specific potentiation of ricin A chain cytotoxicity was observed. It has been demonstrated previously that lectin activit y of the B chain is essential for A chain cytotoxicity, and we conclud e that the fused wild-type B chain was properly folded and maintained lectin activity, These data demonstrate the feasibility of using recom binant ricin B chain in an immunotoxin and of using mammalian cell cul ture for its expression, The use of recombinant hN901-RTB fusion prote in to evaluate the contribution of the lectin activity of ricin B chai n in the penetration of cell membranes by ricin A chain is proposed.