Expression and secretion of a recombinant ricin immunotoxin from murine myeloma cells.
Expression plasmids carrying a humanized N901 immunoglobulin heavy chain 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-RTB delta gly, were constructed. In each case, the fused gene constructions were co-expressed in murine myeloma cells (Sp2/0) with the gene for humanized N901 immunoglobulin light chain to produce the secreted recombinant products hN901-RTB and hN901-RTB delta gly, respectively. When purified by affinity chromatography, both the hN901-RTB and hN901-RTB delta gly products were found to have an apparent molecular mass of 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 retained the full binding affinity and specificity for its cognate antigen, CD56. Moreover, when mixtures of hN901-RTB and native ricin A chain 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 activity of the B chain is essential for A chain cytotoxicity, and we conclude that the fused wild-type B chain was properly folded and maintained lectin activity. These data demonstrate that feasibility of using recombinant ricin B chain in an immunotoxin and of using mammalian cell culture for its expression. The use of recombinant hN901-RTB fusion protein to evaluate the contribution of the lectin activity of ricin B chain in the penetration of cell membranes by ricin A chain is proposed.
Krek
C E
CE
ImmunoGen Inc., Cambridge, MA 02139, USA.
Ladino
C A
CA
Goldmacher
V S
VS
Blättler
W A
WA
Guild
B C
BC
D000595:Amino Acid Sequence; D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D016923:Cell Death; D002460:Cell Line; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D060748:Fetuins; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D000922:Immunotoxins / Q000302:isolation & purification / Q000378:metabolism* / Q000633:toxicity; D007785:Lactose / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D012276:Ricin / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D014407:Tumor Cells, Cultured; D000509:alpha-Fetoproteins / Q000378:metabolism
D001212:Asialoglycoproteins; D060748:Fetuins; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D012276:Ricin; D007785:Lactose