COMPARISON OF RECOMBINANT IMMUNOTOXINS AGAINST LE(Y) ANTIGEN EXPRESSING TUMOR-CELLS - INFLUENCE OF AFFINITY, SIZE, AND STABILITY

Monoclonal antibody B3 (MAb B3) reacts with many epithelial cancers. I t recognizes a carbohydrate antigen (Le(v)) which is expressed in a va riety of solid tumors including breast and colon. We have used the Fab portion of MAE, B3 and a portion of the constant domain of human IgG1 to make recombinant immunotoxins of different compositions. The toxin component employed is a truncated farm of Pseudomonas exotoxin (PE38) . The light chain or Fd of the antibody was cloned from hybridoma RNA and fused to PE38. Immunotoxin (IT) was then expressed in Escherichia coli as a fusion protein and refolded with either the Fd or the light chain. We have also made B3(Fab) immunotoxins of different sizes rangi ng 85-140 kDa, by introducing different portions of the constant domai n of human IgG1 at the junction of Fd and PE38 fusion site. We compare d the properties of the resulting immunotoxins with existing anti-Le(y ) immunotoxins side by side. All recombinant Fab-immunotoxins made in this study were cytotoxic to antigen-positive cancer cell lines. Howev er, in contrast to the B3(scFv) immunotoxin, the B3(Fab) immunotoxins are very stable, retaining 90% of their activity after 24 h of incubat ion in human serum albumin at 37 degrees C. A pharmacokinetics study w ith these immunotoxin molecules showed a longer survival in the circul ation of mice compared to the smaller Fv immunotoxins. The smaller siz e of the Fab immunotoxins compared to B3Lys-PE38 and the increased T-1 /2 value compared to B3(scFv)-PE38 and B3(dsFv)-PE38 make these recomb inant immunotoxins alternative therapeutic agents to treat Le(y) antig en positive cancers.