Idiotope determining regions of a mouse monoclonal antibody and its humanized versions. Identification of framework residues that affect idiotype expression.

The contribution of framework regions (FRs) of antibody-variable domains to idiotype expression was studied by examining the interaction of various "humanized" versions of a mouse anti-TNF alpha monoclonal antibody (mAb78) with polyclonal and two monoclonal antibodies (mAb1G3 and mAb9F1), generated against the mAb78 idiotype. Humanized mAb78, bearing human constant domains and mouse complementarity-determining regions (CDRs) inserted with human FRs, was found to be five to sevenfold less reactive than mAb78 with polyclonal anti-idiotype antibodies and 200 to 300-fold less active in neutralizing TNF alpha. The substitution of heavy-chain FRs residues of the humanized antibody with original mouse residues 28 to 30, 48 to 49, 67 to 68, 70 to 71, 78, 80 and 82 progressively restored the immunoreactivity with polyclonal immunoglobulin Gs to the level of a version having mouse heavy chain and human light chain FRs, and increased 10 to 20-fold the TNF alpha neutralizing activity. This suggests that at least some of these residues are critical for TNF alpha binding as well as for the expression of idiotopes that are strongly immunogenic in syngeneic animals. All antibody versions with either human or mouse FRs were able to bind to various extents mAb1G3, a gamma-type anti-Id antibody that inhibits mAb78/TNF alpha interaction by paratope blockade. At variance, only the antibody versions containing mouse FRs were able to bind mAb9F1, an alpha-type anti-Id antibody unable to block the access of TNF alpha to mAb78 paratopes. Substitution of heavy chain FR residues 28 to 30 markedly decreased the binding of mAb1G3 (100 to 1000-fold). This suggests that these antibodies recognize CDR and FR idiotopes, respectively, that can be drastically modified by changes in the FRs. In conclusion, the results suggest that CDRs as well as FRs markedly contribute to antibody Id expression. Although strongly immunogenic idiotopes are probably located within the CDRs, the results also suggest that some FR residues are critically involved in shaping antibody Id diversity by affecting the structure of CDR-related idiotopes.