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

Angelo Corti, Elena Barbanti, Philip R. Tempest, Frank J. Carr, Fabrizio Marcucci

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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-TNFα 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 TNFα. 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 TNFα neutralizing activity. This suggests that at least some of these residues are critical for TNFα 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 γ-type anti-Id antibody that inhibits mAb78/TNFα interaction by paratope blockade. At variance, only the antibody versions containing mouse FRs were able to bind mAb9F1, an α-type anti-Id antibody unable to block the access of TNFα 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.

Original languageEnglish
JournalJournal of Molecular Biology
Volume235
Issue number1
DOIs
Publication statusPublished - Jan 7 1994

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Antibodies, Monoclonal, Humanized
Complementarity Determining Regions
Antibodies
Antibody Binding Sites
Anti-Idiotypic Antibodies
Antibody Diversity
Monoclonal Antibodies
Immunoglobulins
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Keywords

  • chimeric antibody
  • humanized antibody
  • idiotype
  • monoclonal antibody
  • tumour necrosis factor

ASJC Scopus subject areas

  • Virology

Cite this

Idiotope determining regions of a mouse monoclonal antibody and its humanized versions. Identification of framework residues that affect idiotype expression. / Corti, Angelo; Barbanti, Elena; Tempest, Philip R.; Carr, Frank J.; Marcucci, Fabrizio.

In: Journal of Molecular Biology, Vol. 235, No. 1, 07.01.1994.

Research output: Contribution to journalArticle

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AU - Marcucci, Fabrizio

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N2 - 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-TNFα 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 TNFα. 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 TNFα neutralizing activity. This suggests that at least some of these residues are critical for TNFα 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 γ-type anti-Id antibody that inhibits mAb78/TNFα interaction by paratope blockade. At variance, only the antibody versions containing mouse FRs were able to bind mAb9F1, an α-type anti-Id antibody unable to block the access of TNFα 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.

AB - 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-TNFα 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 TNFα. 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 TNFα neutralizing activity. This suggests that at least some of these residues are critical for TNFα 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 γ-type anti-Id antibody that inhibits mAb78/TNFα interaction by paratope blockade. At variance, only the antibody versions containing mouse FRs were able to bind mAb9F1, an α-type anti-Id antibody unable to block the access of TNFα 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.

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