Framework residue 71 is a major determinant of the position and conformation of the second hypervariable region in the VH domains of immunoglobulins

Tramontano, Anna; Chothia, Cyrus; Lesk, Arthur M.

doi:10.1016/s0022-2836(05)80102-0

Cited by 229 publications

(133 citation statements)

References 23 publications

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“…This demonstrates the plasticity of antibody V domain frameworks belonging to the important IGHV3 subgroup, which makes up a large fraction of all human antibodies (43), and its capacity to tolerate modifications that expand sequence and structure space beyond the limits set by the germline-encoded diversity. Based on the similarities with naturally occurring alterations of loop lengths, our results with insertions and deletions in CDRH1, H2, and L1 of the antibody fragments used in this study, and work on an unrelated scFv with a three-amino acid insertion at the beginning of CDRH1 (10), 3 our conclusion is that both insertions and deletions can be efficiently utilized in antibody engineering to expand the structural space available to human antibodies as long as attention is paid to key residues in the framework (41). As demonstrated by previous studies on murine antibodies, this approach can be used for improving already existing specificities (17,44).…”

Section: Discussionmentioning

confidence: 69%

“…In fact, many of the amino acids that differ between the original AE11F sequence and the grafted sequences are residues that are used to define the canonical structures (27,31). In addition, Tramontano et al (41) have shown that framework residue 80 of the heavy V domain packs against residues in both CDRH1 (position 30) and CDRH2 (position 58) and that it is an important determinant of the conformation of the CDRH2 loop. A subsequent mutational study has also shown that the nature of this residue determines the binding characteristics of an antibody by influencing the conformation of the heavy chain CDR loops (42).…”

Section: Discussionmentioning

confidence: 99%

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Functional Consequences of Insertions and Deletions in the Complementarity-determining Regions of Human Antibodies

Lantto

Ohlin

2002

Journal of Biological Chemistry

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Insertions and deletions of nucleotides in the genes encoding the variable domains of antibodies are natural components of the hypermutation process, which may expand the available repertoire of hypervariable loop lengths and conformations. Although insertion of amino acids has also been utilized in antibody engineering, little is known about the functional consequences of such modifications. To investigate this further, we have introduced single-codon insertions and deletions as well as more complex modifications in the complementaritydetermining regions of human antibody fragments with different specificities. Our results demonstrate that single amino acid insertions and deletions are generally well tolerated and permit production of stably folded proteins, often with retained antigen recognition, despite the fact that the thus modified loops carry amino acids that are disallowed at key residue positions in canonical loops of the corresponding length or are of a length not associated with a known canonical structure. We have thus shown that single-codon insertions and deletions can efficiently be utilized to expand structure and sequence space of the antigen-binding site beyond what is encoded by the germline gene repertoire.Antibodies are highly specific receptors of the immune system that also have a great potential as reagents in biological chemistry and as therapeutic agents. The part of the antibody that makes contact with the antigen is comprised of two variable (V) 1 domains, the heavy (H) and the light (L), which both are made up of a two-␤-sheet framework. From this framework, six complementarity-determining region (CDR) loops, three from the light domain and three from the heavy domain, protrude and make up the antigen-binding site (1, 2). Five of these CDR loops generally adopt only a limited number of backbone conformations, so-called canonical structures (reviewed in Ref.3), which are determined by the lengths of the loops and by the presence of specific key residues. The antigen specificity of the binding site is mainly determined by the sequence and conformation of these CDR loops.Antibody diversity is generated by the imprecise recombination of two or three sets of germline gene segments and by the combination of different heavy and light domains (4). The diversity is further increased by the process of somatic hypermutation (5) and by receptor editing and revision (6). As the germline variable gene repertoire encodes a rather limited number of CDR loop lengths (IMGT, the international ImMunoGeneTics data base, Ref. 7), the number of observed canonical structures is similarly limited. However, it was recently discovered that B cells evolve the genes encoding immunoglobulin V domains not only by nucleotide substitution but also through an additional mechanism of insertion and deletion of nucleotides during the hypermutation process (8 -11). This mechanism has the potential to expand the available repertoire of loop lengths and conformations if the insertions and deletions involve entire codons a...

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Section: Discussionmentioning

confidence: 69%

Section: Discussionmentioning

confidence: 99%

Functional Consequences of Insertions and Deletions in the Complementarity-determining Regions of Human Antibodies

Lantto

Ohlin

2002

Journal of Biological Chemistry

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“…The complexity of CDR-H3 has made such comparisons problematic, but insight has been gained for CDR-H1, CDR-H2, CDR-L1, and CDR-L2, which are encoded entirely by their V gene segments and thus begin in strict germ-line conformity. Each of these V-encoded CDR has been shown to possess one of a small set of main chain conformations termed canonical structures [36][37][38][39]. Each canonical structure is determined by the loop size and by the presence of certain residues at key positions in both the loop and framework regions.…”

Section: Discussionmentioning

confidence: 99%

Categorical selection of the antibody repertoire in splenic B cells

et al. 2007

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In the bone marrow, the passage of developing B cells through critical checkpoints of differentiation is associated with a reduction of specific categories of CDR3 of the Ig heavy chain (CDR-H3), particularly those with excessive hydrophobic or charged amino acids and those with a length of eight or fewer residues. To gain insight into the role of CDR-H3 content in the development of B cells in the spleen, we compared the sequences of V H 7183DJCl transcripts from sorted transitional T1, marginal zone, and follicular B cell subsets to those expressed by immature IgM + IgD -and mature IgM lo IgD hi B cells in the bone marrow. Although differences in V H utilization were noted, the T1 CDR-H3 repertoire showed extensive similarity to that of immature bone marrow B cells, and the follicular CDR-H3 repertoire most resembled that of mature bone marrow B cells. Unlike the splenic follicular and bone marrow mature B cell CDR-H3 repertoires, the marginal zone B cell CDR-H3 repertoire retained both short and highly charged amino acid motifs, including those with two arginines. Our findings suggest that antigen binding sites containing specific categories of CDR-H3 sequence content may inhibit, permit, or even facilitate passage of the host B cell through critical checkpoints in peripheral as well as central development.

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“…We identified V H -71, previously described as a 'key residue' for the CDR-H2 mainchain conformation (Chothia et al, 1989;Tramontano et al, 1990), to be potentially critical for maintaining the structural integrity of the grafted antigen-binding site of the wild-type scFv fragment. To study the effects of framework residue V H -71 on antigen-binding and stability properties of the humanised scFvs, three mutant variants were generated by replacing arginine in the human framework 3 sequence with the murine donor antibody residue alanine (clones 17M, 22M, 4.9M; Table 1).…”

Section: Analysis Of Huhmfg1 Variable Domain Wild-type Sequencementioning

confidence: 99%

Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme

et al. 2004

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Anti-MUC1 single-chain Fv (scFv) fragments generated from the humanised antibody huHMFG1 had adequate antigen-binding properties but very poor stability irrespective of the applied linker or domain orientation. Mutagenesis of heavy-chain framework residue V H -71, previously described as a key residue for maintaining the CDR-H2 main-chain conformation and thus important for antigen binding, markedly stabilised the scFv while having only a minor effect on the binding affinity of the molecule. Because of its improved stability, the engineered fragment exhibited immunoreactivity with tumour cells even after 7 days of incubation in human serum at 371C. It also showed, in contrast to the wild-type scFv, a concentration-dependent binding to the target antigen when displayed on phage. When fusing the scFv to the recombinant ribonuclease rapLRI, only the fusion protein generated with the stable mutant scFv was able to kill MUC1 þ tumour cells with an IC 50 of 80 nM. We expect this novel immunoenzyme to become a promising tool for the treatment of MUC1 þ malignancies.

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Framework residue 71 is a major determinant of the position and conformation of the second hypervariable region in the VH domains of immunoglobulins

Cited by 229 publications

References 23 publications

Functional Consequences of Insertions and Deletions in the Complementarity-determining Regions of Human Antibodies

Functional Consequences of Insertions and Deletions in the Complementarity-determining Regions of Human Antibodies

Categorical selection of the antibody repertoire in splenic B cells

Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme

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