Somatic insertions and deletions shape the human antibody repertoire 1 1Edited by J. Karn

Wildt, R.M.T. de; Venrooij, Walther J. van; Winter, Greg; Hoet, Rene; Tomlinson, Ian M.

doi:10.1006/jmbi.1999.3289

Cited by 78 publications

(79 citation statements)

References 48 publications

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“…Apparently functional antibodies belonging to the IGHV3 subgroup with insertions in CDRH1 and CDRH2 leading to CDR-IMGT loop lengths of 9 amino acids have in fact been described by others (8,34,35). As the deletions at position 58 in CDRH2 of both scFv give rise to loop lengths that are used by other members of the IGHV3 subgroup, it is not entirely unexpected that these modifications are tolerated by the scFv frameworks studied here.…”

Section: Discussionmentioning

confidence: 86%

“…The insertion of one residue in CDRH1 produces a loop length (CDR1-IMGT length 9 amino acids) that occurs naturally within the human IGHV4, but not the IGHV3 subgroup, and which could correspond to canonical structure 2 as judged by the automatic canonical structure classification. This coexistence of canonical structure 2 in CDRH1 with canonical structure 3 in CDRH2 (Table I) does not occur naturally within the human IGHV germline repertoire, although it has been observed in hypermutated antibodies with insertions in CDRH1 (8). In addition, the structure classification also revealed that a large number of the key residue requirements for canonical structure 2 were not fulfilled (27), i.e.…”

Section: Discussionmentioning

confidence: 98%

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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: 86%

Section: Discussionmentioning

confidence: 98%

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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“…It has been estimated that approximately 6.5% of native human antibodies contain insertions or deletions within the variable domains as a consequence of SHM (40), and a number of potential mechanisms for their generation via AID-mediated mutagenesis have been proposed (39,41). The identification of multiple, localized codon insertions within CDR regions in this and other affinity maturation programs substantiates this as a robust feature of AID-directed SHM, and another aspect in which the in vivo process can be reproduced in vitro.…”

Section: Discussionmentioning

confidence: 99%

Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies

Bowers

Horlick

Neben

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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A novel approach has been developed for the isolation and maturation of human antibodies that replicates key features of the adaptive immune system by coupling in vitro somatic hypermutation (SHM) with mammalian cell display. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID), and can be replicated in non-B cells through expression of recombinant AID. A library of human antibodies, based on germline V-gene segments with recombined human D J regions was used to isolate low-affinity antibodies to human β nerve growth factor (hβNGF). These antibodies, initially naïve to SHM, were subjected to AID-directed SHM in vitro and selected using the same mammalian cell display system, as illustrated by the maturation of one of the antibodies to low pM K D . This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs. affinity maturation | mammalian display

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“…The overall process favors single-base transitions over transversions at an ϳ3:1 ratio (7). Insertions and deletions also occur but are considerably less common (8,9). Certain four-base DNA sequence motifs, called hotspots, are correlated with the mutation locations.…”

Section: Trends In Antibody Sequence Changes During the Somatic Hypermentioning

confidence: 99%

Trends in Antibody Sequence Changes during the Somatic Hypermutation Process

Clark

Ganesan

Papp

et al. 2006

The Journal of Immunology

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Probable germline gene sequences from thousands of aligned mature Ab sequences are inferred using simple computational matching to known V(D)J genes. Comparison of the germline to mature sequences in a structural region-dependent fashion allows insights into the methods that nature uses to mature Abs during the somatic hypermutation process. Four factors determine the residue type mutation patterns: biases in the germline, accessibility from single base permutations, location of mutation hotspots, and functional pressures during selection. Germline repertoires at positions that commonly contact the Ag are biased with tyrosine, serine, and tryptophan. These residue types have a high tendency to be present in mutation hotspot motifs, and their abundance is decreased during maturation by a net conversion to other types. The heavy use of tyrosines on mature Ab interfaces is thus a reflection of the germline composition rather than being due to selection during maturation. Potentially stabilizing changes such as increased proline usage and a small number of double cysteine mutations capable of forming disulfide bonds are ascribed to somatic hypermutation. Histidine is the only residue type for which usage increases in each of the interface, core, and surface regions. The net overall effect is a conversion from residue types that could provide nonspecific initial binding into a diversity of types that improve affinity and stability. Average mutation probabilities are ∼4% for core residues, ∼5% for surface residues, and ∼12% for residues in common Ag-contacting positions, excepting the those coded by the D gene.

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Somatic insertions and deletions shape the human antibody repertoire 1 1Edited by J. Karn

Cited by 78 publications

References 48 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

Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies

Trends in Antibody Sequence Changes during the Somatic Hypermutation Process

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