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

Johan Lantto, Mats Ohlin
2002 Journal of Biological Chemistry  
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
more » ... s 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 ImMu-noGeneTics 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 and occur at positions in the sequence that can tolerate such modifications. A number of examples of seemingly functional insertions and deletions in the CDR of both the heavy and light domains of human antibodies have in fact been encountered lately (Refs. 8 and 12 and references therein). Furthermore, we have recently discovered that human IGHV 2 germline genes carry features in CDR1 and CDR2 that make these regions particularly prone to deletions of entire codons (12). The occurrence of insertions and deletions in antibody V genes is not only of fundamental interest but is also of biotechnological importance. It has been known for some time that the topography of the antigen-binding site is related to the size of the antigen (13-15). Three different types of binding sites have been described: cavity, groove, and planar, which roughly correspond to hapten, peptide, and protein, respectively. This relationship has been further investigated by , who have described a correlation between the length of the CDR loops and the antigen recognized. According to these findings, cleft-like binding sites that recognize small molecules are created by long loops (especially the CDRH2 and L1 loops), whereas planar-binding sites that are specific for large molecules are formed by short loops. In other words, by modifying the loop lengths of an antibody-binding site, it may thus be possible to design antibodies optimally suited for recognition of a particular class of antigen. Lamminmäki et al. (17) have in fact used this approach to modify a murine antibody specific for 17␤-estradiol. They introduced additional residues into CDR2 of the heavy domain and were able to improve the recognition of the antigen. This improvement was suggested to be the result of a deeper binding site, created through the extension of CDRH2, which better accommodated the hapten (17). Despite the establishment of insertions and deletions as naturally occurring modifications of antibody sequences and the use of amino acid insertions for antibody engineering, little is still known about the functional consequences of such mod-
doi:10.1074/jbc.m208401200 pmid:12237318 fatcat:zbqk7gqnhrd2dlatesih6wh4dy