V(D)J Rearrangement Is Dispensable for Producing CDR-H3 Sequence Diversity in a Gene Converting Species

Abstract

An important characteristic of chickens is that the antibody repertoire is based on a single framework, with diversity found mainly in the CDRs of the light and heavy chain variable regions. Despite this apparent limitation in the antibody repertoire, high-affinity antibodies can be raised to a wide variety of targets, including those that are highly conserved. Transgenic chickens have previously been generated that express a humanized antibody repertoire, with a single framework that incorporates diversity by the process of gene conversion, as in wild-type chickens. Here, we compare the sequences and antibodies that are generated purely by gene conversion/somatic hypermutation of a pre-rearranged heavy chain, with the diversity obtained by V(D)J rearrangement followed by gene conversion and somatic hypermutation. In a gene converting species, CDR-H3 lengths are more variable with V(D)J rearrangement, but similar levels of amino acid diversity are obtainable with gene conversion/somatic hypermutation alone.

Expression of human lambda expands the repertoire of OmniChickens

“…Wu et al reported the fraction of cysteines out of the total amino acid content of CDR-H3 at 9.4% in wild-type chickens versus 1.6% in humans [12]. Because the V regions of the OmniChicken are entirely human, we rarely observe non-canonical cysteine residues [19]. In fact, deep sequencing of the antibody repertoire of a cohort of OmniChickens showed less than 1.5% cysteine content in CDR-H3.…”

“…Like our previously described Vκ insertion, all noncoding regions of the transgenes were chicken, including enhancer elements and promotor sequences. These Vλ-expressing chickens were then bred to birds with either a pre-rearranged human VH3-23 (SynVH-C) or rearranging VH3-23 (SynVH-SD) at the chicken heavy chain locus, both previously described [19], and combined with knockouts on the other allele for each locus, producing birds with the genotype SynVL/IgLKO; SynVH/IgHKO.…”

“…With each transgene introduced in our OmniChickens, we have assessed immunocompetence by hyperimmunization of a cohort of animals with a model antigen [9,19]. In this way, we are able to evaluate the immune repertoire and compare different transgenes based on responses to the same protein.…”

“…The constant regions were chicken CL for SynVL-D and human CL3 for SynVL-E. The human heavy chain constructs have been described elsewhere [9,19,36].…”

Complementary epitopes and favorable developability of monoclonal anti-LAMP1 antibodies generated using two transgenic animal platforms

“…In contrast, the V H sequence inserted in the SynVH-C OmniChicken strain was derived from a human B cell library and is 86% identical to the closest human germline gene, V H 3-23, and the median identity of the V H s of anti-LAMP1 mAbs was similar. This result was expected considering the 97% human identity of the synthetic human pseudogenes inserted into this OmniChicken strain and the prevalent role of gene conversion in chicken [ 19 ]. No sequence donated by chicken pseudogenes was observed.…”