Molecular mechanisms of avian immunoglobulin gene diversification and prospect for industrial applications

Abstract

Poultry immunoglobulin genes undergo diversification through homologous recombination (HR) and somatic hypermutation (SHM). Most animals share a similar system in immunoglobulin diversification, with the rare exception that human and murine immunoglobulin genes diversify through V(D)J recombination. Poultry possesses only one functional variable gene for each immunoglobulin heavy (HC) and light chains (LC), with clusters of non-productive pseudogenes upstream. During the B cell development, the functional variable gene is overwritten by sequences from the pseudo-variable genes via a process known as gene conversion (GC), a kind of HR. Point mutations caused in the functional variable gene also contribute to immunoglobulin diversification. This review discusses the latest findings on the molecular mechanisms of antibody gene diversification in poultry, using chickens as a model. Additionally, it will outline how these basic research findings have recently been applied especially in the medical field.

Keywords: DNA damage tolerance; gene conversion; homologous recombination; monoclonal antibody; somatic hypermutation; therapeutic antibody.

Figure 1

Chicken immunoglobulin locus and the mechanism of GC and SHM. (A) Genomic structure of chicken immunoglobulin LC and HC loci. (B-F) Schematically represented IgV_λ diversification mechanism in DT40 cell line. (B) Deamination of cytidine followed by the action by UNG generates abasic site (AP) and replicative DNA polymerase stalls at AP site. (C, D) Template is switched to one of the ψV_λ s, resulting HR mediated GC (C) or TLS-mediated SHM (D) events. (E) DNA replication assigns A to U generating dC/dG to dT/dA mutation. (F) TLS assigns G to abasic site, generating dC/dG to dG/dC mutation.

Figure 2

Application of GC and SHM using DT40 cells. (A) Principle of the ADLib system. DT40 cells are cultured with TSA to generate diversified cell-based mAb libraries. Antigen-specific clones can be isolated by, for example, antigen-coated magnetic beads. The isolated cells are expanded, and the antigen-specific monoclonal antibodies are recovered in culture supernatants. (B) Flowchart of the process for ADLib selection. The selection can be conducted using various methods, including antigen-conjugated magnetic beads and fluorescent cell sorting (FACS), among others. Validation of the antibodies can be performed using a range of methods including enzyme-linked immunosorbent assay (ELISA), FACS and other suitable methods. (C) Replacement and insertion of chicken immunoglobulin LC (upper) components (open rectangles) into human counterparts (green rectangles). The endogenous chicken pseudogenes are replaced with designed pseudogenes. Replacement of the chicken immunoglobulin HC (lower) components (open rectangles) with their human counterparts (blue rectangles). Designed pseudogenes are inserted downstream of the chicken pseudogene cluster. (D) Molecular evolution of GFP using DT40 cells. The GFP gene is introduced into immunoglobulin LC locus of DT40 cells and SHM accumulates (left panel). The diversified cells are analyzed by FACS assessing forward scatter (FSC) and GFP fluorescence. Cells with mutant GFP showing enhanced fluorescence are isolated. (E) Introduction of scFv gene to chicken immunoglobulin LC locus. Since scFv does not show homology to chicken ψV_λ s, SHM occurred at introduced scFv gene.