Chickens with humanized immunoglobulin genes generate antibodies with high affinity and broad epitope coverage to conserved targets

Abstract

Transgenic animal platforms for the discovery of human monoclonal antibodies have been developed in mice, rats, rabbits and cows. The immune response to human proteins is limited in these animals by their tolerance to mammalian-conserved epitopes. To expand the range of epitopes that are accessible, we have chosen an animal host that is less phylogenetically related to humans. Specifically, we generated transgenic chickens expressing antibodies from immunoglobulin heavy and light chain loci containing human variable regions and chicken constant regions. From these birds, paired human light and heavy chain variable regions are recovered and cloned as fully human recombinant antibodies. The human antibody-expressing chickens exhibit normal B cell development and raise immune responses to conserved human proteins that are not immunogenic in mice. Fully human monoclonal antibodies can be recovered with sub-nanomolar affinities. Binning data of antibodies to a human protein show epitope coverage similar to wild type chickens, which we previously showed is broader than that produced from rodent immunizations.

Discovery of high affinity, pan-allelic, and pan-mammalian reactive antibodies against the myeloid checkpoint receptor SIRPα

“…In order to generate pan-mammalian and panallelic anti-SIRPα antibodies, we immunized chickens, which are phylogenetically distinct from mice and humans, thereby increasing the probability of generating a productive antibody response against mouse and human immunogens. Using a multi-species and multi-allele immunization schedule and chickens with wildtype or human Ig repertoires, 30,31 we identified a panel of wildtype, chimeric, or fully human anti-SIRPα blocking and nonblocking antibodies with broad specificities directed against the CD47-binding IgV domain of SIRPα. The isolated antibodies exhibited high-affinity binding to SIRPα with affinities ranging from low nM to low pM, and therefore additional affinity maturation was unnecessary.…”

“…Wild-type and two different transgenic chickens were used for immunization: SynVH chickens which contain humanized V H immunoglobulin (Ig) repertoires paired with the natural chicken light chain repertoire, and OmniChickens ® , which contain humanized V H and V L Ig repertoires. 30,31 In total, eight immunized chickens produced high titer antibodies against SIRPα v1, v2 and mouse SIRPα (Table 2, Figure 2(b)) from which splenocytes were harvested, and single antibody- secreting B-lineage cells were screened for reactivity toward SIRPα using a gel-encapsulated microenvironment (GEM) assay. 32 The GEM assay consists of a single-antibody secreting B-cell encapsulated in a droplet containing up to three different fluorescent reporter beads, each coated with a target antigen of interest (Figure 2(a)).…”

“…Specifically, these include fourwildtype White Leghorn chickens, 4 SynVH chickens, which are transgenic chickens containing VH from human and VL from chicken, 31 and 11 chickens with fully human immunoglobulin loci called OmniChicken. 30 The chickens were immunized with varied schedules having alternating doses and boosts of human SIRPα v1, human SIRPα v2 and mouse SIRPα (m129 or NOD). The immunization protocol was approved by the Institutional Animal Care and Use Committee.…”

Golden Gate assembly with a bi-directional promoter (GBid): A simple, scalable method for phage display Fab library creation

“…In addition, chicken immunoglobulins comprise single VH and VL genes, a feature that further simplifies library construction as only one set of oligonucleotide primers is needed for cloning of each antibody chain 38 . On the strength of these attractive features, the use of chicken immune repertoires for discovery of monoclonal antibodies has recently gained in popularity [39][40][41][42] . (ii) The performance of selections in a dual counter-selection/positive selection format that first depletes phage pools of background cell-surface binders on mock-transfected cells prior to incubation with hCD20-transfected cells.…”

Expression of human lambda expands the repertoire of OmniChickens

“…We circumvented the lack of a chicken fusion partner by employing a microdroplet technology to isolate antigen-specific B cells and perform single cell RT-PCR [8]. Further, we recently described the OmniChicken 1 , the first transgenic chicken with fully human V genes at its immunoglobulin loci [9]. The first generation OmniChicken carried prerearranged VK3-15 � 01+JK4 and either a pre-rearranged or a rearranging VH3-23 � 01+D1 +JH4 or JH6 at its light and heavy chain loci, respectively.…”