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.

Keywords: binning; conserved target; epitope; human antibody; transgenic chicken.

Figure 1.

Flow cytometry analysis shows normal B cell development in the OmniChicken and normal levels of surface IgM. A, Wild-type, IgH knockout (IgH KO), and OmniChicken PBMCs were labeled with antibodies against the B cell marker Bu-1 and either IgL or IgM (left), or with antibodies against IgL and IgM (right). B, Flow cytometry analysis of lymphocytes shows normal levels of B and T cells. Wild-type (N = 3 birds) and OmniChicken (N = 12 birds) PBMCs were labeled with antibodies for Bu-1, IgL, IgM and T cell markers TCR1 and TCR2/3.

Figure 2.

Plasma levels of IgM and IgY are reduced in OmniChickens but IgA levels are normal. Immunoglobulins were measured by ELISA in wild-type (N = 7) and OmniChickens (N = 9). Ig concentrations were determined by comparison to standards. Mean values (in μg/mL) and standard deviations are shown below the plots.

Figure 3.

Individual OmniChickens raise specific mAbs to multiple targets. Birds were immunized with a cocktail of mouse IL-13, human transferrin and bovine thrombin, human transferrin, and mouse IL-13. A, Plasma titers to each immunogen from a single bird are shown over multiple draws (top set of graphs). Panels of mAbs to each antigen were identified from that bird, and antigen-specific binding of a single representative mAb for each target is shown (bottom set of graphs). B, epitope binning of eight thrombin antibodies shows three distinct bins. C, phylogenetic tree of the 8 thrombin antibodies, with the epitope bin indicated at right.

Figure 4.

OmniChickens raise specific mAbs to BDNF, a highly conserved protein. A, Plasma titer to human BDNF in one OmniChicken. B, phylogenetic tree for the 6 mAbs against BDNF.

Figure 5.

OmniChickens recognize a wide range of epitopes on PGRN, similar to WT birds. A, Network plot for a merged analysis of 90 OmniChicken clones (red) and 16 WT chicken mAb standards (blue), with subdomain assignments indicated. The mAbs were re-numbered to fit into the circles. The WT mAbs were selected from reference standards. B, iso-affinity chart of kinetics of the mAbs in (A). Human mAbs are in red and WT mAbs are in blue. C, phylogenetic tree of 41 selected human mAbs from the group in (A), with the PGRN subdomain, mouse cross-reactivity, and binding affinity for human PGRN of each antibody indicated at right. A box is drawn around a pair of mAbs from different birds that show convergent sequences. D, the CDR sequences from the mAb pair boxed in (C). Where it was possible to determine pseudogene usage in generating the CDR sequences, the pseudogenes are indicated below.