Expression of human lambda expands the repertoire of OmniChickens

Abstract

Most of the approved monoclonal antibodies used in the clinic were initially discovered in mice. However, many targets of therapeutic interest are highly conserved proteins that do not elicit a robust immune response in mice. There is a need for non-mammalian antibody discovery platforms which would allow researchers to access epitopes that are not recognized in mammalian hosts. Recently, we introduced the OmniChicken ® , a transgenic animal carrying human VH3-23 and VK3-15 at its immunoglobulin loci. Here, we describe a new version of the OmniChicken which carries VH3-23 and either VL1-44 or VL3-19 at its heavy and light chain loci, respectively. The Vλ-expressing birds showed normal B and T populations in the periphery. A panel of monoclonal antibodies demonstrated comparable epitope coverage of a model antigen compared to both wild-type and Vκ-expressing OmniChickens. Kinetic analysis identified binders in the picomolar range. The Vλ-expressing bird increases the antibody diversity available in the OmniChicken platform, further enabling discovery of therapeutic leads.

Common light chain chickens produce human antibodies of high affinity and broad epitope coverage for the engineering of bispecifics

“…In OmniChicken lines, these heavy chains are paired with the human V-kappa transgene (SynVK-CK; pre-rearranged VK3-15/JK4), 20 or a human V-lambda transgene (SynVL-E-CL; VL1-44/JL3). 32 The heavy chains are designed in a similar fashion to the light chains, each with a single functional human variable region, an array of upstream human pseudogenes for gene conversion, and splicing to the chicken constant regions for efficient engagement of Fc receptors and B cell receptor complexes in the bird. Chickens contain one light chain and one heavy chain.…”

“…Birds were immunized with human progranulin protein (PGRN), a modular protein that we have used for comparison Top line, the CmLC1 transgene was inserted into the chicken light chain locus using phiC31 integrase, as previously described for the V-kappa and V-lambda OmnChicken light chains. 20,32 The transgene contains a human pre-rearranged variable region (VK3-15/JK1) transcriptionally driven by the chicken IgL promoter and enhancer elements (IgL enh). Upstream of the promoter lie six pseudogenes with the same sequence as the functional V region, in opposite orientation as shown by the direction of the arrowhead shapes.…”

“…of immune responses across various genotypes of OmniChickens and wild-type chickens. 19,20,32 PGRN is composed of 7.5 domains that can be used for identifying epitope binning patterns in a cohort of antibodies. Six OmniClic birds were immunized with human PGRN, three with the SynVH-C heavy chain and three with the SynVH-SD heavy chain, on a two-week interval of intramuscular boosts, with serum titers determined on the in-between weeks.…”

“…All the antibodies were analyzed on the Carterra LSA instrument for kinetics and epitope binning, as done previously on cohorts of OmniChicken-derived mAbs. 20,32 Figure 4a shows a sequence dendrogram for the clones lined up with a column indicating the PGRN subdomain targeted by each clone, and a column indicating whether the clone binds to the mouse ortholog of PGRN. Clone names highlighted in green contain germline light chain.…”

“…31 We have engineered chickens to express human V region antibodies (OmniChickens®). 20,32 The constructs in these birds are designed with human pseudogenes containing diverse CDRs, to produce large repertoires of human sequences in the B cell population of these birds. 20,33,34 Here, we pursued the opposite goal, at least for the light chain: a chicken with a non-mutating, common light chain that can pair with any heavy chain in the bird, for discovery of antibodies that can be developed into bispecifics.…”