Project Summary Genetically-modified mouse models have proven to be essential for the production of antibody-related biological drugs (biologics). To date, the majority of biologics originate from mouse models, and small animal models are used not only to generate the antibodies, but also as a platform for further optimization and testing of the biologics. Camelid-based antibodies, which have superior antigen binding and physicochemical properties (stability, hydrophilicity, etc.) have not realized their full potential, to the same extent that conventional antibodies have. This is founded in the logistic and financial hurdles immunization of camelids pose for monoclonal heavy-chain antibody (HCAb) production and the fact that in vitro technologies cannot fully recapitulate the exceptional natural selection towards extremely diversified, high-affinity binders that occurs in animals. In this SBIR project we propose to develop genetic platforms in a murine host for the discovery and development of partially humanized hybrid HCAbs (and their products). Since their discovery in the early 1990s, HCAbs have generated progressive interest in the biotech, diagnostic and therapeutic fields due to their intrinsic properties and adaptability. Apart from a small size paired with robustness and superior access to difficult epitopes, HCAbs can be easily processed into, and utilized as, single domain binding units (VHH) while preserving their affinity towards antigens (in contrast to conventional antibodies). The proposed targeted mouse models carrying an engineered immunoglobulin locus will potentiate the production of high affinity HCAbs by serving as an alternative, hybrid Ab host. It will allow natural, in vivo affinity-maturation of antigen-specific HCAbs in a small animal platform, one amenable to further genetic manipulation. It will enable larger cohort sizes than the natural camelid hosts, and streamline HCAb generation, thus providing further potential for the development of HCAb and VHH domains for downstream applications. In our Aim 1, we focus on honing and characterizing our hybrid camelid immunoglobulin locus by adding more camelid VHHs and introducing modified human VHs into the locus while also evaluating B-cell development and antibody affininty and diversity. In Aim 2, our focus is to benchmark the the repertoire and efficiency of the Ab response with competing technologies by using disease-relevant, difficult antigens and progress promising hits to hybridoma development and larger scale antibody production. In accomplishing these milestone based Aims, we will be able to develop our business and begin licensing of the platforms to individual labs and established pharmaceutical companies to support discovery of novel antibodies for high-value targets.