Multispecific Antibody Development Platform Based on Human Heavy Chain Antibodies

Abstract

Heavy chain-only antibodies (HCAbs) do not associate with light chains and their VH regions are functional as single domains, forming the smallest active antibody fragment. These VH regions are ideal building blocks for a variety of antibody-based biologics because they tolerate fusion to other molecules and may also be attached in series to construct multispecific antibodies without the need for protein engineering to ensure proper heavy and light chain pairing. Production of human HCAbs has been impeded by the fact that natural human VH regions require light chain association and display poor biophysical characteristics when expressed in the absence of light chains. Here, we present an innovative platform for the rapid development of diverse sets of human HCAbs that have been selected in vivo. Our unique approach combines antibody repertoire analysis with immunization of transgenic rats, called UniRats, that produce chimeric HCAbs with fully human VH domains in response to an antigen challenge. UniRats express HCAbs from large transgenic loci representing the entire productive human heavy chain V(D)J repertoire, mount robust immune responses to a wide array of antigens, exhibit diverse V gene usage and generate large panels of stable, high affinity, antigen-specific molecules.

Anti-BCMA chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains

“…The potential for decreased immunogenicity of heavy-chain-only binding domains compared with scFvs and other advantages of CARs with heavy-chain-only binding domains led us to develop fully human heavy-chain-only CARs targeting BCMA. We previously reported novel rats transgenic for the complete functional human heavy-chain variable gene repertoire 30 . The rats are referred to as Unirats 30 .…”

“…We previously reported novel rats transgenic for the complete functional human heavy-chain variable gene repertoire 30 . The rats are referred to as Unirats 30 . Unirats produce Uniabs that consist of two covalently linked antibody heavy chains.…”

“…Unirats produce Uniabs that consist of two covalently linked antibody heavy chains. Unirats were immunized with human BCMA protein in adjuvant 30 . RNA was isolated from draining lymph nodes, and high-abundance heavychain variable region sequences were expressed as proteins 30,39 .…”

“…Unirats were immunized with human BCMA protein in adjuvant 30 . RNA was isolated from draining lymph nodes, and high-abundance heavychain variable region sequences were expressed as proteins 30,39 . These heavy-chain variable region domains were tested for BCMA recognition 30,39 .…”

“…Immunoglobulin-like molecules with antigen-binding domains made up of only heavy chains without light chains were first described in camelids and cartilaginous fish [25][26][27][28] . Binding domains made up of only a single immunoglobulin heavy-chain variable region domain have been reported to exhibit strong and specific antigen binding 25,[29][30][31][32][33] . Unlike a scFv, single heavychain-only binding domains have no need for a potentially immunogenic linker to connect the heavy chain to the light chain.…”