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.
Highlights
Heavy chain only antibodies (HCAbs) occur naturally in camelids (VHH) and cartilaginous fish (VNAR), where they form a functional paratope using only the heavy chain variable domain without light chain pairing [1,2,3,4]
The surface hydrophobicity of the UniAb VH was similar to that of the llama VHHs despite the UniAb lacking the hallmark framework 2 mutations known to increase the hydrophilicity of camelid VHHs (Figure 5D) [1, 15, 39, 40]. These data suggest that antigen-specific UniAbs fold identically to classic human H2L2 VH domains but may exhibit a hydrophobicity profile more closely aligned to naturally evolved Heavy chain-only antibodies (HCAbs) rather than that of conventional human VH domains. It is well-established that conventional human antibody repertoires can be expressed in engineered rodents, and that HCAbs are spontaneously produced if both CH1 domains and light chains are absent [17, 22, 41]
We built on these results in the creation of UniRats, animals which lack all endogenous Ig expression and efficiently produce chimeric human/rat IgH molecules containing human VH, D, and JH sequences on rat constant regions deleted for CH1
Summary
Heavy chain only antibodies (HCAbs) occur naturally in camelids (VHH) and cartilaginous fish (VNAR), where they form a functional paratope using only the heavy chain variable domain without light chain pairing [1,2,3,4]. HCAbs are smaller than standard Ig molecules since they lack light chains, which may facilitate targeting of epitopes not readily accessible to classic antibodies, including narrow protein clefts and enzyme active sites [1, 9, 10]. Multispecific Antibody Development Platform as single domain proteins. VH domains from HCAbs are attractive building blocks for multispecific biologics, facilitating easy combination of different antigen-specific variable regions within a single molecule [10,11,12,13,14]
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