Abstract
Several technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and variable light (VL) domains in combinatorial display libraries or isolation of cognate pairs of VH and VL domains from human B cells or from transgenic mice carrying human immunoglobulin loci followed by single-cell sorting, single-cell RT-PCR, and bulk cloning of isolated natural VH–VL pairs. Heavy-chain-only antibodies (HCAbs) that naturally occur in camelids require only heavy immunoglobulin chain cloning. Here, we present an automatable novel, high-throughput technology for rapid direct cloning and production of fully human HCAbs from sorted population of transgenic mouse plasma cells carrying a human HCAb locus. Utility of the technique is demonstrated by isolation of diverse sets of sequence unique, soluble, high-affinity influenza A strain X-31 hemagglutinin-specific HCAbs.
Highlights
Camelidae produce conventional antibodies, composed of two heavy and two light chains (H2L2), and antibodies composed of heavy chains only
Immunization of Transgenic Mice Leads to Successful Production of Antigen-Specific Heavy-chain-only antibodies (HCAbs)
Our approach is based on capturing the antibody repertoire of antibody-secreting plasma cells (PCs) from both bone marrow and spleen of immunized transgenic mice
Summary
Camelidae produce conventional antibodies, composed of two heavy and two light chains (H2L2), and antibodies composed of heavy chains only. In the conventional antibodies both chains contribute to the antigen binding site, the antigen binding site of camelid heavy-chain-only antibodies (HCAbs) is formed by single heavy chain variable domain (VHH) [1, 2]. We have previously generated transgenic mice containing hybrid llama-human antibody loci with two llama variable VHH regions and human D, J, and Cμ and/or Cγ constant regions. Such loci rearrange productively and rescue B cell development efficiently [3]. HCAb production does not require an IgM stage for effective pre-B cell signaling, and antigen-specific heavy-chain-only IgGs are produced upon immunization [3]. Camelid VHH segments are soluble and this is attributed to the presence of a germ line-encoded tetrad of specific hydrophilic amino acid substitutions at the hydrophobic interface of the conventional VH domain that normally interacts with a variable light
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