Generation of heavy-chain-only antibodies in mice

Abstract

We have generated transgenic mice containing hybrid llama͞ human antibody loci that contain two llama variable regions and the human D, J, and C and͞or C␥ constant regions. Such loci rearrange productively and rescue B cell development efficiently without LC rearrangement. Heavy-chain-only antibodies (HCAb) are expressed at high levels, provided that the CH1 domain is deleted from the constant regions. HCAb production does not require an IgM stage for effective pre-B cell signaling. Antigenspecific heavy-chain-only IgM or IgGs are produced upon immunization. The IgG is dimeric, whereas IgM is multimeric. The chimeric HCAb loci are subject to allelic exclusion, but several copies of the transgenic locus can be rearranged and expressed successfully on the same allele in the same cell. Such cells are not subject to negative selection. The mice produce a full antibody repertoire and provide a previously undescribed avenue to produce specific human HCAb in the future.immunoglobulin rearrangement ͉ transgenic C onventional antibodies contain two heavy and light chains (LC) coded for by heavy and LC loci. B cell development and antibody production starts in the bone marrow (BM) by heavy chain (HC) VDJ recombination and expression of IgM associated with a surrogate LC on the cell surface. In a second round of recombination, one of the LC rearranges in pre-B cells. If successful, the B cells undergo selection, affinity maturation, and switching to different HC constant regions to result in B cells, which express tetrameric antibodies of different isotypes (IgA, IgG, and IgE). Normally absence of HC or LC expression leads to arrest of B cell development. However, some species produce HC-only antibodies (HCAb) as part of their normal B cell development and repertoire. The best-known HCAb (i.e., no LC) are IgG2 and IgG3 in camelids (1). They undergo antigen-mediated selection and affinity maturation, and their variable domains are subject to somatic hypermutation (2, 3). HCAb are thought to recognize unusual epitopes, such as clefts on the antigen surface (4). The first domain of the constant region, CH1, is spliced out because of the loss of a consensus splice signal (5, 6). CH1 exon loss also has been described in other mammals, albeit associated with disease, e.g., in mouse myelomas (7) and human HC disease (HCD) (8-10).Camelid HCAbs contain a complete VDJ region. Its size, stability, specificity, and solubility have generated considerable biotechnological interest. The antigen-binding site, a single-variable domain (VHH), resembles VH of conventional Abs. However, differences in FR2 and CDR3 prevent VHH to pair with a variable LC, whereas hydrophilic amino acids provide solubility (11). HCAb of the IgM class have not been found in camelids, suggesting that the IgM ϩ stage of HCAb formation is very transient and͞or circumvented.Murine NSO myeloma cells can express a rearranged camelid VHH-␥2a gene (12) and, recently, the same gene was expressed in transgenic mice (13). Here, we describe transgenic mice containing various...

Tetrameric and Homodimeric Camelid IgGs Originate from the Same IgH Locus

“…Previous studies using mice transgenic for chimeric dromedary or llama/human IgH loci reported that homodimeric IgG2 can be expressed at the cell surface of developing and mature B cells and can replace IgM in its function during B cell development and as an Ag sensor (33,34). These data could be interpreted as evidence that camelid B cells expressing homodimeric IgGs develop without passing through an IgM ϩ stage.…”

Heavy chain-only antibodies and tetravalent bispecific antibody neutralizing Staphylococcus aureus leukotoxins

“…Transgenic mice containing a llama/human hybrid Ig heavy chain locus (26) were immunized with recombinant LukS-PV and LukF-PV proteins. Using phage display, sdAb libraries were made from immunized animals.…”

“…Five GΔ transgenic mice containing hybrid llama/human Ig loci (26) were immunized with 20 μg of recombinant LukS-PV or LukF-PV protein five times in 2-wk intervals. The immunization protocol, phage display library, ELISAs, and antibody production after transfection in HEK cells were performed as described in SI Appendix.…”

“…Consequently, HCAbs are attractive therapeutics, but would have to be humanized. Here, we evaluate the effect of one humanized anti-LukS-PV and three anti-LukF-PV HCAbs derived following antigen challenge of transgenic mice containing llama/human heavy chain Ig loci (26). Furthermore, we demonstrate that an engineered tetravalent bispecific antibody comprising both antiLukS-PV and anti-LukF-PV humanized VH binding domains retains the functionalities of the parent HCAbs.…”

Immunoglobulin Polygeny: An Evolutionary Perspective

“…The best known examples are the naturally occurring single chain antibodies of the camelid group and some sharks (HamersCasterman et al, 1993;De Genst et al, 2006;Dooley et al, 2003;Diaz et al, 2002;Nguyen et al, 2002). Based on the convenience of producing single chain antibodies from these species for therapy and the evidence that the HCDR3 domainplays the major role in forming the antibody binding site (see Section 6.2) there have also been various attempts to developsynthetic single chain antibodies or "camelized" antibodies (Janssens et al, 2006;Reiter et al, 1999;Davies & Riechmann,1995). Among the camelids, single chain antibodies use a separate set of VH genes (called VHH) that encode a much largerportion of the binding sites than the conventional VH genes which compensates for the lack of a light chain.…”