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...

Panton-Valentine leukocidin (PVL) is a pore-forming toxin associated with current outbreaks of community-associated methicillin-resistant strains and implicated directly in the pathophysiology of Staphylococcus aureus-related diseases. Humanized heavy chain-only antibodies (HCAb) were generated against S. aureus PVL from immunized transgenic mice to neutralize toxin activity. The active form of PVL consists of the two components, LukS-PV and LukF-PV, which induce osmotic lysis following pore formation in host defense cells. One anti-LukS-PV HCAb, three anti-LukF-PV HCAbs with affinities in the nanomolar range, and one engineered tetravalent bispecific HCAb were tested in vitro and in vivo, and all prevented toxin binding and pore formation. Anti-LukS-PV HCAb also binds to γ-hemolysin C (HlgC) and inhibits HlgC/HlgB pore formation. Experiments in vivo in a toxin-induced rabbit endophthalmitis model showed that these HCAbs inhibit inflammatory reactions and tissue destruction, with the tetravalent bispecific HCAb performing best. Our findings show the therapeutic potential of HCAbs, and in particular, bispecific antibodies.

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.

The value of anti-CTLA-4 antibodies in cancer therapy is well established. However, the broad application of currently available anti-CTLA-4 therapeutic antibodies is hampered by their narrow therapeutic index. It is therefore challenging and attractive to develop the next generation of anti-CTLA-4 therapeutics with improved safety and efficacy. To this end, we generated fully human heavy chain–only antibodies (HCAbs) against CTLA-4. The hIgG1 Fc domain of the top candidate, HCAb 4003-1, was further engineered to enhance its regulatory T (T reg ) cell depletion effect and to decrease its half-life, resulting in HCAb 4003-2. We tested these HCAbs in in vitro and in vivo experiments in comparison with ipilimumab and other anti-CTLA4 antibodies. The results show that human HCAb 4003-2 binds human CTLA-4 with high affinity and potently blocks the binding of B7-1 (CD80) and B7-2 (CD86) to CTLA-4. The results also show efficient tumor penetration. HCAb 4003-2 exhibits enhanced antibody-dependent cellular cytotoxicity function, lower serum exposure, and more potent anti-tumor activity than ipilimumab in murine tumor models, which is partly driven by a substantial depletion of intratumoral T reg s. Importantly, the enhanced efficacy combined with the shorter serum half-life and less systemic drug exposure in vivo potentially provides an improved therapeutic window in cynomolgus monkeys and preliminary clinical applications. With its augmented efficacy via T reg depletion and improved safety profile, HCAb 4003-2 is a promising candidate for the development of next generation anti-CTLA-4 therapy.