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Antibodies expressed in mice, humans, and most mammals consist of paired heavy (H) and light (L) chains. Cellular release of single H-chains without L-chains is prevented by chaperone association and retention in the endoplasmic reticulum. Consequently, H-chain-only antibodies are absent, except in pathological conditions known as Heavy Chain Disease, where they occur in mutated form. An exception to this is found in the serum of Camelidae. Homodimeric H-chain antibodies in camelids lack the first constant region domain (CH1) of the H-chain but retain an otherwise intact H-chain polypeptide, thus maintaining antigen specificity and effector functions. In this review, we summarize how H-chain antibodies can be expressed in mice, and whether normal developmental processes leading to antibody expression without L-chain are retained.

In mature B cells of mice and most mammals, cellular release of single H chain Abs without L chains is prevented by H chain association with Ig-specific chaperons in the endoplasmic reticulum. In precursor B cells, however, surface expression of mu-H chain in the absence of surrogate and conventional L chain has been identified. Despite this, Ag-specific single H chain Ig repertoires, using mu-, gamma-, epsilon-, or alpha-H chains found in conventional Abs, are not produced. Moreover, removal of H chain or, separately, L chain (kappa/lambda) locus core sequences by gene targeting has prevented B cell development. In contrast, H chain-only Abs are produced abundantly in Camelidae as H2 IgG without the C(H)1 domain. To test whether H chain Abs can be produced in mice, and to investigate how their expression affects B cell development, we introduced a rearranged dromedary gamma2a H chain into the mouse germline. The dromedary transgene was expressed as a naturally occurring Ag-specific disulphide-linked homodimer, which showed that B cell development can be instigated by expression of single H chains without L chains. Lymphocyte development and B cell proliferation was accomplished despite the absence of L chain from the BCR complex. Endogenous Ig could not be detected, although V(D)J recombination and IgH/L transcription was unaltered. Furthermore, crossing the dromedary H chain mice with mice devoid of all C genes demonstrated without a doubt that a H chain-only Ab can facilitate B cell development independent of endogenous Ig expression, such as mu- or delta-H chain, at early developmental stages.

Whereas functional heavy (H)-chain antibodies devoid of light (L)- chains account for about half of the circulating immunoglobulins in Camelidae, H-chain only antibodies (HCAbs) are not produced in other healthy mammals including rodents and humans. To test the feasibility of expressing single chain antibodies in the mouse, which on account of their small size and antigen-recognition properties would have a major impact on antibody engineering strategies, we constructed a rearranged dromedary H-chain gene encoding the immunoglobulin G2a (IgG2a) isotype with specificity for hen-egg lysozyme (HEL). This IgG2a H-chain gene was introduced into mouse myeloma cells not expressing endogenous immunoglobulin H- or L-chains. Unexpectedly the mouse cells processed and expressed the introduced H-chain as naturally occurring dromedary antibody. For this the first constant (C) region exon was proficiently removed from the recombinant H-chain transcript. This resulted in specific H-chain antibodies of the correct molecular weight (2 x 50 000 MW) secreted as disulfide-linked homodimers and displayed on the mouse cell surface as glycosyl-phosphatidyl-inositol-linked B-cell receptor. The results indicate that antibody expression and maturation without immunoglobulin L-chain is feasible and paves the way for the generation of transgenic single chain antibody repertoires.

Silencing individual C (constant region) lambda genes in a kappa(-/-) background reduces mature B cell levels, and L chain-deficient (lambda(-/-)kappa(-/-)) mice attain a complete block in B cell development at the stage when L chain rearrangement, resulting in surface IgM expression, should be completed. L chain deficiency prevents B cell receptor association, and L chain function cannot be substituted (e.g., by surrogate L chain). Nevertheless, precursor cell levels, controlled by developmental progression and checkpoint apoptosis, are maintained, and B cell development in the bone marrow is fully retained up to the immature stage. L chain deficiency allows H chain retention in the cytoplasm, but prevents H chain release from the cell, and as a result secondary lymphoid organs are B cell depleted while T cell levels remain normal.