Unique single-domain antigen binding fragments derived from naturally occurring camel heavy-chain antibodies

Abstract

The humoral immune response of camels, dromedaries and llamas includes functional antibodies formed by two heavy chains and no light chains. The amino acid sequence of the variable domain of the naturally occurring heavy-chain antibodies reveals the necessary adaptations to compensate for the absence of the light chain. In contrast to the conventional antibodies, a large proportion of the heavy-chain antibodies acts as competitive enzyme inhibitors. Studies on the dromedary immunoglobulin genes start to shed light on the ontogeny of these heavy-chain antibodies. The presence of the heavy-chain antibodies and the possibility of immunizing a dromedary allows for the production of antigen binders consisting of a single domain only. These minimal antigen-binding fragments are well expressed in bacteria, bind the antigen with affinity in the nM range and are very stable. We expect that such camelid single domain antibodies will find their way into a number of biotechnological or medical applications. The structure of the camelid single domain is homologous to the human VH, however, the antigen-binding loop structures deviate fundamentally from the canonical structures described for human or mouse VHs. This has two additional advantages: (1) the camel or llama derived single domain antibodies might be an ideal scaffold for anti-idiotypic vaccinations; and (2) the development of smaller peptides or peptide mimetic drugs derived from of the antigen binding loops might be facilitated due to their less complex antigen binding site.

A naturally occurring NAR variable domain binds the Kgp protease from Porphyromonas gingivalis

“…This arrangement, and particularly the single variable domain, is very similar to the V H H antibodies found in camelid species in a clear case of convergent evolution at the molecular level [3,4]. V H Hs are capable of binding a wide range of protein, hapten and peptide targets and represent a signi¢cant proportion of the camelid immune response [5,6]. While a similar function for the NAR in the shark immune response awaits formal proof, there is strong evidence that NARs are functional antibodies.…”

“…With an a⁄nity for the target antigen of V130 nM, these single domains have antigen speci¢city comparable to recombinant forms of the camelid V H H single domain antibodies, where the a⁄nity varies between 2 and 300 nM [5,6]. However, NARs encompass this a⁄nity in two, rather than three CDR loops, as the CDR2 region is severely truncated [3,9].…”

Efficient Cancer Therapy with a Nanobody-Based Conjugate

“…Binding affinity to CEA ranged from 0.34 to 55 nM, which is within the affinity range of other single-domain antibodies (16,27) To assess the potential of nanobodies as vehicles to selectively deliver toxic principles to tumors, we fused ␤-lactamase from E. cloacae P99 to the high-affinity binder cAb-CEA5. This particular ␤-lactamase was chosen because it effectively converts many substrates into potent cytotoxic compounds (20).…”

Protein studies in dysferlinopathy patients using llama-derived antibody fragments selected by phage display

“…12 Camelideae-derived single-domain VHH antibody fragments exhibit unique properties, 13 including small size, good solubility, stability and a high level of specificity and affinity. A nonimmune llama phage antibody library was therefore selected against two dysferlin fragments.…”