Clefts on protein surfaces are avoided by antigen-combining sites of conventional antibodies, in contrast to heavy-chain antibodies (HCAbs) of camelids that seem to be attracted by enzymes' substrate pockets. The explanation for this pronounced preference of HCAbs was investigated. Eight single domain antigen-binding fragments of HCAbs (VHH) with nanomolar affinities for lysozyme were isolated from three immunized dromedaries. Six of eight VHHs compete with small lysozyme inhibitors. This ratio of active site binders is also found within the VHH pool derived from polyclonal HCAbs purified from the serum of the immunized dromedary. The crystal structures of six VHHs in complex with lysozyme and their interaction surfaces were compared to those of conventional antibodies with the same antigen. The interface sizes of VHH and conventional antibodies to lysozyme are very similar as well as the number and chemical nature of the contacts. The main difference comes from the compact prolate shape of VHH that presents a large convex paratope, predominantly formed by the H3 loop and interacting, although with different structures, into the concave lysozyme substrate-binding pocket. Therefore, a single domain antigen-combining site has a clear structural advantage over a conventional dimeric format for targeting clefts on antigenic surfaces.antibody-lysozyme structures ͉ camel single domain antibody ͉ enzyme inhibitor ͉ epitope-paratope interactions S ix hypervariable antigen-binding loops constitute the antigen-combining sites of conventional antibodies. These loops, three (H1-H3) from the variable domain of the heavy chain (VH), three (L1-L3) from the variable domain of the light chain (VL) are juxtaposed forming a continuous surface (paratope) that is complementary to a surface on the antigen (epitope) (1). The paratope is essentially planar for protein antigens and forms a groove or cavity to interact with peptides and haptens (2, 3). The loops L1-L3 and H1-H2 fold into a limited number of canonical structure classes, determined by the loop length and the presence of conserved residues at key positions within the hypervariable and framework regions (4, 5). The extreme length and sequence variability of H3 makes the structure prediction of this loop extremely difficult (6).The structures of antigen-binding sites and loops, as well as the canonical loop determining residues, are well established (1,4,5). In contrast, the elucidation of the molecular basis for the recognition of particular epitopes by antibodies remains a major challenge. Our knowledge and paradigms of protein-epitope recognition by antibodies is largely based on the analysis of the immune response toward hen egg white lysozyme (HEWL). This is due to the high antigenicity, the large number of natural variants of HEWL (7), and the availability of eleven different crystal structures of Fab or Fv antibody fragments (8-10) in complex with lysozyme, collected over the last two decades. Six structures represent Fabs or Fvs that are clearly clonally unrelated (8)...

Nanobodies, single-domain antigen-binding fragments of camelid-specific heavy-chain only antibodies offer special advantages in therapy over classic antibody fragments because of their smaller size, robustness, and preference to target unique epitopes. A Nanobody differs from a human heavy chain variable domain in about ten amino acids spread all over its surface, four hallmark Nanobody-specific amino acids in the framework-2 region (positions 42, 49, 50, and 52), and a longer third antigen-binding loop (H3) folding over this area. For therapeutic applications the camelid-specific amino acid sequences in the framework have to be mutated to their human heavy chain variable domain equivalent, i.e. humanized. We performed this humanization exercise with Nanobodies of the subfamily that represents close to 80% of all dromedary-derived Nanobodies and investigated the effects on antigen affinity, solubility, expression yield, and stability. It is demonstrated that the humanization of Nanobody-specific residues outside framework-2 are neutral to the Nanobody properties. Surprisingly, the Glu-49 3 Gly and Arg-50 3 Leu humanization of hallmark amino acids generates a single domain that is more stable though probably less soluble. The other framework-2 substitutions, Phe-42 3 Val and Gly/Ala-52 3 Trp, are detrimental for antigen affinity, due to a repositioning of the H3 loop as shown by their crystal structures. These insights were used to identify a soluble, stable, well expressed universal humanized Nanobody scaffold that allows grafts of antigen-binding loops from other Nanobodies with transfer of the antigen specificity and affinity.Minimizing the size of antigen-binding entities from a multidomain protein such as a monoclonal antibody to a singlechain variable fragment or even a single domain has been one of the primary goals of antibody engineering. For drug therapy, these smaller formats can be beneficial in various aspects such as immunogenicity, biodistribution, renal clearance, serum half-life, tissue penetration, and target retention. However, the minimal sized antibody fragments need to retain sufficiently high antigen specificity and affinity, be expressed in high yields, and should have a low tendency to aggregate so as to maintain maximal potency and reduce possible risks of immunogenicity. Moreover functionality in adverse environments such as high concentrations of denaturant or elevated temperatures, and a concomitant increased shelf-life are additional assets.A significant proportion of the functional antibodies within species of the Camelidae are devoid of light chains. These immunoglobulins are referred to as heavy-chain antibodies (1), and their antigen-binding fragment is reduced to a single domain (referred to as VHH or Nanobody), with a molecular size of only ϳ15 kDa, which is smaller in comparison to singlechain variable fragment fragments (30 kDa), Fab fragments (60 kDa), and whole antibodies (150 kDa). All Nanobodies belong to the same sequence family, closely related to that of the human VH 3...

No abstract