Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies

Abstract

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)...

Mathematical analysis of the pharmacokinetic–pharmacodynamic (PKPD) behaviour of monoclonal antibodies: Predicting in vivo potency

“…Clearly, these conditions can be fulfilled in two ways, namely by L 0 being large relative to k out , k off and k e(L) (as we have already considered) or by k on being large relative to k out , k off and k e(L) (with no corresponding requirement that L 0 also be large). In this second case, the results given by (19) and (20) also hold. We note that even for a large (fixed) value of L 0 , if K D becomes sufficiently large, then k on L 0 will no longer be large relative to k off and so this approximation fails.…”

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…65 It was recognized from early structural studies of anti-lysozyme V H Hs 6 and V NAR s 7 that these molecules interacted with the enzyme in unusual fashion, probing deeply into its active site using extended complementarity-determining region (CDR)3 loops. These results were later replicated independently using additional V H s, 51 V H Hs 11,47,48 and V NAR s 12 directed against the active site of lysozyme, as well as with active site-binding V H Hs against α-amylase, 31,32 carbonic anhydrase, 32 and urokinase. 62,63 Inhibition of α-amylase was achieved by one V H H through penetration of the active site cleft with its CDR2 loop, 31 demonstrating that CDR3-centric binding is not the only mechanism of competitive enzyme inhibition by sdAbs.…”

“…105 By contrast, sdAb paratopes can clearly adopt both flat 106,107 and convex 11 topologies, although possibly only inefficiently adopt concave ones. The CDR1 and CDR2 loops of V H Hs depart from the typical canonical structures of conventional antibodies (Figure 2A), potentially through somatic mutation since germline human V H and camelid V H H repertoires appear to have similar canonical structures.…”

“…66,67 This hypothesis is supported by experiments using purified polyclonal immunoglobulin (Ig)Gs from enzyme-immunized dromedaries showing that competitive inhibition was a feature of heavy chain-only IgGs, but not of conventional IgGs. 11,32 It remains unclear why immunization with some enzymes yields mostly sdAbs with planar paratopes and bind outside the active site, achieving allosteric or no inhibition, although tolerance mechanisms may play a role.…”

“…With hindsight, the notion that sdAbs might preferentially target particular types of antigenic structures may not seem totally unexpected, given their recombination from distinct repertoires of V, D and J genes (see Box 1 ), 8 their potential ontogeny from separate B-cell precursors, 9 and for camelid V H Hs, their specialized constant regions bearing very long hinge regions. 10 However, the specific mechanisms of sdAb antigen recognition ( e.g ., the tertiary structures and physicochemical properties of sdAb:antigen interfaces, which may differ fundamentally from those of conventional antibody:antigen interfaces) remain unclear, although several studies have suggested protein cleft recognition as a general function for both V H Hs 11 and V NAR s. 12 Over time, the idea that sdAbs can target ‘cryptic’ epitopes (so-called because they are inaccessible to conventional antibodies, either for steric reasons or due to their fundamental antigenic properties) has become entrenched, and although several case studies have supported it, its generality and implications are questionable. Several excellent recent reviews and opinion pieces have alluded to the nature of sdAb paratopes and their interactions with antigens, but have either not been rigorous in their approach or have incompletely addressed the topic, analyzing the properties of sdAb paratopes only, with no comparison to those of conventional antibodies.…”