Structural Basis of Epitope Recognition by Heavy-Chain Camelid Antibodies

Truncated versions of heavy-chain antibodies (HCAbs) from camelids, also termed nanobodies, comprise only one-tenth the mass of conventional antibodies, yet retain similar, high binding affinities for the antigens. Here we analyze a large data set of nanobody–antigen crystal structures and investigate how nanobody–antigen recognition compares to the one by conventional antibodies. We find that nanobody paratopes are enriched in aromatic residues just like conventional antibodies, but additionally, they also bear a more hydrophobic character. Most striking differences were observed in the characteristics of the antigen's epitope. Unlike conventional antibodies, nanobodies bind to more rigid, concave, conserved and structured epitopes enriched with aromatic residues. Nanobodies establish fewer interactions with the antigens compared to conventional antibodies, and we speculate that high binding affinities are achieved due to less unfavorable conformational and more favorable solvation entropy contributions. We observed that interactions with antigen are mediated not only by three CDR loops but also by numerous residues from the nanobody framework. These residues are not distributed uniformly; rather, they are concentrated into four structurally distinct regions and mediate mostly charged interactions. Our findings suggest that in some respects nanobody–antigen interactions are more similar to the general protein–protein interactions rather than antibody–antigen interactions.     

An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

Conventional anti-hapten antibodies typically bind low-molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain-only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti-hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an anti-methotrexate VHH (anti-MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti-MTX VHH CDR1-3 Graft revealed a micromolar binding affinity, while the crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1-3 graft, which resulted in a dramatic 1000-fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti-MTX CDR1-4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti-MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low-molecular weight ligands with high affinity and specificity, despite their reduced interface.     

靶向PSMA多价纳米抗体的制备及其生物学活性表征*

目的:构建原核表达系统,制备靶向前列腺特异性膜抗原(prostate-specific membrane antigen,PSMA)多价纳米抗体并初步评价其生物学活性。方法:Bglbrick法构建多价纳米抗体表达载体,转化至大肠杆菌表达并利用亲和层析法纯化。联合蛋白质电泳和Western blot验证纯化产物,BCA法检测表达量。通过免疫荧光和流式细胞术定性评估PSMA特异性亲和能力,细胞ELISA法定量检测PSMA亲和水平,流式细胞术检测内吞效率。结果:成功构建靶向PSMA单价、二价、三价和四价纳米抗体大肠杆菌表达菌株。发酵结果表明四种纳米抗体均能在摇瓶水平实现高效可溶表达,其中二价纳米抗体表达量最高[(259.14±23.56) mg/L],单价纳米抗体表达量最低[(100.58±6.27) mg/L]。亲和实验结果证实四种纳米抗体均能特异性识别并结合PSMA阳性肿瘤细胞,与单价纳米抗体相比,二价、三价和四价纳米抗体对PSMA亲和能力分别提高了3.32倍、2.29倍和2.03倍。最后的内吞实验显示四种纳米抗体均能被PSMA阳性肿瘤细胞高效摄取,30 min内的摄取率均在80%以上。结论:靶向PSMA的多价纳米抗体,尤其是二价纳米抗体,具有比单价纳米抗体更高的产量和亲和水平,且具备不亚于单价纳米抗体的内吞效率,是未来基于PSMA肿瘤诊疗试剂开发的重要候选。     

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.     

Role of the non‐hypervariable FR3 D‐E loop in single‐domain antibody recognition of haptens and carbohydrates

Single‐domain antibodies (sdAbs), the variable domains of camelid heavy chain‐only antibodies, are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However, the structures of anti‐methotrexate, anti‐triclocarban and anti‐cortisol sdAbs revealed unexpected contributions of the non‐hypervariable “CDR4” loop, formed between β‐strands D and E of framework region 3, in binding. Here, we investigated the potential role of CDR4 in sdAb binding to a hapten, 15‐acetyl‐deoxynivalenol (15‐AcDON), and to carbohydrates. We constructed and panned a phage‐displayed library in which CDR4 of the 15‐AcDON‐specific sdAb, NAT‐267, was extended and randomized. From this library, we identified one sdAb, MA‐232, bearing a 14‐residue insertion in CDR4 and showing improved binding to 15‐AcDON by ELISA and surface plasmon resonance. On the basis of these results, we constructed a second set of phage‐displayed libraries in which the CDR4 and other regions of three hapten‐ or carbohydrate‐binding sdAbs were diversified. With the goal of identifying sdAbs with novel glycan‐binding specificities, we panned the library against four tumor‐associated carbohydrate antigens but were unable to enrich binding phages. Thus, we conclude that while CDR4 may play a role in binding of some rare hapten‐specific sdAbs, diversifying this region through molecular engineering is probably not a general solution to sdAb carbohydrate recognition in the absence of a paired V_L domain.     

Equilibrium and kinetic parameters for the interaction of a monoclonal antibody with liposomes bearing fluorescent haptens

We have obtained equilibrium and rate constants for the interaction of monoclonal IgG and its monovalent Fab fragment with a hapten (fluorescein) attached to the surface of a liposome. Binding was detected at nanomolar hapten concentrations by the quenching of the hapten's fluorescence on antibody binding. The binding parameters were computed from nonlinear least squares fits, using mass-action models. Crypticity of the hapten was observed and interpreted as an equilibrium between two states, extended and sequestered, the latter representing haptens associated with the membrane surface. Depending on the lipid composition of the liposomes, the fraction of sequestered hapten ranged from 0.25 to 0.975; transitions between the two states took place on the time scale of minutes. Fab interactions with extended hapten on the membrane were similar to interactions with water-soluble hapten. The ability of IgG to bind bivalently to membrane gave it an avidity two to six times the affinity for purely monovalent binding. However, the equilibrium constant for the monovalent-bivalent binding equilibrium was effectively four to five orders of magnitude less than that for the initial binding step. This probably reflects steric penalties for the simultaneous binding of two haptens on a membrane.     

Exploring single-domain antibody thermostability by molecular dynamics simulation

Abstract

Single-domain antibodies also known as nanobodies are recombinant antigen-binding domains that correspond to the heavy-chain variable region of camelid antibodies. Previous experimental studies showed that the nanobodies have stable and active structures at high temperatures. In this study, the thermal stability and dynamics of nanobodies have been studied by employing molecular dynamics simulation at different temperatures. Variations in root mean square deviation, native contacts, and solvent-accessible surface area of the nanobodies during the simulation were calculated to analyze the effect of different temperatures on the overall conformation of the nanobody. Then, the thermostability mechanism of this protein was studied through calculation of dynamic cross-correlation matrix, principal component analyses, native contact analyses, and root mean square fluctuation. Our results manifest that the side chain conformation of some residues in the complementarity-determining region 3 (CDR3) and also the interaction between α-helix region of CDR3 and framework2 play a critical role to stabilize the protein at a high temperature.

Communicated by Ramaswamy H. Sarma     

Effects of charged groups in haptens on adsorption equilibrium of hapten antibody

Antisera against charged (p-azobenzoate and p-azoben zenesulfonate) and uncharged (dinitrophenyl) haptenic groups were produced in rabbits, and the equilibrium characteristics of hapten-antibody were measured by use of immunoadsorbents. The antibody to the uncharged hapten formed a stable binding with the hapten to the changes in ionic strength and pH. On the other hand, the antibodies to the charged haptens showed affinities sensitive to the changes in pH and ionic strength. Therefore, the effect of the pK(a) of ionizable haptens on the pH dependence of the hapten-antibody binding was studied by comparing the interactions between a series of para-substituted benzoic acids and the anti-p-azobenzoate antibody. The pH dependence of the interactions was strongly affected by the pK(a) of ionizable groups in haptens. Furthermore, the equilibrium characteristics of anti-p-aminobenzoyl dipeptides were compared. The characteristics of interactions were affected by the features of amino acid residues.     

Lateral recognition of a dye hapten by a llama VHH domain

Camelids, camels and llamas, have a unique immune system able to produce heavy-chain only antibodies. Their VH domains (VHHs) are the smallest binding units produced by immune systems, and therefore suitable for biotechnological applications through heterologous expression. The recognition of protein antigens by these VHHs is rather well documented, while less is known about the VHH/hapten interactions. The recently reported X-ray structure of a VHH in complex with a copper-containing azo-dye settled the ability of VHH to recognize haptens by forming a cavity between the three complementarity-determining regions (CDR). Here we report the structures of a VHH (VHH A52) free or complexed with an azo-dye, RR1, without metal ion. The structure of the complex illustrates the involvement of CDR2, CDR3 and a framework residue in a lateral interaction with the hapten. Such a lateral combining site is comparable to that found in classical antibodies, although in the absence of the VL.     

Resurfaced cell‐penetrating nanobodies: A potentially general scaffold for intracellularly targeted protein discovery

By virtue of their size, functional group diversity, and complex structure, proteins can often recognize and modulate disease‐relevant macromolecules that present a challenge to small‐molecule reagents. Additionally, high‐throughput screening and evolution‐based methods often make the discovery of new protein binders simpler than the analogous small‐molecule discovery process. However, most proteins do not cross the lipid bilayer membrane of mammalian cells. This largely limits the scope of protein therapeutics and basic research tools to those targeting disease‐relevant receptors on the cell surface or extracellular matrix. Previously, researchers have shown that cationic resurfacing of proteins can endow cell penetration. However, in our experience, many proteins are not amenable to such extensive mutagenesis. Here, we report that nanobodies—a small and stable protein that can be evolved to recognize virtually any disease‐relevant receptor—are amenable to cationic resurfacing, which results in cell internalization. Once internalized, these nanobodies access the cytosol. Polycationic resurfacing does not appreciably alter the structure, expression, and function (target recognition) of a previously reported GFP‐binding nanobody, and multiple nanobody scaffolds are amenable to polycationic resurfacing. Given this, we propose that polycationic resurfaced cell‐penetrating nanobodies might represent a general scaffold for intracellularly targeted protein drug discovery.     

By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro.

By display of antibody repertoires on the surface of a filamentous bacteriophage and selection of the phage by binding to antigen, we can mimic immune selection. Recently, by tapping the repertoire of rearranged V-genes from the peripheral blood lymphocytes of unimmunised donors, we succeeded in making human antibody fragments with different specificities, including both haptens and proteins, from the same library of phage. Now we have built a repertoire of human VH genes from 49 human germline VH gene segments rearranged in vitro to create a synthetic third complementarity determining region (CDR) of five or eight residues. The rearranged VH genes were cloned with a human V lambda 3 light chain as single chain Fv fragments for phage display, and the library of phage panned by binding to each of two haptens, 2-phenyl-5-oxazolone (phOx) or 3-iodo-4-hydroxy-5-nitrophenyl-acetate (NIP) coupled to bovine serum albumin (BSA). Many different antibody fragments were isolated which bound specifically to hapten, some with affinities in the micromolar range. The in vitro "immune response" to the hapten NIP was dominated by the 9-1 segment (VH3 family), and that to phOx by the VH26 segment (VH3 family) with an invariant aromatic residue (Tyr, Phe, Trp) at residue 97 of CDR3. However, the isolation of phage against protein antigens proved more elusive, with a single phage binding to human tumour necrosis factor, and none to bovine serum albumin, turkey egg-white lysozyme or human thyroglobulin. Nevertheless, the work shows that human antibody fragments with specific binding activities can be made entirely in vitro.     

Nanobody Mediated Inhibition of Attachment of F18 Fimbriae Expressing Escherichia coli

Post-weaning diarrhea and edema disease caused by F18 fimbriated E. coli are important diseases in newly weaned piglets and lead to severe production losses in farming industry. Protective treatments against these infections have thus far limited efficacy. In this study we generated nanobodies directed against the lectin domain of the F18 fimbrial adhesin FedF and showed in an in vitro adherence assay that four unique nanobodies inhibit the attachment of F18 fimbriated E. coli bacteria to piglet enterocytes. Crystallization of the FedF lectin domain with the most potent inhibitory nanobodies revealed their mechanism of action. These either competed with the binding of the blood group antigen receptor on the FedF surface or induced a conformational change in which the CDR3 region of the nanobody displaces the D″-E loop adjacent to the binding site. This D″-E loop was previously shown to be required for the interaction between F18 fimbriated bacteria and blood group antigen receptors in a membrane context. This work demonstrates the feasibility of inhibiting the attachment of fimbriated pathogens by employing nanobodies directed against the adhesin domain.     

Camelid heavy-chain variable domains provide efficient combining sites to haptens

Camelids can produce antibodies devoid of light chains and CH1 domains (Hamers-Casterman, C. et al. (1993) Nature 363, 446-448). Camelid heavy-chain variable domains (VHH) have high affinities for protein antigens and the structures of two of these complexes have been determined (Desmyter, A. et al. (1996) Nature Struc. Biol. 3, 803-811; Decanniere, K. et al. (1999) Structure 7, 361-370). However, the small size of these VHHs and their monomeric nature bring into question their capacity to bind haptens. Here, we have successfully raised llama antibodies against the hapten azo-dye Reactive Red (RR6) and determined the crystal structure of the complex between a dimer of this hapten and a VHH fragment. The surface of interaction between the VHH and the dimeric hapten is large, with an area of ca. 300 A(2); this correlates well with the low-dissociation constant of 22 nM measured for the monomer. The VHH fragment provides an efficient combining site to the RR6, using its three CDR loops. In particular, CDR1 provides a strong interaction to the hapten through two histidine residues bound to its copper atoms. VHH fragments might, therefore, prove to be valuable tools for selecting, removing, or capturing haptens. They are likely to play a role in biotechnology extending beyond protein recognition alone.     

The testosterone binding mechanism of an antibody derived from a naïve human scFv library

A testosterone binding scFv antibody was isolated from a naïve human library with a modest size of 10⁸ clones. The crystal structure of the Fab fragment form of the 5F2 antibody clone complexed with testosterone determined at 1.5 Å resolution shows that the hapten is bound deeply in the antibody binding pocket. In addition to the interactions with framework residues only CDR‐L3 and CDR‐H3 loops interact with testosterone and the heavy chain forms the majority of the contacts with the hapten. The testosterone binding site of the 5F2 antibody with a high abundance of aromatic amino acid residues shows similarity with an in vitro affinity matured antibody having around 300 times higher affinity. The moderate affinity of the 5F2 antibody originates from the different orientation of the hapten and few light chain contacts. This is the first three‐dimensional structure of a human steroid hormone binding antibody that has been isolated from a naïve human repertoire. Copyright © 2010 John Wiley & Sons, Ltd.     

Targeting high affinity and epitope-distinct oligoclonal nanobodies to HER2 over-expressing tumor cells

Modern anti-HER2 antibody therapy tends to exploit a panel of different antibodies against different epitopes on the antigen. For this aim, nanobodies are very striking targeting agents and can be easily produced against any cell-specific membrane antigen. The oligoclonal nanobodies can be used to block more than one functional epitope on a target antigen and inhibit the generation of escape variants associated with cancer therapy. In this study, 12 nanobody clones selected from an immune camel library were examined for their ability to differ between tumor markers. These oligoclonal nanobodies targeted breast cancer cells better than each individual nanobody. In epitope mapping, several nanobodies overlapped in the epitope recognized by trastuzumab and some of the non-overlapping nanobodies could affect the binding of trastuzumab to HER2. This study demonstrates that the oligoclonal nanobodies are potential therapeutic tools that can be used instead of, or in combination with trastuzumab to assess tumor viability during treatment.     

Mechanistic analysis of allosteric and non-allosteric effects arising from nanobody binding to two epitopes of the dihydrofolate reductase of Escherichia coli

Although allosteric effector antibodies are used widely as modulators of receptors and enzymes, experimental analysis of their mechanism remains highly challenging. Here, we investigate the molecular mechanisms of allosteric and non-allosteric effector antibodies in an experimentally tractable system, consisting of single-domain antibodies (nanobodies) that target the model enzyme dihydrofolate reductase (DHFR) from Escherichia coli. A panel of thirty-five nanobodies was isolated using several strategies to increase nanobody diversity. The nanobodies exhibit a variety of effector properties, including partial inhibition, strong inhibition and stimulation of DHFR activity. Despite these diverse effector properties, chemical shift perturbation NMR epitope mapping identified only two epitope regions: epitope α is a new allosteric site that is over 10 Å from the active site, while epitope β is located in the region of the Met20 loop. The structural basis for DHFR allosteric inhibition or activation upon nanobody binding to the α epitope was examined by solving the crystal structures of DHFR in complex with Nb113 (an allosteric inhibitor) and Nb179 (an allosteric activator). The structures suggest roles for conformational constraint and altered protein dynamics, but not epitope distortion, in the observed allosteric effects. The crystal structure of a β epitope region binder (ca1698) in complex with DHFR is also reported. Although CDR3 of ca1698 occupies the substrate binding site, ca1698 displays linear mixed inhibition kinetics instead of simple competitive inhibition kinetics. Two mechanisms are proposed to account for this apparent anomaly. Evidence for structural convergence of ca1698 and Nb216 during affinity maturation is also presented.     

Domain swapping of a llama VHH domain builds a crystal-wide beta-sheet structure

Among mammals, camelids have a unique immunological system since they produce functional antibodies devoid of light chains and CH1 domains. To bind antigens, whether they are proteins or haptens, camelids use the single domain VH from their heavy chain (VHH). We report here on such a llama VHH domain (VHH-R9) which was raised against a hapten, the RR6 red dye. This VHH possesses the shortest complementarity determining region 3 (CDR3) among all the known VHH sequences and nevertheless binds RR6 efficiently with a K(d) value of 83 nM. However, the crystal structure of VHH-R9 exhibits a striking feature: its CDR3 and its last beta-strand (beta9) do not follow the immunoglobulin VH domain fold, but instead extend out of the VHH molecular boundary and associate with a symmetry-related molecule. The two monomers thus form a domain-swapped dimer which establishes further contacts with symmetry-related molecules and build a crystal-wide beta-sheet structure. The driving force of the dimer formation is probably the strain induced by the short CDR3 together with the cleavage of the first seven residues.     

Affinity enhancement of nanobody binding to EGFR: in silico site-directed mutagenesis and molecular dynamics simulation approaches

Epidermal growth factor receptor (EGFR), a transmembrane glycoprotein, is overexpressed in many cancers such as head-neck, breast, prostate, and skin cancers for this reason it is a good target in cancer therapy and diagnosis. In nanobody-based cancer diagnosis and treatment, nanobodies with high affinity toward receptor (e.g. EGFR) results in effective treatment or diagnosis of cancer. In this regard, the main aim of this study is to develop a method based on molecular dynamic (MD) simulations for designing of 7D12 based nanobody with high affinity compared with wild-type nanobody. By surveying electrostatic and desolvation interactions between different residues of 7D12 and EGFR, the critical residues of 7D12 that play the main role in the binding of 7D12 to EGFR were elucidated and based on these residues, five logical variants were designed. Following the 50 ns MD simulations, pull and umbrella sampling simulation were performed for 7D12 and all its variants in complex with EGFR. Binding free energy of 7D12 (and all its variants) with EGFR was obtained by weighted histogram analysis method. According to binding free energy results, GLY101 to GLU mutation showed the highest binding affinity but this variant is unstable after 50 ns MD simulations. ALA100 to GLU mutation shows suitable binding enhancement with acceptable structural stability. Suitable position and orientation of GLU in residue 100 of 7D12 against related amino acids of EGFR formed some extra hydrogen and electrostatic interactions which resulted in binding enhancement.     

Engineered high-affinity nanobodies recognizing staphylococcal Protein A and suitable for native isolation of protein complexes

In addition to its high affinity for antibody Fc domains, staphylococcal Protein A has been shown to bind certain Fab domains. We investigated this in order to develop a small, recombinant Protein A-binding alternative to immunoglobulin G (IgG) from nanobodies, single-domain antibodies derived from a camelid variant IgG’s variable region. We engineered a nanobody with affinity solely for Protein A as well as a dimerized version of higher affinity for typical multidomain Protein A constructs. Because this recombinant nanobody can be immobilized using a cleavable crosslinker, it has proven to be suitable for the isolation and mild elution of protein complexes in native conditions.     

Resonance Raman-spectroscopic studies of the hapten features involved in the binding of 2,4-dinitrophenyl haptens by the mouse myeloma proteins MOPC 315 and MOPC 460.

The binding of four dinitrophenyl haptens to the mouse myeloma proteins MOPC 315 IgA (immunoglobulin A) and MOPC 460IgA was studied by resonance Raman spectroscopy. Isotopic substitution with 15N and 2H was used to assign features in the resonance Raman spectra of the free haptens. Changes in each of these features on binding to the proteins could then be attributed to interactions of the proteins' binding sites with either the p-NO2 or the o-NO2/amine regions of the haptens. The interactions between a given hapten and MOPC 315 IgA are often quite distinct from those between the same hapten and MOPC 460 IgA. Moreover, for both antibodies the nature of the R side chain in a Dnp-NHR (Dnp, 2,4-dinitrophenyl) compound appears to modify the interactions between the Dnp chromophore and the protein. Thus, with the haptens studied, there is no unique set of contacts between the Dnp group and the binding site. The contacts expected between epsilon-2,4-dinitrophenyl-L-lysine and the site on MOPC 315 IgA, on the basis of a recent model for this site [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977) Nature (London) 266, 31--37] were not detected. However, the contacts between this hapten and the site on MOPC 460 IgA were closer to those predicted by the model for MOPC 315 IgA.