Dissection of the IgNAR V Domain: Molecular Scanning and Orthologue Database Mining Define Novel IgNAR Hallmarks and Affinity Maturation Mechanisms

The shark antigen-binding V_NAR domain has the potential to provide an attractive alternative to traditional biotherapeutics based on its small size, advantageous physiochemical properties, and unusual ability to target clefts in enzymes or cell surface molecules. The V_NAR shares many of the properties of the well-characterised single-domain camelid V_HH but is much less understood at the molecular level. We chose the hen-egg-lysozyme-specific archetypal Type I V_NAR 5A7 and used ribosome display in combination with error-prone mutagenesis to interrogate the entire sequence space. We found a high level of mutational plasticity across the V_NAR domain, particularly within the framework 2 and hypervariable region 2 regions. A number of residues important for affinity were identified, and a triple mutant combining A1D, S61R, and G62R resulted in a K_D of 460 pM for hen egg lysozyme, a 20-fold improvement over wild-type 5A7, and the highest K_D yet reported for V_NAR-antigen interactions. These findings were rationalised using structural modelling and indicate the importance of residues outside the classical complementarity determining regions in making novel antigen contacts that modulate affinity. We also located two solvent-exposed residues (G15 and G42), distant from the V_NAR paratope, which retain function upon mutation to cysteine and have the potential to be exploited as sites for targeted covalent modification. Our findings with 5A7 were extended to all known NAR structures using an in-depth bioinformatic analysis of sequence data available in the literature and a newly generated V_NAR database. This study allowed us to identify, for the first time, both V_NAR-specific and V_NAR/Ig V_L/TCR V_α overlapping hallmark residues, which are critical for the structural and functional integrity of the single domain. Intriguingly, each of our designated V_NAR-specific hallmarks align precisely with previously defined mutational ‘cold spots’ in natural nurse shark cDNA sequences. These findings will aid future V_NAR engineering and optimisation studies towards the development of V_NAR single-domain proteins as viable biotherapeutics.     

An anti-urokinase plasminogen activator receptor (uPAR) antibody: crystal structure and binding epitope

Human urokinase-type plasminogen activator receptor (uPAR/CD87) is expressed at the invasive interface of the tumor-stromal microenvironment in many human cancers and interacts with a wide array of extracellular molecules. An anti-uPAR antibody (ATN615) was prepared using hybridoma technology. This antibody binds to uPAR in vitro with high affinity (K(d) approximately 1 nM) and does not interfere with uPA binding to uPAR. Here we report the crystal structure of the Fab fragment of ATN615 at 1.77 A and the analysis of ATN615-suPAR-ATF structure that was previously determined, emphasizing the ATN615-suPAR interaction. The complementarity determining regions (CDRs) of ATN615 consist of a high percentage of aromatic residues, and form a relatively flat and undulating surface. The ATN615 Fab fragment recognizes domain 3 of suPAR. The antibody-antigen recognition involves 11 suPAR residues and 12 Fab residues from five CDRs. Structural data suggest that Pro188, Asn190, Gly191, and Arg192 residues of uPAR are the key residues for the antibody recognition, while Pro189 and Arg192 render specificity of ATN615 for human uPAR. Interestingly, this antibody-antigen interface has a small contact area, mainly polar interaction with little hydrophobic character, yet has high binding strength. Furthermore, several solvent molecules (assigned as polyethylene glycols) were clearly visible in the binding interface between antibody and antigen, suggesting that solvent molecules may be important for the maximal binding between suPAR and ATN615 Fab. ATN615 undergoes small but noticeable changes in its CDR region upon antigen binding.     

Immunochemical Features of Complementarity Determining Region (CDR) Peptide in Anti Hemin Monoclonal Antibody

Messenger RNA purified from the anti hemin monoclonal antibody (1D3) secreting hybridoma was amplified by RT‐PCR and the nueleotide and amino acid sequences of the antibody were determined. The role of complementarity determining regions (CDRs) in porphyrin recognition and its immunochemical feature of the antibody were investigated by using ELISA, fluorescence measurement and computational calculation of the conformation. All CDR peptides of the heavy chain of the antibody were synthesized and their affinity constants to porphyrins were determined. The value of CDR2 of heavy chain (CDRH2) of 1D3 was 1.5×10⁵/M for protoporphyrin and 7×10⁷/M for TCPP, respectively, while that of the whole antibody showed to be 1.2×10⁷/M for TCPP. Though CDRH2 is a 17 meric peptide, it showed higher affinity than the whole antibody (1D3). Porphyrins can be considered to firmly bind with CDRH2, while CDRH3 is not involved in the antigen binding. CDR‐1 may participate in the recognition with a small contribution. By the computational analysis of steric conformation, it was suggested that CDRH1 and CDRH2 co‐operatively function in the recognition of porphyrin. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Analyzing the "degree of humanness" of antibody sequences

Genetically engineered mouse antibodies are now commonly in clinical use. However, their development is limited because the human immune system tends to regard them as foreign and this triggers an immune response. The solution is to make engineered antibodies appear more human. Here, we propose a method to assess the "degree of humanness" of antibody sequences providing a tool that may contribute to predictions of antigenicity. We analyzed sequences of antibodies belonging to various chains/classes in human and mouse. Our analysis of metrics based on percentage sequence identity between antibody sequences shows distinct differences between human and mouse sequences. Based on mean sequence identity and standard deviation, we calculated Z-scores for data sets of antibody sequences extracted from the Kabat database. We applied the analysis to a set of humanized and chimeric antibodies and to human germline sequences. We conclude that this approach may aid in the selection of more suitable mouse variable domains for antibody engineering to render them more human but in general, we find that typicality of a sequence compared with the expressed human repertoire is not well correlated with antigenicity. We have provided a Web server allowing humanness to be assigned for a sequence.     

Single MHC mutation eliminates enthalpy associated with T cell receptor binding

The keystone of the adaptive immune response is T cell receptor (TCR) recognition of peptide presented by major histocompatibility complex (pMHC) molecules. The crystal structure of AHIII TCR bound to MHC, HLA-A2, showed a large interface with an atypical binding orientation. MHC mutations in the interface of the proteins were tested for changes in TCR recognition. From the range of responses observed, three representative HLA-A2 mutants, T163A, W167A, and K66A, were selected for further study. Binding constants and co-crystal structures of the AHIII TCR and the three mutants were determined. K66 in HLA-A2 makes contacts with both peptide and TCR, and has been identified as a critical residue for recognition by numerous TCR. The K66A mutation resulted in the lowest AHIII T cell response and the lowest binding affinity, which suggests that the T cell response may correlate with affinity. Importantly, the K66A mutation does not affect the conformation of the peptide. The change in affinity appears to be due to a loss in hydrogen bonds in the interface as a result of a conformational change in the TCR complementarity-determining region 3 (CDR3) loop. Isothermal titration calorimetry confirmed the loss of hydrogen bonding by a large loss in enthalpy. Our findings are inconsistent with the notion that the CDR1 and CDR2 loops of the TCR are responsible for MHC restriction, while the CDR3 loops interact solely with the peptide. Instead, we present here an MHC mutation that does not change the conformation of the peptide, yet results in an altered conformation of a CDR3.     

The specificity of cross-reactivity: promiscuous antibody binding involves specific hydrogen bonds rather than nonspecific hydrophobic stickiness

Proteins are renowned for their specificity of function. There is, however, accumulating evidence that many proteins, from enzymes to antibodies, are functionally promiscuous. Promiscuity is of considerable physiological importance. In the immune system, cross‐reactive or multispecific antibodies are implicated in autoimmune and allergy conditions. In most cases, however, the mechanism behind promiscuity and the relationship between specific and promiscuous activities are unknown. Are the two contradictory? Or can a protein exhibit several unrelated activities each of which is highly specific? To address these questions, we studied a multispecific IgE antibody (SPE7) elicited against a 2,4‐dinitrophenyl hapten (DNP). SPE7 is able to distinguish between closely related derivatives such as NP (nitrophenol) and DNP, yet it can also bind a number of unrelated ligands. We find that, like DNP, the cross‐reactants are themselves bound specifically—close derivatives of these cross‐reactants show very low or no binding to SPE7. It has been suggested that cross‐reactivity is simply due to “hydrophobic stickiness”, nonspecific interactions between hydrophobic ligands and binding sites. However, partitioning experiments reveal that affinity for SPE7 is unrelated to ligand hydrophobicity. These data, combined with crystal structures of SPE7 in complex with four different ligands, demonstrate that each cross‐reactant is bound specifically, forming different hydrogen bonds dependant upon its particular chemistry and the availability of complementary antibody residues. SPE7 is highly homologous to the germline antinitrophenol (NP) antibody B1–8. By comparing the sequences and binding patterns of SPE7 and B1–8, we address the relationship between affinity maturation, specificity, and cross‐reactivity.     

Exploring the capacity of minimalist protein interfaces: interface energetics and affinity maturation to picomolar KD of a single-domain antibody with a flat paratope

A major architectural class in engineered binding proteins ("antibody mimics") involves the presentation of recognition loops off a single-domain scaffold. This class of binding proteins, both natural and synthetic, has a strong tendency to bind a preformed cleft using a convex binding interface (paratope). To explore their capacity to produce high-affinity interfaces with diverse shape and topography, we examined the interface energetics and explored the affinity limit achievable with a flat paratope. We chose a minimalist paratope limited to two loops found in a natural camelid heavy-chain antibody (VHH) that binds to ribonuclease A. Ala scanning of the VHH revealed only three "hot spot" side chains and additional four residues important for supporting backbone-mediated interactions. The small number of critical residues suggested that this is not an optimized paratope. Using selection from synthetic combinatorial libraries, we enhanced its affinity by >100-fold, resulting in variants with Kd as low as 180 pM with no detectable loss of binding specificity. High-resolution crystal structures revealed that the mutations induced only subtle structural changes but extended the network of interactions. This resulted in an expanded hot spot region including four additional residues located at the periphery of the paratope with a concomitant loss of the so-called "O-ring" arrangement of energetically inert residues. These results suggest that this class of simple, single-domain scaffolds is capable of generating high-performance binding interfaces with diverse shape. More generally, they suggest that highly functional interfaces can be designed without closely mimicking natural interfaces.     

Identification and characterization of single chain anti-cocaine catalytic antibodies

Cocaine is a powerful and addictive stimulant whose abuse remains a prevalent health and societal crisis. Unfortunately, no pharmacological therapies exist and therefore alternative protein-based therapies have been examined. One such approach is immunopharmacotherapy, wherein antibodies are utilized to either bind or hydrolyze cocaine thereby blocking it from exerting its euphoric effect. Towards this end, antibodies capable of binding and hydrolyzing cocaine were identified by phage display from a biased single chain antibody library generated from the spleens of mice previously immunized with a cocaine phosphonate transition state analog hapten. Two classes of antibodies emerged based on sequence homology and mode of action. Alanine scanning mutagenesis and kinetic analysis revealed that residues H97, H99, and L96 are crucial for antibodies 3F5 and 3H9 to accelerate the hydrolysis of cocaine. Antibodies 3F1 through 3F4, which are similar to our previously identified 3A6 class of antibodies, catalyze hydrolysis through transition state stabilization by tyrosine or histidine residues H50 and L94. Mutation of either one or both tyrosine residues to histidine conferred hydrolytic activity on previously inactive antibody 3F4. Mutational analysis of residue H50 of antibody 3F3 resulted in a glutamine mutant with a rate enhancement three times greater than wild-type. A double mutant, containing glutamineH50 and lysineH52, showed a tenfold rate enhancement over wild-type. These results indicate the power of initial selection of catalytic antibodies from a biased antibody library in both rapid generation and screening of mutants for improved catalysis.     

AbRSA: A robust tool for antibody numbering

The remarkable progress in cancer immunotherapy in recent years has led to the heat of great development for therapeutic antibodies. Antibody numbering, which standardizes a residue index at each position of an antibody variable domain, is an important step in immunoinformatic analysis. It provides an equivalent index for the comparison of sequences or structures, which is particularly valuable for antibody modeling and engineering. However, due to the extremely high diversity of antibody sequences, antibody‐numbering tools cannot work in all cases. This article introduces a new antibody‐numbering tool named AbRSA, which integrates heuristic knowledge of region‐specific features into sequence mapping to enhance the robustness. The benchmarks demonstrate that, AbRSA exhibits robust performance in numbering sequences with diverse lengths and patterns compared with the state‐of‐the‐art tools. AbRSA offers a user‐friendly interface for antibody numbering, complementarity‐determining region delimitation, and 3D structure rendering. It is freely available at http://cao.labshare.cn/AbRSA .     

Modulating the binding properties of an anti-17beta-estradiol antibody by systematic mutation combinations

The anti-17beta-estradiol antibody 57-2 has been a subject for several protein engineering studies that have produced a number of mutants with improved binding properties. Here, we generated a set of 16 antibody 57-2 variants by systematically combining mutations previously identified from phage display-derived improved antibody mutants. These mutations included three point mutations in the variable domain of the light-chain and a heavy-chain variant containing a four-residue random insertion in complementarity determining region CDR-H2. The antibody variants were expressed as Fab fragments, and they were characterized for affinity toward estradiol, for cross-reactivity toward three related steroids, and for dissociation rate of the Fab/estradiol complex by using time-resolved fluorescence based immunoassays. The double-mutant cycle method was used to address the cooperativity effects between the mutations. The experimental data were correlated with structural information by using molecular modeling and visual analysis of the previously solved antibody 57-2 crystal structures. These analyses provided information about the steroid-binding mode of the antibody, the potential mechanisms of individual mutations, and their mutual interactions. Furthermore, several combinatorial mutants with improved affinity and specificity were obtained. The capacity of one of these mutants to detect estradiol concentrations at a clinically relevant range was proved by establishing a time-resolved fluorescence based immunoassay.     

Intracellular ribosome display via SecM translation arrest as a selection for antibodies with enhanced cytosolic stability

Ribosome display is a powerful approach for affinity and stability maturation of recombinant antibodies. However, since ribosome display is performed entirely in vitro, there are several limitations to this approach including technical challenges associated with: (i) efficiently expressing and stalling antibodies on ribosomes using cell-free translation mixtures; and (ii) folding of antibodies in buffers where the concentration and composition of factors varies from that found in the intracellular milieu. We have developed a novel method for intracellular ribosome display that takes advantage of the recently discovered Escherichia coli SecM translation arrest mechanism. Specifically, we provide the first evidence that the encoding mRNA of SecM-stalled heterologous proteins remains stably attached to ribosomes, thereby enabling creation of stalled antibody-ribosome-mRNA (ARM) complexes entirely inside of living cells. Since ARM complexes faithfully maintain a genotype-phenotype link between the arrested antibody and its encoding mRNA, we demonstrate that this method is ideally suited for isolating stability-enhanced single-chain variable fragment (scFv) antibodies that are efficiently folded and functional in the bacterial cytoplasm.     

Modulation of the antigenic peptide transporter TAP by recombinant antibodies binding to the last five residues of TAP1

The transporter associated with antigen processing (TAP) plays a pivotal role in the major histocompatibility complex (MHC) class I mediated immune response against infected or malignantly transformed cells. It belongs to the ATP-binding cassette (ABC) superfamily and consists of TAP1 (ABCB2) and TAP2 (ABCB3), each of which possesses a transmembrane and a nucleotide-binding domain (NBD). Here we describe the generation of recombinant Fv and Fab antibody fragments to human TAP from a hybridoma cell line expressing the TAP1-specific monoclonal antibody mAb148.3. The epitope of the antibody was mapped to the very last five C-terminal amino acid residues of TAP1 on solid-supported peptide arrays. The recombinant antibody fragments were heterologously expressed in Escherichia coli and purified to homogeneity from periplasmic extracts by affinity chromatography. The monoclonal and recombinant antibodies bind with nanomolar affinity to the last five C-terminal amino acid residues of TAP1 as demonstrated by ELISA and surface plasmon resonance. Strikingly, the recombinant antibody fragments confer thermal stability to the heterodimeric TAP complex. At the same time TAP is arrested in a peptide transport incompetent conformation, although ATP and peptide binding to TAP are not affected. Based on our results we suggest that the C terminus of TAP1 modulates TAP function presumably as part of the dimer interface of the NBDs.     

A disulfide-free single-domain V(L) intrabody with blocking activity towards huntingtin reveals a novel mode of epitope recognition

We present the crystal structure and biophysical characterization of a human V_L [variable domain immunoglobulin (Ig) light chain] single-domain intrabody that binds to the huntingtin (Htt) protein and has been engineered for antigen recognition in the absence of its intradomain disulfide bond, otherwise conserved in the Ig fold. Analytical ultracentrifugation demonstrated that the αHtt-V_L 12.3 domain is a stable monomer under physiological conditions even at concentrations > 20 μM. Using peptide SPOT arrays, we identified the minimal binding epitope to be EKLMKAFESLKSFQ, comprising the N-terminal residues 5-18 of Htt and including the first residue of the poly-Gln stretch. X-ray structural analysis of αHtt-V_L both as apo protein and in the presence of the epitope peptide revealed several interesting insights: first, the role of mutations acquired during the combinatorial selection process of the αHtt-V_L 12.3 domain—initially starting from a single-chain Fv fragment—that are responsible for its stability as an individually soluble Ig domain, also lacking the disulfide bridge, and second, a previously unknown mode of antigen recognition, revealing a novel paratope. The Htt epitope peptide adopts a purely α-helical structure in the complex with αHtt-V_L and is bound at the base of the complementarity-determining regions (CDRs) at the concave β-sheet that normally gives rise to the interface between the V_L domain and its paired V_H (variable domain Ig heavy chain) domain, while only few interactions with CDR-L1 and CDR-L3 are formed. Notably, this noncanonical mode of antigen binding may occur more widely in the area of in vitro selected antibody fragments, including other Ig-like scaffolds, possibly even if a V_H domain is present.     

Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation

Rheumatoid factors (RF) are autoantibodies that recognize epitopes in the Fc region of immunoglobulin (Ig) G and that correlate with the clinical severity of rheumatoid arthritis (RA). Here we report the X-ray crystallographic structure, at 3 A resolution, of a complex between the Fc region of human IgG1 and the Fab fragment of a monoclonal IgM RF (RF61), derived from an RA patient and with a relatively high affinity for IgG Fc. In the complex, two Fab fragments bind to each Fc at epitopes close to the C terminus, and each epitope comprises residues from both Cgamma3 domains. A central role in the unusually hydrophilic epitope is played by the side-chain of Arg355, accounting for the subclass specificity of RF61, which recognizes IgG1,-2, and -3 in preference to IgG4, in which the corresponding residue is Gln355. Compared with a previously determined complex of a lower affinity RF (RF-AN) bound to IgG4 Fc, in which only residues at the very edge of the antibody combining site were involved in binding, the epitope bound by RF61 is centered in classic fashion on the axis of the V(H):V(L) beta-barrel. The complementarity determining region-H3 loop plays a key role, forming a pocket in which Arg355 is bound by two salt-bridges. The antibody contacts also involve two somatically mutated V(H) residues, reinforcing the suggestion of a process of antigen-driven maturation and selection for IgG Fc during the generation of this RF autoantibody.     

Evolution of an interloop disulfide bond in high-affinity antibody mimics based on fibronectin type III domain and selected by yeast surface display: molecular convergence with single-domain camelid and shark antibodies

The 10th human fibronectin type III domain ((10)Fn3) is one of several protein scaffolds used to design and select families of proteins that bind with high affinity and specificity to macromolecular targets. To date, the highest affinity (10)Fn3 variants have been selected by mRNA display of libraries generated by randomizing all three complementarity-determining region -like loops of the (10)Fn3 scaffold. The sub-nanomolar affinities of such antibody mimics have been attributed to the extremely large size of the library accessible by mRNA display (10(12) unique sequences). Here we describe the selection and affinity maturation of (10)Fn3-based antibody mimics with dissociation constants as low as 350 pM selected from significantly smaller libraries (10(7)-10(9) different sequences), which were constructed by randomizing only 14 (10)Fn3 residues. The finding that two adjacent loops in human (10)Fn3 provide a large enough variable surface area to select high-affinity antibody mimics is significant because a smaller deviation from wild-type (10)Fn3 sequence is associated with a higher stability of selected antibody mimics. Our results also demonstrate the utility of an affinity-maturation strategy that led to a 340-fold improvement in affinity by maximizing sampling of sequence space close to the original selected antibody mimic. A striking feature of the highest affinity antibody mimics selected against lysozyme is a pair of cysteines on adjacent loops, in positions 28 and 77, which are critical for the affinity of the (10)Fn3 variant for its target and are close enough to form a disulfide bond. The selection of this cysteine pair is structurally analogous to the natural evolution of disulfide bonds found in new antigen receptors of cartilaginous fish and in camelid heavy-chain variable domains. We propose that future library designs incorporating such an interloop disulfide will further facilitate the selection of high-affinity, highly stable antibody mimics from libraries accessible to phage and yeast surface display methods.     

X-ray structures of fragments from binding and nonbinding versions of a humanized anti-CD18 antibody: Structural indications of the key role of VH residues 59 to 65

X-ray crystal structures of fragments from two different humanized antiCD18 antibodies are reported. The Fv fragment of the nonbinding version has been refined in space group C2 with a=64.2 Å, b=61.3 Å, c=51.8 Å, and β=99° to an R-value of 18.0% at 1.9 Å, and the Fab fragment of the tight-binding version has been refined in space group P3 with a=101. Å and c=45.5 Å to an R-value of 17.8% at 3.0 Å resolution. The very large difference in their binding affinity (>1000-fold) is attributed to large and local structural differences in the C-terminal part of CDR-H2, and from this we conclude there is direct contact between this region and antigen when they combine. X-ray structures of antibody–antigen complexes available in the literature have yet to show this part of CDR-H2 in contact with antigen, despite its hypervariable sequence. Implications of this result for antibody humanization are discussed. © 1994 John Wiley & Sons, Inc.     

A new clustering of antibody CDR loop conformations

Previous analyses of the complementarity-determining regions (CDRs) of antibodies have focused on a small number of “canonical” conformations for each loop. This is primarily the result of the work of Chothia and coworkers, most recently in 1997. Because of the widespread utility of antibodies, we have revisited the clustering of conformations of the six CDR loops with the much larger amount of structural information currently available. In this work, we were careful to use a high-quality data set by eliminating low-resolution structures and CDRs with high B-factors or high conformational energies. We used a distance function based on directional statistics and an effective clustering algorithm with affinity propagation. With this data set of over 300 nonredundant antibody structures, we were able to cover 28 CDR-length combinations (e.g., L1 length 11, or “L1-11” in our CDR-length nomenclature) for L1, L2, L3, H1, and H2. The Chothia analysis covered only 20 CDR-lengths. Only four of these had more than one conformational cluster, of which two could easily be distinguished by gene source (mouse/human; κ/λ) and one could easily be distinguished purely by the presence and the positions of Pro residues (L3-9). Thus, using the Chothia analysis does not require the complicated set of “structure-determining residues” that is often assumed. Of our 28 CDR-lengths, 15 have multiple conformational clusters, including 10 for which the Chothia analysis had only one canonical class. We have a total of 72 clusters for non-H3 CDRs; approximately 85% of the non-H3 sequences can be assigned to a conformational cluster based on gene source and/or sequence. We found that earlier predictions of “bulged” versus “nonbulged” conformations based on the presence or the absence of anchor residues Arg/Lys94 and Asp101 of H3 have not held up, since all four combinations lead to a majority of conformations that are bulged. Thus, the earlier analyses have been significantly enhanced by the increased data. We believe that the new classification will lead to improved methods for antibody structure prediction and design.     

Cross-reactivity studies of an anti-Plasmodium vivax apical membrane antigen 1 monoclonal antibody: binding and structural characterisation

Apical membrane antigen 1 (AMA1) has an important, but as yet uncharacterised, role in host cell invasion by the malaria parasite, Plasmodium. The protein, which is quite conserved between Plasmodium species, comprises an ectoplasmic region, a single transmembrane segment and a small cytoplasmic domain. The ectoplasmic region, which can induce protective immunity in animal models of human malaria, is a leading vaccine candidate that has entered clinical trials. The monoclonal antibody F8.12.19, raised against the recombinant ectoplasmic region of AMA1 from Plasmodium vivax, cross-reacts with homologues from Plasmodium knowlesi, Plasmodium cynomolgi, Plasmodium berghei and Plasmodium falciparum, as shown by immunofluorescence assays on mature schizonts. The binding of F8.12.19 to recombinant AMA1 from both P. vivax and P. falciparum was measured by surface plasmon resonance, revealing an apparent affinity constant that is about 100-fold weaker for the cross-reacting antigen when compared to the cognate antigen. Crystal structure analysis of Fab F8.12.19 complexed to AMA1 from P. vivax and P. falciparum shows that the monoclonal antibody recognises a discontinuous epitope located on domain III of the ectoplasmic region, the major component being a loop containing a cystine knot. The structures provide a basis for understanding the cross-reactivity. Antibody contacts are made mainly to main-chain and invariant side-chain atoms of AMA1; contact antigen residues that differ in sequence are located at the periphery of the antigen-binding site and can be accommodated at the interface between the two components of the complex. The implications for AMA1 vaccine development are discussed.     

Prediction of efficient virological response to pegylated interferon/ribavirin combination therapy by NS5A sequences of hepatitis C virus and anti-NS5A antibodies in pre-treatment sera

A considerable number of patients infected with Hepatitis C virus subtype 1b (HCV-1b) do not respond to pegylated interferon/ribavirin combination therapy. In this study we explored a useful factor(s) to predict treatment outcome. A total of 47 HCV-1b-infected patients were treated with pegylated interferon/ ribavirin for 48 weeks. Sera of the patients were examined for the entire NS5A sequence of the HCV genome, HCV RNA titers and anti-NS5A antibodies. According to their responses, the patients were divided into two groups, early viral responders who cleared the virus by week 16 (EVR[16w]) and those who did not (Non-EVR[16w]). The mean number of mutations in the V3 region (aa 2356 to 2379) or that in the V3 region plus its N-terminally flanking region, which we refer to as interferon/ribavirin resistancedetermining region (IRRDR; aa 2334 to 2379), of NS5A obtained from the pretreatment sera was signifi-cantly larger for EVR(16w) compared with Non-EVR(16w). Moreover, HCV-1b isolates with > or =5 mutations in V3 or those with > or =6 mutations in IRRDR were almost exclusively found in EVR(16w). Also, the presence of detectable levels of anti-NS5A antibodies in the pretreatment sera was closely associated with EVR(16w). In conclusion, a high degree of sequence variation in V3 (> or =5) or IRRDR (> or =6) and the presence of detectable levels of anti-NS5A antibodies in the pretreatment sera would be useful factors to predict EVR(16w). On the other hand, a less diverse sequence in V3 (< or =4) or IRRDR (< or =5) together with the absence of detectable anti-NS5A antibodies could be a predictive factor for Non-EVR(16w).     

Molecular modeling of the complex between Torpedo acetylcholine receptor and anti-MIR Fab198

Myasthenia gravis is a neuromuscular disorder caused by an antibody-mediated autoimmune response to the muscle-type nicotinic acetylcholine receptor (AChR). The majority of monoclonal antibodies (mAbs) produced in rats immunized with intact AChR compete with each other for binding to an area of the alpha-subunit called the main immunogenic region (MIR). The availability of a complex between the AChR and Fab198 (Fab fragment of the anti-MIR mAb198) would help understand how the antigen and antibody interact and in designing improved antibody fragments that protect against the destructive activity of myasthenic antibodies. In the present study, we modeled the Torpedo AChR/Fab198 complex, based primarily on the recent 4A resolution structure of the Torpedo AChR. In order to computationally dock the two structures, we used the ZDOCK software. The total accessible surface area change of the complex compared to those of experimentally determined antigen-antibody complexes indicates an intermediate size contact surface. CDRs H3 and L3 seem to contribute most to the binding, while L2 seems to contribute least. These data suggest mutagenesis experiments aimed at validating the model and improving the binding affinity of Fab198 for the AChR.