Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires

Studies of antibodies of known three-dimensional structure have revealed that insertion and deletion of amino acids at the hypervariable loops change the canonical structures, thus generating differences in the antigen-binding site topography. Such differences determine the size of the antigen with which the antibody interacts. Here, 59 unique antibodies determined at a resolution of 3.0 A or below, including 19 in complex with proteins, 18 with peptides and 22 with haptens, were analyzed to identify and characterize differences in the residues that are directly involved in the interaction with antigen, so-called specificity-determining residues (SDRs). It was found that antibodies use a similar number of SDRs to recognize proteins and peptides but contact haptens with five SDRs less. By using a score of SDR usage, differences in the location of the SDRs, depending on the type of antigen recognized, were then identified with precision. An analysis of the surface generated by the SDRs usage indicates that the differences found correlate well with the size of the antigen. Anti-protein antibodies have the largest SDR surface, with SDRs of high usage located in the edge of the surface. The SDR surface of anti-hapten antibodies is the smallest, with hot spots of contacts in the interior of the binding surface and buried in the V(L):V(H) interface. The SDR surface of anti-peptide antibodies has a size in between anti-protein and anti-hapten antibodies, with the SDRs of high usage located in the interior of the antigen-binding site but do not buried as in anti-hapten antibodies. These findings led to a fine-tuning of the model correlating differences in the antigen-binding site topography with its preference to recognize antigens of different size. Therefore, it is discussed how this knowledge should help to design antibody repertoires biased toward the recognition of antigens of predefined size.     

Analysis of antibodies of known structure suggests a lack of correspondence between the residues in contact with the antigen and those modified by somatic hypermutation

Forty unique murine antibody-antigen complexes determined at 2.5 A or less resolution are analyzed to determine whether the residues in direct contact with the antigen are modified by somatic hypermutation. This was done by taking advantage of the recent characterization of the pool of Vkappa germline genes of the mouse. The average number of residues in contact with the antigen in the V(L) gene, which contains the CDRL-1, CDRL-2, and all but one residue of CDRL-3, was six. The average number of somatic mutations was similar (around five). However, as many as 53% of the antibodies did not show somatic replacements of residues in contact with the antigen. Another 28% had only one. Overall, the frequency of antibodies with increasing number of somatic replacements in residues in contact with the antigen decreased exponentially. A possible explanation of this finding is that mutations in the contacting residues have an adverse effect on the antigen-antibody interaction. This implies that most of the observed mutations are those remaining after negative (purifying) selection. Therefore, efficient strategies of site-directed mutagenesis to improve the affinity of antibodies should be focused on residues other than those directly interacting with the antigen.     

Mutational analysis of the affinity maturation of antibody 48G7

The affinity maturation of antibody 48G7 from its germline predecessor 48G7g has been studied at a molecular level through a combination of structural and biochemical means. Each of the nine somatic mutations accumulated during affinity maturation has been assessed for gain or loss of function in both the germline and affinity-matured antibodies. Individual somatic mutations were found to be either positive or neutral in their effects on affinity for hapten JWJ1, with a marked context-dependence for some sites of mutation. In a number of cases significant cooperativity was found between pairs of somatically mutated residues. Interpretation of the structural changes introduced by many of the point mutations has been possible due to the availability of high-resolution crystal structures of 48G7g and 48G7, and mechanisms by which these structural changes may result in enhanced affinity for hapten have been identified. Precise dissection of structure-function relationships in this system provides additional insights into the role of cooperativity in the evolution of antibody affinity. Comparison of 48G7 with previously characterized systems provides a varied view of the structure-function mechanisms by which the humoral immune system produces large increases in affinity.     

Human Germline Antibody Gene Segments Encode Polyspecific Antibodies

Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three V_H gene segments. Panels of somatically mutated antibodies encoded by a common V_H gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the V_H gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.     

Defining the Recognition Elements of Lewis Y-Reactive Antibodies

Antibody response to carbohydrate antigens is often independent of T cells and the process of affinity/specificity improvement is considered strictly dependent on the germinal centers. Antibodies induced during a T cell-independent type 2 (TI-2) response are less variable and less functionally versatile than those induced with T cell help. The antigen specificity consequences of accumulation of somatic mutations in antibodies during TI-2 responses of Marginal Zone (MZ) B cells is a fact that still needs explanation. Germline genes that define carbohydrate-reactive antibodies are known to sculpt antibody-combining sites containing innate, key side-chain contacts that define the antigen recognition step. However, substitutions associated with MZ B cell derived antibodies might affect the mobility and polyspecificity of the antibody. To examine this hypothesis, we analyzed antibodies reactive with the neolactoseries antigen Lewis Y (LeY) to define the residue subset required for the reactive repertoire for the LeY antigen. Our molecular simulation studies of crystallographically determined and modeled antibody-LeY complexes suggests that the heavy-chain germline gene VH7183.a13.20 and the light-chain Vκ cr1 germline gene are sufficient to account for the recognition of the trisaccharide-H determinant Types 1–4, while the specificity for LeY is driven by the CDR3 backbone conformation of the heavy chain and not the side chain interactions. These results confirm that these monoclonals use germline-encoded amino acids to recognize simple carbohydrate determinants like trisaccharide-H but relies on somatic mutations in the periphery of the combining site to modify affinity for LeY through electrostatic interactions that leads to their optimized binding. These observations bring further attention to the role of mutations in T-cell independent antibodies to distinguish self from non-self carbohydrate antigens.     

Unraveling the effect of changes in conformation and compactness at the antibody VL‐VH interface upon antigen binding

We have analyzed conformational changes that occur at the interface between the light (V_L) and heavy (V_H) chains in antibody variable fragments upon binding to antigens. We wrote and applied the Tiny Probe program that computes the buried atomic contact surface area of three‐dimensional structures to evaluate changes in compactness of the V_L–V_H interface between bound and unbound antibodies. We found three categories of these changes, which correlated with the size of the antigen. Upon binding, medium‐sized nonprotein antigens cause an opening of the V_L–V_H interface (less compact), small antigens or haptens cause a closure of the interface (more compact), whereas large protein antigens have little effect on the compactness of the V_L–V_H interface. The largest changes in the atomic buried contact surface area at the V_L–V_H interface occur in residue pairs providing two ‘shock absorbers’ between the edge β‐strands of the V_L and V_H β‐sheets forming the antibody binding site. Importantly, the correlation between the size of antigens and conformational changes indicates that the V_L–V_H interface in antibodies plays a significant role in the antigen binding process. Furthermore, as the energy involved in such a motion is significant (up to 3 kcal/mol), these results provide a general mechanism for how residues distant from the combining site can significantly alter the affinity of an antibody for its antigen. Thus, mutations introduced at the V_L–V_H interface can be used to change antibody binding affinity with antigens. Due to the tightly packed V_L–V_H interface, the introduction of random mutations is not advisable. Rather our analysis suggests that concerted mutations of residues preceding CDRL2 and following CDRH3 or residues preceding CDRH2 and at the end of CDRL3 are most likely to alter or improve antigen binding affinity. Copyright © 1999 John Wiley & Sons, Ltd.     

Humanization of a murine monoclonal antibody by simultaneous optimization of framework and CDR residues.

Optimal protein function often depends on co-operative interactions between amino acid residues distant in the protein primary sequence yet spatially near one another following protein folding. For example, antibody affinity is influenced by interactions of framework residues with complementarity-determining region (CDR) residues. However, despite the abundance of antibody structural information and computational tools the humanization of rodent antibodies for clinical use often results in a significant loss of affinity. To date, antibody engineering efforts have focused either on optimizing CDR residues involved in antigen binding or on optimizing antibody framework residues that serve critical roles in preserving the conformation of CDRs. In the present study a new approach which permits the rapid identification of co-operatively interacting framework and CDR residues was used to simultaneously humanize and optimize a murine antibody directed against CD40. Specifically, a combinatorial library that examined eight potentially important framework positions concomitantly with focused CDR libraries consisting of variants containing random single amino acid mutations in the third CDR of the heavy and light chains was expressed. Multiple anti-CD40 Fab variants containing as few as one murine framework residue and displaying up to approximately 500-fold higher affinity than the initial chimeric Fab were identified. The higher affinity humanized variants demonstrated a co-operative interaction between light chain framework residue Y49 and heavy chain CDR3 residue R/K101 (coupling energy, DeltaGI=0.9 kcal/mol). Screening of combinatorial framework-CDR libraries permits identification of monoclonal antibodies (mAb) with structures optimized for function, including instances in which the antigen induces conformational changes in the mAb. Moreover, the enhanced humanized variants contain fewer murine framework residues and could not be identified by sequential in vitro humanization and affinity muturation strategies. This approach to identifying co-operatively interacting residues is not restricted to antibody-antigen interactions and consequently, may be used broadly to gain insight into protein structure-function relationships, including proteins that serve as catalysts.     

Somatic mutation, affinity maturation and the antibody repertoire: a computer model

Somatic mutation has been implicated as a significant and possibly primary factor in the maturation of antibody affinity in the humoral immune response. B cells stimulated by antigen experience a hyper-mutation in the gene segments that code for the antigen-binding site of the antibody, creating antibody specificities that did not exist at the time of immunization. Although most of the mutations are likely to be disadvantageous, new specificities with a higher affinity for the antigen are sometimes created. These higher-affinity cells are preferentially selected for proliferation and eventual antibody secretion, resulting in a progressively higher average affinity over time. In this paper we present the results of an investigation of somatic mutation through the use of a computer model. At the basis of the model is a large repertoire of discrete antibodies and antigens, having three-dimensional structures, that exhibit properties similar to those of the real populations. The key factor is that the binding strength between any antibody/antigen pair can be calculated as a function of the complementarity of the (a) size, (b) shape and (c) functional groups that comprise the two structures. The created repertoires are imbedded in a dynamical system model of the immune response to directly evaluate the affect of somatic mutation on affinity maturation. We also present an expanded hypothesis of clonal selection and development to explain how the mutational restrictions imposed by the genetic code and the structure of the antibody repertoire, along with antigen concentration, affinity, and probabilistic factors may interact and contribute to the expansion of specific clones as the response develops over time.     

Structure of an IgNAR-AMA1 complex: targeting a conserved hydrophobic cleft broadens malarial strain recognition

Apical membrane antigen 1 (AMA1) is essential for invasion of erythrocytes and hepatocytes by Plasmodium parasites and is a leading malarial vaccine candidate. Although conventional antibodies to AMA1 can prevent such invasion, extensive polymorphisms within surface-exposed loops may limit the ability of these AMA1-induced antibodies to protect against all parasite genotypes. Using an AMA1-specific IgNAR single-variable-domain antibody, we performed targeted mutagenesis and selection against AMA1 from three P. falciparum strains. We present cocrystal structures of two antibody-AMA1 complexes which reveal extended IgNAR CDR3 loops penetrating deep into a hydrophobic cleft on the antigen surface and contacting residues conserved across parasite species. Comparison of a series of affinity-enhancing mutations allowed dissection of their relative contributions to binding kinetics and correlation with inhibition of erythrocyte invasion. These findings provide insights into mechanisms of single-domain antibody binding, and may enable design of reagents targeting otherwise cryptic epitopes in pathogen antigens.     

Structural insights into parallel strategies for germline antibody recognition of lipopolysaccharide from Chlamydia

The structure of the antigen-binding fragment from the monoclonal antibody S64-4 in complex with a pentasaccharide bisphosphate fragment from chlamydial lipopolysaccharide has been determined by x-ray diffraction to 2.6 ? resolution. Like the well-characterized antibody S25-2, S64-4 displays a pocket formed by the residues of germline sequence corresponding to the heavy and light chain V gene segments that binds the terminal Kdo residue of the antigen; however, although S64-4 shares the same heavy chain V gene segment as S25-2, it has a different light chain V gene segment. The new light chain V gene segment codes for a combining site that displays greater affinity, different specificity, and allows a novel antigen conformation that brings a greater number of antigen residues into the combining site than possible in S25-2. Further, while antibodies in the S25-2 family use complementarity determining region (CDR) H3 to discriminate among antigens, S64-4 achieves its specificity via the new light chain V gene segment and resulting change in antigen conformation. These structures reveal an intriguing parallel strategy where two different combinations of germline-coded V gene segments can act as starting points for the generation of germline antibodies against chlamydial antigens and show how anti-carbohydrate antibodies can exploit the conformational flexibility of this class of antigens to achieve high affinity and specificity independently of CDR H3.     

Enhancement and destruction of antibody function by somatic mutation: unequal occurrence is controlled by V gene combinatorial associations.

We examined the positive and negative effects of somatic mutation on antibody function using saturation mutagenesis in vitro to mimic the potential of the in vivo process to diversify antibodies. Identical mutations were introduced into the second complementarity determining region of two anti-phosphocholine antibodies, T15 and D16, which share the same germline VH gene sequence. T15 predominates in primary responses and does not undergo affinity maturation. D16 is representative of antibodies that co-dominate in memory responses and do undergo affinity maturation. We previously reported that > 50% of T15 mutants had decreased antigen binding capacity. To test if this high frequency of binding loss was unique to T15 or a consequence of random point mutations applicable to other combining sites, we analyzed the same mutations in D16. We show that D16 suffers a similar loss of function, indicating an equally high potential for B-cell wastage. However, only D16 displayed the capacity for somatic mutation to improve antigen binding, which should enhance its persistence in memory responses. Mutation of residues contacting the haptenic group, as determined by molecular modeling, did not improve binding. Instead, productive mutations occurred in residues that either contacted carrier protein or were distant from the antigen binding site, possibly increasing binding site flexibility through long-range effects. Targeting such residues for mutation should aid in the rational design of improved antibodies.     

Localization of a 215-kDa tyrosine-phosphorylated protein that cross-reacts with tensin antibodies.

Tyrosine phosphorylation of cytoskeletal proteins at adhesive junctions has been speculated to play a role in the regulation of cell signaling at these sites. Previously, monoclonal antibodies were generated against phosphotyrosine-containing proteins from Rous sarcoma virus-transformed chick embryo fibroblasts, resulting in two antibodies which recognized antigens of 76 and 215 kDa that localized to focal contacts. We have now localized the 215-kDa antigen to a number of adhesive junctions in vivo, including the zonula adherens, intercalated discs, and myotendinous and neuromuscular junctions. In sections of skeletal muscle and in isolated myofibrils, the 215-kDa protein was localized to the I-band. By immunoprecipitation and immunoblot analysis, we determined that the 215-kDa antigen cross-reacts with a polyclonal anti-tensin antibody.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to Yersinia lipopolisacharydes and Yop proteins by ELISA

The antibodies against the somatic antigens of Y. enterocolitica O3, O8, O9, O5,27,Y. pseudotuberculosis I, and released proteins Yop were detected using the ELISA in 1634 serum samples and 84 synovial fluids collected from 1290 persons suspected for yersiniosis, as well as 200 serum samples from healthy individuals (blood donors). The presence of antibody in diagnostically significant titres for somatic antigens of Yersinia were detected by ELISA in 20.5% and 50.6%, antibodies for released proteins Yop in 11.5% and 28.4% respectively of blood donors and patients suspected for yersiniosis. The antibody against the O3 antigen of Y. enterocolitica was the most frequently detected antibody while the most infrequent was the antibody for the antigen from the 08 serologic group. The results of the study showed that the humoral response picture to Yersinia antigens in the course of yersiniosis in humans is dependent on the age and sex of the patient, duration of the infection, and clinical manifestations. Most frequently the elevated antibody levels were detected among patients with erythema nodosum and patients with gastrointestinal symptoms. The frequency of occurrence of antibodies for most antigens of Yersinia, together with age increased reaching its peak, on the average, among individuals aged 21 - 40 years. Analysis of individual cases showed that by the end of the first week of infection, elevated levels of antibodies for somatic antigens of Yersinia are evident. On the other hand, antibodies for released proteins Yop as a matter of rule appear in the second week from the onset of clinical symptoms. Within this early phase of infection immunoglobulins of the A and M classes dominate reaching their highest level in the second to third week of the infection. In majority of the individuals studied antibodies of the IgG class reached their highest level much later in relation to those of the IgA and IgM classes. Significant differences were found in IgA antibody detection among individuals with clinical manifestations of stomachaches and arthritis. Nevertheless, among individuals with clinical symptoms of stomachaches, these immunoglobulins as a matter of principle disappear with a period of 2-3 months from the onset of clinical symptoms. In individuals with arthritis however the aforementioned immunoglobulins maintained at considerable levels even after a year. In joint-fluid samples obtained from patients with arthritis antibodies for Yersinia antigens were detected in similar levels just as obtained simultaneously serum from those individuals.     

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.     

New surface contacts formed upon reductive lysine methylation: Improving the probability of protein crystallization

Surface lysine methylation (SLM) is a technique for improving the rate of success of protein crystallization by chemically methylating lysine residues. The exact mechanism by which SLM enhances crystallization is still not clear. To study these mechanisms, and to analyze the conditions where SLM will provide the optimal benefits for rescuing failed crystallization experiments, we compared 40 protein structures containing N,N-dimethyl-lysine (dmLys) to a nonredundant set of 18,972 nonmethylated structures from the PDB. By measuring the relative frequency of intermolecular contacts (where contacts are defined as interactions between the residues in proximity with a distance of 3.5 Å or less) of basic residues in the methylated versus nonmethylated sets, dmLys-Glu contacts are seen more frequently than Lys-Glu contacts. Based on observation of the 10 proteins with both native and methylated structures, we propose that the increased rate of contact for dmLys-Glu is due to both a slight increase in the number of amine-carboxyl H-bonds and to the formation of methyl C–H···O interactions. By comparing the relative contact frequencies of dmLys with other residues, the mechanism by which methylation of lysines improves the formation of crystal contacts appears to be similar to that of Lys to Arg mutation. Moreover, analysis of methylated structures with the surface entropy reduction (SER) prediction server suggests that in many cases SLM of predicted SER sites may contribute to improved crystallization. Thus, tools that analyze protein sequences and mark residues for SER mutation may identify proteins with good candidate sites for SLM.     

Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen.

The Fab portion of a humanized antibody (Fab-12; IgG form known as rhuMAb VEGF) to vascular endothelial growth factor (VEGF) has been affinity-matured through complementarity-determining region (CDR) mutation, followed by affinity selection using monovalent phage display. After stringent binding selections at 37 degrees C, with dissociation (off-rate) selection periods of several days, high affinity variants were isolated from CDR-H1, H2, and H3 libraries. Mutations were combined to obtain cumulatively tighter-binding variants. The final variant identified here, Y0317, contained six mutations from the parental antibody. In vitro cell-based assays show that four mutations yielded an improvement of about 100-fold in potency for inhibition of VEGF-dependent cell proliferation by this variant, consistent with the equilibrium binding constant determined from kinetics experiments at 37 degrees C. Using X-ray crystallography, we determined a high-resolution structure of the complex between VEGF and the affinity-matured Fab fragment. The overall features of the binding interface seen previously with wild-type are preserved, and many contact residues are maintained in precise alignment in the superimposed structures. However, locally, we see evidence for improved contacts between antibody and antigen, and two mutations result in increased van der Waals contact and improved hydrogen bonding. Site-directed mutants confirm that the most favorable improvements as judged by examination of the complex structure, in fact, have the greatest impact on free energy of binding. In general, the final antibody has improved affinity for several VEGF variants as compared with the parental antibody; however, some contact residues on VEGF differ in their contribution to the energetics of Fab binding. The results show that small changes even in a large protein-protein binding interface can have significant effects on the energetics of interaction.     

Three-dimensional structure determination of an anti-2-phenyloxazolone antibody: the role of somatic mutation and heavy/light chain pairing in the maturation of an immune response.

The three-dimensional structure of the Fab fragment of an anti-2-phenyloxazolone monoclonal antibody (NQ10/12.5) in its native and complexed forms has been determined at 2.8 and 3.0 A resolution, respectively. Identification of hapten-contacting residues has allowed us to evaluate the contribution of individual somatic point mutations to maturation of the immune response. In particular, amino acid residues 34 and 36 of the light chain, which are frequently mutated in antibodies with increased affinity for 2-phenyloxazolone, are shown to interact directly with the hapten. We propose that the strict maintenance of certain amino acid sequences at the potentially highly variable VL-JL and VH-D-JH junctions observed among anti-2-phenyloxazolone antibodies is due largely to structural constraints related to antigen recognition. Finally, the three-dimensional model of NQ10/12.5, which uses the typical light chain of primary response anti-2-phenyloxazolone antibodies but a different heavy chain, allows an understanding of how, by preserving key contact residues, a given heavy chain may be replaced by another, apparently unrelated one, without loss of hapten binding activity and why the V kappa Ox1 germline gene is so frequently selected amongst the other known members of this family.     

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies.     

Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering

By combining the knowledge gained from an analysis of the biophysical properties of natural antibody variable domains, the effects of mutations obtained in directed evolution experiments, and the detailed structural comparison of antibodies, it has now become possible to engineer antibodies for higher thermodynamic stability and more efficient folding. This is particularly important when antibodies are to be used under conditions where the disulfide bonds cannot form, i.e., in intracellular applications (as "intrabodies"). We describe in detail two methods for the knowledge-based improvement of antibody stability and folding efficiency. While CDR grafting from a non-human to the most closely related human antibody framework is an established technique to reduce the immunogenicity of a therapeutic antibody, CDR grafting for stabilization implies the use of a more distantly related acceptor framework with superior biophysical characteristics. The use of such dissimilar frameworks requires particular attention to antigen contact residues outside the classical CDR definition and to residues capable of indirectly affecting the conformation of the antigen binding site. As a second alternative, the stability of a suboptimal framework can be improved by the introduction of point mutations designed to optimize key residue interactions. We describe the analysis methods used to identify such point mutations, which can be introduced all at once, while maintaining the framework features necessary for antigen binding. These rational approaches render the continued "rediscovery" of certain mutations by directed evolution unnecessary, but they can also be used in conjunction with such methods to discover even better molecules.     

Biochemical and structural characterization of lysophosphatidic Acid binding by a humanized monoclonal antibody

Lysophosphatidic acid (LPA) is a common product of glycerophospholipid metabolism and an important mediator of signal transduction. Aberrantly high LPA concentrations accompany multiple disease states. One potential approach for treatment of these diseases, therefore, is the therapeutic application of antibodies that recognize and bind LPA as their antigen. We have determined the X-ray crystal structure of an anti-LPA antibody (LT3015) Fab fragment in its antigen-free form to 2.15 Å resolution and in complex with two LPA isotypes (14:0 and 18:2) to resolutions of 1.98 and 2.51 Å, respectively. The variable CDR (complementarity-determining region) loops at the antigen binding site adopt nearly identical conformations in the free and antigen-bound crystal structures. The crystallographic models reveal that the LT3015 antibody employs both heavy- and light-chain CDR loops to create a network of eight hydrogen bonds with the glycerophosphate head group of its LPA antigen. The head group is almost completely excluded from contact with solvent, while the hydrocarbon tail is partially solvent-exposed. In general, mutation of amino acid residues at the antigen binding site disrupts LPA binding. However, the introduction of particular mutations chosen strategically on the basis of the structures can positively influence LPA binding affinity. Finally, these structures elucidate the exquisite specificity demonstrated by an anti-lipid antibody for binding a structurally simple and seemingly unconstrained target molecule.