Comprehensive analysis of the factors contributing to the stability and solubility of autonomous human VH domains

We report a comprehensive analysis of sequence features that allow for the production of autonomous human heavy chain variable (V(H)) domains that are stable and soluble in the absence of a light chain partner. Using combinatorial phage-displayed libraries and conventional biophysical methods, we analyzed the entire former light chain interface and the third complementarity determining region (CDR3). Unlike the monomeric variable domains of camelid heavy chain antibodies (V(H)H domains), in which autonomous behavior depends on interactions between the hydrophobic former light chain interface and CDR3, we find that the stability of many in vitro evolved V(H) domains is essentially independent of the CDR3 sequence and instead derives from mutations that increase the hydrophilicity of the former light chain interface by replacing exposed hydrophobic residues with structurally compatible hydrophilic substitutions. The engineered domains can be expressed recombinantly at high yield, are predominantly monomeric at high concentrations, unfold reversibly, and are even more thermostable than typical camelid V(H)H domains. Many of the stabilizing mutations are rare in natural V(H) and V(H)H domains and thus could not be predicted by studying natural sequences and structures. The results demonstrate that autonomous V(H) domains with structural properties beyond the scope of natural frameworks can be derived by using non-natural mutations, which differ from those found in camelid V(H)H domains. These findings should enable the development of libraries of synthetic V(H) domains with CDR3 diversities unconstrained by structural demands.     

Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions

A structure-based approach was used to design libraries of synthetic heavy chain complementarity determining regions (CDRs). The CDR libraries were displayed as either monovalent or bivalent single-chain variable fragments (scFvs) with a single heavy chain variable domain scaffold and a fixed light chain variable domain. Using the structure of a parent antibody as a guide, we restricted library diversity to CDR positions with significant exposure to solvent. We introduced diversity with tailored degenerate codons that ideally only encoded for amino acids commonly observed in natural antibody CDRs. With these design principles, we reasoned that we would produce libraries of diverse solvent-exposed surfaces displayed on stable scaffolds with minimal structural perturbations. The libraries were sorted against a panel of proteins and yielded multiple unique binding clones against all six antigens tested. The bivalent library yielded numerous unique sequences, while the monovalent library yielded fewer unique clones. Selected scFvs were converted to the Fab format, and the purified Fab proteins retained high affinity for antigen. The results support the view that synthetic heavy chain diversity alone may be sufficient for the generation of high-affinity antibodies from phage-displayed libraries; thus, it may be possible to dispense with the light chain altogether, as is the case in natural camelid immunoglobulins.     

A structure-based database of antibody variable domain diversity

The diversity of natural antibodies is limited by the genetic mechanisms that engender diversity and the functional requirements of antigen binding. Using an in vitro-evolved autonomous heavy chain variable domain (V(H)H-RIG), we have investigated the limits of structurally-tolerated diversity in the three complementarity-determining regions and a fourth loop within the third framework region. We determined the X-ray crystal structure of the V(H)H-RIG domain at 1.9A resolution and used it to guide the design of phage-displayed libraries encompassing the four loops. The libraries were subjected to selections for structural stability, and a database of structurally-tolerated sequences was compiled from the sequences of approximately 1000 unique clones. The results reveal that all four loops accommodate significantly greater diversity than is observed in nature. Thus, it appears that most sequence biases in the natural immune repertoire arise from factors other than structural constraints and, consequently, it should be possible to enhance the functions of antibodies significantly through in vitro evolution.     

Crystal structure of HEL4, a soluble, refoldable human V(H) single domain with a germ-line scaffold

The antigen binding site of antibodies usually comprises associated heavy (V(H)) and light (V(L)) chain variable domains, but in camels and llamas, the binding site frequently comprises the heavy chain variable domain only (referred to as V(HH)). In contrast to reported human V(H) domains, V(HH) domains are well expressed from bacteria and yeast, are readily purified in soluble form and refold reversibly after heat-denaturation. These desirable properties have been attributed to highly conserved substitutions of the hydrophobic residues of V(H) domains, which normally interact with complementary V(L) domains. Here, we describe the discovery and characterisation of an isolated human V(H) domain (HEL4) with properties similar to those of V(HH) domains. HEL4 is highly soluble at concentrations of > or =3 mM, essentially monomeric and resistant to aggregation upon thermodenaturation at concentrations as high as 56 microM. However, in contrast to V(HH) domains, the hydrophobic framework residues of the V(H):V(L) interface are maintained and the only sequence changes from the corresponding human germ-line segment (V3-23/DP-47) are located in the loops comprising the complementarity determining regions (CDRs). The crystallographic structure of HEL4 reveals an unusual feature; the side-chain of a framework residue (Trp47) is flipped into a cavity formed by Gly35 of CDR1, thereby increasing the hydrophilicity of the V(H):V(L) interface. To evaluate the specific contribution of Gly35 to domain properties, Gly35 was introduced into a V(H) domain with poor solution properties. This greatly enhanced the recovery of the mutant from a gel filtration matrix, but had little effect on its ability to refold reversibly after heat denaturation. Our results confirm the importance of a hydrophilic V(H):V(L) interface for purification of isolated V(H) domains, and constitute a step towards the design of isolated human V(H) domains with practical properties for immunotherapy.     

High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries

We have previously established a minimalist approach to antibody engineering by using a phage-displayed framework to support complementarity determining region (CDR) diversity restricted to a binary code of tyrosine and serine. Here, we systematically augmented the original binary library with additional levels of diversity and examined the effects. The diversity of the simplest library, in which only heavy chain CDR positions were randomized by the binary code, was expanded in a stepwise manner by adding diversity to the light chain, by diversifying non-paratope residues that may influence CDR conformations, and by adding additional chemical diversity to CDR-H3. The additional diversity incrementally improved the affinities of antibodies raised against human vascular endoethelial growth factor and the structure of an antibody-antigen complex showed that tyrosine side-chains are sufficient to mediate most of the interactions with antigen, but a glycine residue in CDR-H3 was critical for providing a conformation suitable for high-affinity binding. Using new high-throughput procedures and the most complex library, we produced multiple high-affinity antibodies with dissociation constants in the single-digit nanomolar range against a wide variety of protein antigens. Thus, this fully synthetic, minimalist library has essentially recapitulated the capacity of the natural immune system to generate high-affinity antibodies. Libraries of this type should be highly useful for proteomic applications, as they minimize inherent complexities of natural antibodies that have hindered the establishment of high-throughput procedures. Furthermore, analysis of a large number of antibodies derived from these well-defined and simplistic libraries allowed us to uncover statistically significant trends in CDR sequences, which provide valuable insights into antibody library design and into factors governing protein-protein interactions.     

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.     

Analysis and modeling of the variable region of camelid single-domain antibodies

Camelids have a special type of Ab, known as heavy chain Abs, which are devoid of classical Ab light chains. Relative to classical Abs, camelid heavy chain Abs (cAbs) have comparable immunogenicity, Ag recognition diversity and binding affinities, higher stability and solubility, and better manufacturability, making them promising candidates for alternate therapeutic scaffolds. Rational engineering of cAbs to improve therapeutic function requires knowledge of the differences of sequence and structural features between cAbs and classical Abs. In this study, amino acid sequences of 27 cAb variable regions (V(H)H) were aligned with the respective regions of 54 classical Abs to detect amino acid differences, enabling automatic identification of cAb V(H)H CDRs. CDR analysis revealed that the H1 often (and sometimes the H2) adopts diverse conformations not classifiable by established canonical rules. Also, although the cAb H3 is much longer than classical H3 loops, it often contains common structural motifs and sometimes a disulfide bond to the H1. Leveraging these observations, we created a Monte Carlo-based cAb V(H)H structural modeling tool, where the CDR H1 and H2 loops exhibited a median root-mean-square deviation to natives of 3.1 and 1.5 ?, respectively. The protocol generated 8-12, 14-16, and 16-24 residue H3 loops with a median root-mean-square deviation to natives of 5.7, 4.5, and 6.8 ?, respectively. The large deviation of the predicted loops underscores the challenge in modeling such long loops. cAb V(H)H homology models can provide structural insights into interaction mechanisms to enable development of novel Abs for therapeutic and biotechnological use.     

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.     

Crystal structure of a 3B3 variant—A broadly neutralizing HIV‐1 scFv antibody

We present the crystal structure determination of an anti‐HIV‐1 gp120 single‐chain variable fragment antibody variant, 3B3, at 2.5 Å resolution. This 3B3 variant was derived from the b12 antibody, using phage display and site‐directed mutagenesis of the variable heavy chain (V_H) complementary‐determining regions (CDRs). 3B3 exhibits enhanced binding affinity and neutralization activity against several cross‐clade primary isolates of HIV‐1 by interaction with the recessed CD4‐binding site on the gp120 envelope protein. Comparison with the structures of the unbound and bound forms of b12, the 3B3 structure closely resembles these structures with minimal differences with two notable exceptions. First, there is a reorientation of the CDR‐H3 of the V_H domain where the primary sequences evolved from b12 to 3B3. The structural changes in CDR‐H3 of 3B3, in light of the b12‐gp120 complex structure, allow for positioning an additional Trp side chain in the binding interface with gp120. Finally, the second region of structural change involves two peptide bond flips in CDR‐L3 of the variable light (V_L) domain triggered by a point mutation in CDR‐H3 of Q100eY resulting in changes in the intramolecular hydrogen bonding patterning between the V_L and V_H domains. Thus, the enhanced binding affinities and neutralization capabilities of 3B3 relative to b12 probably result from higher hydrophobic driving potential by burying more aromatic residues at the 3B3‐gp120 interface and by indirect stabilization of intramolecular contacts of the core framework residues between the V_L and V_H domains possibly through more favorable entropic effect through the expulsion of water.     

Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach

In a systematic study of V gene families carried out with consensus V(H) and V(L) domains alone and in combinations in the scFv format, we found comparatively low expression yields and lower cooperativity in equilibrium unfolding in antibody fragments containing V(H) domains of human germline families 2, 4, and 6. From an analysis of the packing of the hydrophobic core, the completeness of charge clusters, the occurrence of unsatisfied hydrogen bonds, and residues with low beta-sheet propensities, positive Phi angles, and exposed hydrophobic side chains, we pinpointed residues potentially responsible for the unsatisfactory properties of these germline-encoded sequences. Several of those are in common between the domains of the even-numbered subgroups, but do not occur in the odd-numbered ones. In this study, we have systematically exchanged those residues alone and in combination in two different scFvs using the V(H)6 framework, and we describe their effect on equilibrium stability and folding yield. We improved the stability by 20.9 kJ/mol and the expression yield by a factor of 4 and can now use these data to rationally engineer antibodies derived from this and similar germline families for better biophysical properties. Furthermore, we provide an improved design for libraries exploiting the significant additional diversity provided by these frameworks. Both antibodies studied here completely retain their binding affinity, demonstrating that the CDR conformations were not affected.     

Primary structures and chain dominance of anti-DNA antibodies

Using several anti-DNA autoantibodies, we analyzed the relative involvement of heavy and light chains in their interactions with DNA. We previously obtained eight hybridomas producing monoclonal anti-DNA autoantibodies by fusing spleen cells from an MRL-lpr/lpr mouse with myeloma cells. The chain dominance was analyzed by UV cross-linking experiments, in which the antibodies were covalently cross-linked with radioisotope-labeled oligonucleotides by short-wavelength UV-light, and the cross-linked H and L chains were analyzed by SDS-PAGE and densitometric scanning. Among these, three were found to be heavy chain dominant antibodies in which heavy chains are dominantly involved in DNA binding. The other five were co-dominant antibodies in which both heavy and light chains are involved in DNA binding. To determine the factor(s) that can explain the chain dominance in DNA binding, we determined the amino acid sequences of the variable regions of both heavy (VH) and light (VL) chains of all eight monoclonal antibodies. By analyzing the data, we were able to draw the following conclusions: (1) The arginine residues are found in the CDR3 regions of both VH and VL of the co-dominant antibodies; whereas, the same residues are found only in the CDR3s of VH, but not in VL, of the heavy chain dominant antibodies. (2) The net charges of the V regions affect the chain dominance. From the results of this study it is suggested that the presence of arginine residue in CDR3 is a critical factor in determining chain-dominance, as well as DNA binding of anti-DNA antibodies in general.     

Using a heavy chain-loss hybridoma 26.4.1LL for studying the structural basis of immunoglobulin chain association

One of the mechanisms contributing to antibody diversity is created by the association of different heavy and light chains. The combinability of heavy and light chains has been studied previously in two systems: in vitro chain recombination and hybrid hybridoma. Here, a novel in vivo chain combination assay system involving a heavy chain-loss variant, 26.4.1LL, producing two kappa light chains (L(DEX) and L(MPC)) different in size is described. In conjunction with DNA transfection, immunoprecipitation and SDS-PAGE, the structural basis of noncovalent interaction between heavy and light chains can be elucidated systematically by examining the relative association tendency of a heavy chain with two light chains. To demonstrate the usefulness of this system, three stably transfected 26.4.1LL cell lines expressing gamma2b heavy chains, designated as H(DEX), H(CHI) and H(ARS), respectively, with structural interrelated variable regions were generated: H(DEX) differs from H(CHI) only in framework regions whereas H(CHI) differs from H(ARS) in complementarity-determining regions. The relative amounts (R values) of L(DEX) and L(MPC) associated with the heavy chains H(DEX), H(CHI) and H(ARS) in the assembled immunoglobulin molecules were found to be 1.02, 0.64 and 0.05, respectively, suggesting that the complementarity-determining regions and framework regions contribute equally to the V(L)-V(H) interaction. This conclusion is consistent with previous observations based on calculation of the buried area in the V(L)-V(H) interface, thus demonstrating the usefulness of this system.     

Construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain

Currently, almost all U.S. Food and Drug Administration-approved therapeutic antibodies and the vast majority of those in clinical trials are full-size antibodies mostly in an immunoglobulin G1 format of about 150 kDa in size. Two fundamental problems for such large molecules are their poor penetration into tissues (e.g., solid tumors) and poor or absent binding to regions on the surface of some molecules [e.g., on the human immunodeficiency virus envelope glycoprotein (Env)] that are accessible by molecules of smaller size. We have identified a phage-displayed heavy chain-only antibody by panning of a large (size, ∼ 1.5 × 10¹⁰) human naive Fab (antigen-binding fragment) library against an Env and found that the heavy chain variable domain (V_H) of this antibody, designated as m0, was independently folded, stable, highly soluble, monomeric, and expressed at high levels in bacteria. m0 was used as a scaffold to construct a large (size, ∼ 2.5 × 10¹⁰), highly diversified phage-displayed human V_H library by grafting naturally occurring complementarity-determining regions (CDRs) 2 and 3 of heavy chains from five human antibody Fab libraries and by randomly mutating four putative solvent-accessible residues in CDR1 to A, D, S, or Y. The sequence diversity of all CDRs was determined from 143 randomly selected clones. Most of these V_Hs were with different CDR2 origins (six of seven groups of V_H germlines) or CDR3 lengths (ranging from 7 to 24 residues) and could be purified directly from the soluble fraction of the Escherichia coli periplasm. The quality of the library was also validated by successful selection of high-affinity V_Hs against viral and cancer-related antigens; all selected V_Hs were monomeric, easily expressed, and purified with high solubility and yield. This library could be a valuable source of antibodies targeting size-restricted epitopes and antigens in obstructed locations where efficient penetration could be critical for successful treatment.     

Crystal structure of the HIV-2 neutralizing Fab fragment 7C8 with high specificity to the V3 region of gp125

7C8 is a mouse monoclonal antibody specific for the third hypervariable region (V3) of the human immunodeficiency virus type 2 (HIV-2)-associated protein gp125. The three-dimensional crystal structure of the Fab fragment of 7C8, determined to 2.7 Å resolution, reveals a deep and narrow antigen-binding cleft with architecture appropriate for an elongated epitope. The highly hydrophobic cleft is bordered on one side by the negatively charged second complementarity determining region (CDR2) and the unusually long positively charged CDR3 of the heavy chain and, on the other side, by the CDR1 of the light chain. Analysis of 7C8 in complex with molecular models of monomeric and trimeric gp125 highlights the importance of a conserved stretch of residues FHSQ that is localized centrally on the V3 region of gp125. Furthermore, modeling also indicates that the Fab fragment neutralizes the virus by sterically impairing subsequent engagement of the gp125 trimer with the co-receptor on the target cell.     

Antibody CDR H3 modeling rules: extension for the case of absence of Arg H94 and Asp H101

The third complementary determining region of the immunoglobulin heavy chain (CDR H3) is one of the more difficult structures to model due to genetic reasons. However, the conformation of proximal to beta-framework ("torso") part of the CDR H3 is very predictable. Current "CDR's canonical classes" theory is based on identifying the key positions, H94 and H101. We can determine the CDR H3 "torso" structure if arginine or lysine is present in the H94 position and/or aspartic acid in the H101 position. We target the case characterized by the absence of key residues in both the H94 and H101 positions. There has not been discussion on this case in the literature. 51 CDR H3 structures of this nature are analyzed and we established new sequence-structure rules. These rules contribute to more accurate modeling of the antibody's structure.     

Structural Basis for the Recognition of Human Cytomegalovirus Glycoprotein B by a Neutralizing Human Antibody

Human cytomegalovirus (HCMV) infections are life-threating to people with a compromised or immature immune system. Upon adhesion, fusion of the virus envelope with the host cell is initiated. In this step, the viral glycoprotein gB is considered to represent the major fusogen. Here, we present for the first time structural data on the binding of an anti-herpes virus antibody and describe the atomic interactions between the antigenic domain Dom-II of HCMV gB and the Fab fragment of the human antibody SM5-1. The crystal structure shows that SM5-1 binds Dom-II almost exclusively via only two CDRs, namely light chain CDR L1 and a 22-residue-long heavy chain CDR H3. Two contiguous segments of Dom-II are targeted by SM5-1, and the combining site includes a hydrophobic pocket on the Dom-II surface that is only partially filled by CDR H3 residues. SM5-1 belongs to a series of sequence-homologous anti-HCMV gB monoclonal antibodies that were isolated from the same donor at a single time point and that represent different maturation states. Analysis of amino acid substitutions in these antibodies in combination with molecular dynamics simulations show that key contributors to the picomolar affinity of SM5-1 do not directly interact with the antigen but significantly reduce the flexibility of CDR H3 in the bound and unbound state of SM5-1 through intramolecular side chain interactions. Thus, these residues most likely alleviate unfavorable binding entropies associated with extra-long CDR H3s, and this might represent a common strategy during antibody maturation. Models of entire HCMV gB in different conformational states hint that SM5-1 neutralizes HCMV either by blocking the pre- to postfusion transition of gB or by precluding the interaction with additional effectors such as the gH/gL complex.     

Length of the antibody heavy chain complementarity determining region 3 as a specificity-determining factor

The antigen binding site of an antibody is made up of residues residing in six hypervariable loops of the heavy and light chains. In most cases several or all of these loops are required for the establishment of the antigen-binding surface. Five of these loops display a limited diversity in length and sequence while the third complementarity determining region (CDR) of the heavy chain is highly different between antibodies not only with respect to sequence but also with respect to length. Its extensive diversity is a key component in the establishment of binding sites allowing for the recognition of essentially any antigen by humoral immunity. The relative importance of its sequence vs its length diversity in this context is however, not very well established. To investigate this matter further we have used an approach employing combinatorial antibody libraries and antigen-specific selection in the search for CDRH3 length and sequence diversity compatible with a given antigen specificity, the major antigenic determinant on the tumour-associated antigen mucin-1. In this way we have now defined heavy chain CDR3 length as a critical parameter in the creation of an antigen-specific binding site. We also propose that this may reflect a dependence of a particular structure of this hypervariable loop, the major carrier of diversity in the binding site, for establishment of a given specificity.