Global conformational changes in IgG-Fc upon mutation of the FcRn-binding site are not associated with altered antibody-dependent effector functions.

Antibody engineering is important for many diagnostic and clinical applications of monoclonal antibodies. We recently reported a series of fragment crystallizable (Fc) mutations targeting the neonatal Fc receptor (FcRn) site on a Lewis Y (Ley) binding IgG1, hu3S193. The hu3S193 variants displayed shortened in vivo half-lives and may have potential for radioimaging or radiotherapy of Ley-positive tumors. Here, we report Fc crystal structures of wild-type hu3S193, seven FcRn-binding site variants, and a variant lacking C1q binding or complement-dependent cytotoxicity (CDC) activity. The Fc conformation of the FcRn-binding sites was similar for wild-type and all mutants of hu3S193 Fc, which suggests that FcRn interactions were directly affected by the amino acid substitutions. The C1q-binding site mutant Fc was nearly identical with the wild-type Fc. Surprisingly, several hu3S193 Fc variants showed large changes in global structure compared with wild-type Fc. All hu3S193 Fc mutants had similar antibody-dependent cellular cytotoxicity, despite some with conformations expected to diminish Fc gamma receptor binding. Several hu3S193 variants displayed altered CDC, but there was no correlation with the different Fc conformations. All versions of hu3S193, except the C1q-binding site mutant, bound C1q, suggesting that the altered CDC of some variants could result from different propensities to form IgG hexamers after engaging Ley on target cells. Overall, our findings support the concept that the antibody Fc is both flexible and mobile in solution. Structure-based design approaches should take into account the conformational plasticity of the Fc when engineering antibodies with optimal effector properties.

Structural basis for Fc gammaRIIa recognition of human IgG and formation of inflammatory signaling complexes.

The interaction of Abs with their specific FcRs is of primary importance in host immune effector systems involved in infection and inflammation, and are the target for immune evasion by pathogens. FcγRIIa is a unique and the most widespread activating FcR in humans that through avid binding of immune complexes potently triggers inflammation. Polymorphisms of FcγRIIa (high responder/low responder [HR/LR]) are linked to susceptibility to infections, autoimmune diseases, and the efficacy of therapeutic Abs. In this article, we define the three-dimensional structure of the complex between the HR (arginine, R134) allele of FcγRIIa (FcγRIIa-HR) and the Fc region of a humanized IgG1 Ab, hu3S193. The structure suggests how the HR/LR polymorphism may influence FcγRIIa interactions with different IgG subclasses and glycoforms. In addition, mutagenesis defined the basis of the epitopes detected by FcR blocking mAbs specific for FcγRIIa (IV.3), FcγRIIb (X63-21), and a pan FcγRII Ab (8.7). The epitopes detected by these Abs are distinct, but all overlap with residues defined by crystallography to contact IgG. Finally, crystal structures of LR (histidine, H134) allele of FcγRIIa and FcγRIIa-HR reveal two distinct receptor dimers that may represent quaternary states on the cell surface. A model is presented whereby a dimer of FcγRIIa-HR binds Ag-Ab complexes in an arrangement that possibly occurs on the cell membrane as part of a larger signaling assembly.

A conserved host and pathogen recognition site on immunoglobulins: structural and functional aspects.

A common site in the constant region (Fc) of immunoglobulins is recognized by host receptors and is a frequent target of proteins expressed by pathogens. This site is located at the junction of two constant domains in the antibody heavy chains and produces a large shallow cavity formed by loops of the CH2 and CH3 domains in IgG and IgA (CH3 and CH4 domains in IgM). Crystal structures have been determined for complexes of IgG-Fc and IgA-Fc with a structurally diverse set of host, pathogen and in vitro selected ligands. While pathogen proteins may directly block interactions with the immunoglobulins thereby evading host immunity, it is likely that the same pathogen molecules also interact with other host factors to carry out their primary biological function. Herein we review the structural and functional aspects of host and pathogen molecular recognition of the common site on the Fc of immunoglobulins. We also propose that some pathogen proteins may promote virulence by affecting the bridging between innate and adaptive immunity.

Crystal structure of a glycosylated Fab from an IgM cryoglobulin with properties of a natural proteolytic antibody.

The 2.6 A (1 A=0.1 nm) resolution structure has been determined for the glycosylated Fab (fragment antigen binding) of an IgM (Yvo) obtained from a subject with Waldenström's macroglobulinaemia. Dynamic light scattering was used to estimate the gel point and monitor the formation of an ordered hydroscopic gel of Yvo IgM upon cooling. If a cryoglobulin forms gels in peripheral tissues and organs, the associated swelling and damage to microvasculature can result in considerable morbidity and mortality. The three-dimensional structure of the branched N-linked oligosaccharide associated with the CH1 domain (first constant domain of heavy chain) is reported. The carbohydrate may act to shield part of the lateral surface of the CH1 domain and crowd the junction between the CH1 and CH2 domains, thereby limiting the segmental flexibility of the Fab arms in intact Yvo IgM, especially at low temperatures. Recently, Yvo IgM was shown to have the properties of a naturally occurring proteolytic antibody [Paul, Karle, Planque, Taguchi, Salas, Nishiyama, Handy, Hunter, Edmundson and Hanson (2004) J. Biol. Chem. 279, 39611-39619; Planque, Bangale, Song, Karle, Taguchi, Poindexter, Bick, Edmundson, Nishiyama and Paul (2004) J. Biol Chem. 279, 14024-14032]. The Yvo protein displayed the ability to cleave, by a nucleophilic mechanism, the amide bonds of a variety of serine protease substrates and the gp120 coat protein of HIV. An atypical serine, arginine and glutamate motif is located in the middle of the Yvo antigen-binding site and displays an overall geometry that mimics the classical serine, histidine and aspartate catalytic triad of serine proteases. Our present findings indicate that pre-existing or natural antibodies can utilize at least one novel strategy for the cleavage of peptide bonds.

Cross-species analysis of Fc engineered anti-Lewis-Y human IgG1 variants in human neonatal receptor transgenic mice reveal importance of S254 and Y436 in binding human neonatal Fc receptor.

IgG has a long half-life through engagement of its Fc region with the neonatal Fc receptor (FcRn). The FcRn binding site on IgG1 has been shown to contain I253 and H310 in the CH2 domain and H435 in the CH3 domain. Altering the half-life of IgG has been pursued with the aim to prolong or reduce the half-life of therapeutic IgGs. More recent studies have shown that IgGs bind differently to mouse and human FcRn. In this study we characterize a set of hu3S193 IgG1 variants with mutations in the FcRn binding site. A double mutation in the binding site is necessary to abrogate binding to murine FcRn, whereas a single mutation in the FcRn binding site is sufficient to no longer detect binding to human FcRn and create hu3S193 IgG1 variants with a half-life similar to previously studied hu3S193 F(ab')2 (t1/2ß, I253A, 12.23 h; H310A, 12.94; H435A, 12.57; F(ab')2, 12.6 h). Alanine substitutions in S254 in the CH2 domain and Y436 in the CH3 domain showed reduced binding in vitro to human FcRn and reduced elimination half-lives in huFcRn transgenic mice (t1/2ß, S254A, 37.43 h; Y436A, 39.53 h; wild-type, 83.15 h). These variants had minimal effect on half-life in BALB/c nu/nu mice (t1/2ß, S254A, 119.9 h; Y436A, 162.1 h; wild-type, 163.1 h). These results provide insight into the interaction of human Fc by human FcRn, and are important for antibody-based therapeutics with optimal pharmacokinetics for payload strategies used in the clinic.

Structural convergence of antibody binding of carbohydrate determinants in Lewis Y tumor antigens.

Antibodies targeting human epithelial carcinomas bearing Lewis Y (Le(y)) carbohydrate antigens provide a striking illustration of convergent immune recognition. We report a 1.9A resolution crystal structure of the Fab of a humanized antibody (hu3S193) in complex with the Le(y) tetrasaccharide, Fuc(alpha 1-->2)Gal(beta 1-->4)[Fuc(alpha 1-->3)]GlcNAc. Comparisons of the hu3S193 and BR96 antibodies bound to Le(y) tumor antigens revealed extremely similar mechanisms for recognition of the carbohydrate epitopes. Solvent plays a critical role in hu3S193 antibody binding to the Le(y) carbohydrate epitope. Specificity for Le(y) is maintained because a conserved pocket accepts an N-acetyl group of the core Gal(beta 1-->4)GlcNAc disaccharide. Closely related blood-group determinants (Le(a) and Le(b)) cannot enter the specificity pocket, making the Le(y) antibodies promising candidates for immunotherapy of epithelial cancer.

Cold-induced precipitation of a monoclonal IgM: a negative activation enthalpy reaction.

Cold-induced precipitation of a monoclonal IgM cryoglobulin isolated from a patient with Waldenström's macroglobulinemia was observed to have a negative activation enthalpy. The rate of the reaction increased, as the temperature decreased. Differential scanning calorimetry of the monoclonal IgM showed precipitation as an inverted peak during a downward temperature scan. The transition temperature was between 14 and 15 °C and was possibly concentration dependent. At temperatures below the transition the precipitation was best described by second-order kinetics. The difference in change in enthalpy between precipitation and disassociation suggests that cold-induced precipitation had a fast precipitation stage followed by a slower consolidation reaction. Negligible curvature of the Eyring plot suggested the precipitation reaction was dominated by van der Waal forces and hydrogen bonding. Conversely, during an upward temperature scan, disassociation was observed as a positive enthalpy peak. This reaction had two stages, a reaction undoing consolidation followed by heat-induced disassociation that had first-order kinetics.

Carbohydrate-mimetic peptides: structural aspects of mimicry and therapeutic implications.

INTRODUCTION: The existence of specific carbohydrates on the surface of a wide range of cells provides the opportunity for the development of highly targeted therapeutic agents. The potential applications of such agents are diverse, and include vaccines against pathogenic microorganisms, cancer and HIV, and anti-rejection agents for organ transplantation. However, the use of carbohydrates as either therapeutic agents or immunogens is frequently problematic, as they are often rapidly metabolized and poorly immunogenic. Therefore, the search for carbohydrate-mimetic agents is of considerable therapeutic value, for the potential of such agents to both interfere with carbohydrate-protein interactions and to generate carbohydrate-specific immune responses. AREAS COVERED: The review discusses recent examples of carbohydrate-mimetic peptides with regard to the structural and functional aspects of mimicry and the implications of peptide mimicry for application in therapeutics. The reader will gain knowledge of the various mechanisms of peptide carbohydrate mimicry, and the potential importance of these mechanisms in targeted therapeutic design. EXPERT OPINION: Peptide carbohydrate mimicry is manifested by distinct mechanisms, any one of which may be relevant to specific protein targets. As structural information becomes available for a wider variety of systems, the questions about mimicry will be more effectively addressed.

CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity.

CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n=16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained by the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.

Construction of single-chain Fv with two possible CDR3H conformations but similar inter-molecular forces that neutralize bovine herpesvirus 1.

Bovine herpesvirus 1 (BoHV-1) causes respiratory and genital diseases in cattle for which available vaccines do not confer adequate protection. Since passive immunization with antibodies permits disease prevention, single-chain fragment variable (scFv), originating from a monoclonal bovine IgG1 antibody against BoHV-1, were constructed and expressed in Pichia pastoris in V(lambda)-V(H) orientation via a flexible seven-amino acid linker. Similar to the intact IgG, the purified recombinant scFv neutralized BoHV-1 in vitro and recognized viral antigens in BoHV-1 infected MDBK cells by immunofluorescence. Homology modeling of the Fv predicts two distinct conformations for CDR3H. Firstly, a long protruding CDR3H conformation where no disulfide linkage occurred between two "non-canonical" Cys residues resulted in a large binding cavity between V(lambda) and V(H). Secondly, a smaller potential antigen-binding cavity is predicted with a disulfide linkage between the two Cys residues of CDR3H creating a six-membered loop in the ascending polypeptide, which fitted into the space between V(lambda) and V(H). Despite such potential configurational diversity of the antigen-binding site, the electrostatic surface potentials that would interact with the BoHV-1 epitope are largely similar for both the topographies where salt-bridge type electrostatic interactions likely occur at the edges of the binding site. Given that IgG1 antibody against BoHV-1 is clonally selected, it is likely that disulfide-stabilized broader and flatter surface topography is specifically generated to accommodate the predicted carbohydrate neutralizing B-epitope on BoHV-1. The specificity and neutralizing capacity for BoHV-1 of the scFv should make this bovine antibody fragment a useful diagnostic and potential therapeutic candidate for an important viral pathogen in cattle.

An altered and more efficient mechanism of CCR5 engagement contributes to macrophage tropism of CCR5-using HIV-1 envelopes.

While CCR5 is the principal coreceptor used by macrophage (M)-tropic HIV-1, not all primary CCR5-using (R5) viruses enter macrophages efficiently. Here, we used functionally-diverse R5 envelope (Env) clones to characterize virus-cell interactions important for efficient CCR5-mediated macrophage entry. The magnitude of macrophage entry by Env-pseudotyped reporter viruses correlated with increased immunoreactivity of CD4-induced gp120 epitopes, increased ability to scavenge low levels of cell-surface CCR5, reduced sensitivity to the CCR5 inhibitor maraviroc, and increased dependence on specific residues in the CCR5 ECL2 region. These results are consistent with an altered and more efficient mechanism of CCR5 engagement. Structural studies revealed potential alterations within the gp120 V3 loop, the gp41 interaction sites at the gp120 C- and N-termini, and within the gp120 CD4 binding site which may directly or indirectly lead to more efficient CCR5-usage. Thus, enhanced gp120-CCR5 interactions may contribute to M-tropism of R5 HIV-1 strains through different structural mechanisms.

A possible role for metallic ions in the carbohydrate cluster recognition displayed by a Lewis Y specific antibody.

BACKGROUND: Lewis Y (Le(y)) is a blood group-related carbohydrate that is expressed at high surface densities on the majority of epithelial carcinomas and is a promising target for antibody-based immunotherapy. A humanized Le(y)-specific antibody (hu3S193) has shown encouraging safety, pharmacokinetic and tumor-targeting properties in recently completed Phase I clinical trials. METHODOLOGY/PRINCIPAL FINDINGS: We report the three-dimensional structures for both the free (unliganded) and bound (Le(y) tetrasaccharide) hu3S193 Fab from the same crystal grown in the presence of divalent zinc ions. There is no evidence of significant conformational changes occurring in either the Le(y) carbohydrate antigen or the hu3S193 binding site, which suggests a rigid fit binding mechanism. In the crystal, the hu3S193 Fab molecules are coordinated at their protein-protein interface by two zinc ions and in solution aggregation of Fab can be initiated by zinc, but not magnesium ions. Dynamic light scattering revealed that zinc ions could initiate a sharp transition from hu3S193 Fab monomers to large multimeric aggregates in solution. CONCLUSIONS/SIGNIFICANCE: Zinc ions can mediate interactions between hu3S193 Fab in crystals and in solution. Whether metallic ion mediated aggregation of antibody occurs in vivo is not known, but the present results suggest that similar clustering mechanisms could occur when hu3S193 binds to Le(y) on cells, particularly given the high surface densities of antigen on the target tumor cells.

Non-canonical anchor motif peptides bound to MHC class I induce cellular responses.

The major histocompatibility complex (MHC) on the surface of antigen presenting cells functions to display peptides to the T cell receptor (TCR). Recognition of peptide-MHC by T cells initiates a cascade of signals, which results in the initiation of a T cell dependent immune response. An understanding of how peptides bind to MHC molecules is important for determining the structural basis for T cell dependent immune responses and facilitates the structure-based design of peptides as candidate vaccines to elicit a specific immune response. To date, crystal structures, immunogenicity and in vivo biological relevance have mainly been characterized for high affinity peptide-MHC interactions. From the crystal structures of numerous peptide-MHC complexes it became apparent what canonical sequence features were required for high affinity binding, which led to the ability to predict in most instances peptides with high affinity for MHC. We previously identified the crystal structures of non-canonical peptides in complex with MHC class I (one bound with low affinity and the other with high affinity, but utilizing novel peptide anchors and MHC pockets). It is becoming increasingly evident that other non-canonical peptides can also bind, such as long-, short- and glyco-peptides. However, the in vivo role of non-canonical peptides is not clear and we present here the immunogenicity of two non-canonical peptides and their affinity when bound to MHC class I, H2K(b). Comparison of the three-dimensional structures in complex with MHC suggests major differences in hydrogen bonding patterns with H2K(b), despite sharing similar binding modes, which may account for the differences in affinity and immunogenicity. These studies provide further evidence for the diverse range of peptide ligands that can bind to MHC and be recognized by the TCR, which will facilitate approaches to peptide-based vaccine design.

Structural biology of carbohydrate xenoantigens.

Transplantation of organs across species (xenotransplantation) is being considered to overcome the shortage of human donor organs. However, unmodified pig organs undergo an antibody-mediated hyperacute rejection that is brought about by the presence of natural antibodies to Galalpha(1,3)Gal, which is the major carbohydrate xenoantigen. Genetic modification of pig organs to remove most of the Galalpha(1,3)Gal epitopes has been achieved, but the human immune system may still recognize residual lipid-linked Galalpha(1,3)Gal carbohydrates, new (cryptic) carbohydrates or additional non-Galalpha(1,3)Gal carbohydrate xenoantigens. The structural basis for lectin and antibody recognition of Galalpha(1,3)Gal carbohydrates is starting to be understood and is discussed in this review. Antibody binding to Galalpha(1,3)Gal carbohydrates is predicted to primarily involve end-on insertion of the terminal alphaGal residue, but it is possible that groove-type binding can occur, as for some lectins. It is likely that similar antibody and lectin recognition will occur with other non-Galalpha(1,3)Gal xenoantigens, which potentially represent new barriers for pig-to-human xenotransplantation.

Dissimilar aggregation processes govern precipitation and gelation of human IgM cryoglobulins.

Cryoglobulinemia is associated with a range of diseases including rheumatoid arthritis, B-cell malignancies, and chronic viral infections. This "cold-sensitivity" condition is caused by cryoglobulins that precipitate, gel, or occasionally crystallize in the cold. Clinical manifestations vary widely in severity, depending on many factors, including the type of cryoglobulin (monoclonal or mixed immunoglobulins) and the physical nature of the aggregates (precipitate, gel, or crystal). Dynamic light scattering (DLS) was used to examine the cold-induced precipitation or gelation of two human cryoglobulins, namely, Pot IgM and Yvo IgM. The DLS assay was highly reproducible, sensitive, and had low intra-assay variations for both IgM cryoglobulins. Distinct processes were revealed to contribute to precipitation and gelation of cryoglobulins. The precipitation of Pot IgM displayed a rapid transition from solution to solid phases, with a wide distribution of aggregate sizes. In contrast, the gelation of Yvo IgM progressed gradually across a broad temperature range to produce a relatively uniform gel matrix. Initial cryoglobulin concentrations determined the kinetics and critical temperatures for both precipitation and gelation. Moreover, the Yvo IgM was observed to have a distinct relationship between concentrations and mean hydrodynamic diameters or particle sizes. Concentration-dependent effects on particle sizes were present, but not as pronounced for the Pot IgM. Precipitation and gelation of cryoglobulins were also found to be differentially responsive to changes in the aqueous environment. Our results indicate that DLS is a rapid, reliable, and sensitive method for characterizing the nature of disease-associated cryoglobulins.

Structural basis for evasion of IgA immunity by Staphylococcus aureus revealed in the complex of SSL7 with Fc of human IgA1.

Infection by Staphylococcus aureus can result in severe conditions such as septicemia, toxic shock, pneumonia, and endocarditis with antibiotic resistance and persistent nasal carriage in normal individuals being key drivers of the medical impact of this virulent pathogen. In both virulent infection and nasal colonization, S. aureus encounters the host immune system and produces a wide array of factors that frustrate host immunity. One in particular, the prototypical staphylococcal superantigen-like protein SSL7, potently binds IgA and C5, thereby inhibiting immune responses dependent on these major immune mediators. We report here the three-dimensional structure of the complex of SSL7 with Fc of human IgA1 at 3.2 A resolution. Two SSL7 molecules interact with the Fc (one per heavy chain) primarily at the junction between the Calpha2 and Calpha3 domains. The binding site on each IgA chain is extensive, with SSL7 shielding most of the lateral surface of the Calpha3 domain. However, the SSL7 molecules are positioned such that they should allow binding to secretory IgA. The key IgA residues interacting with SSL7 are also bound by the leukocyte IgA receptor, FcalphaRI (CD89), thereby explaining how SSL7 potently inhibits IgA-dependent cellular effector functions mediated by FcalphaRI, such as phagocytosis, degranulation, and respiratory burst. Thus, the ability of S. aureus to subvert IgA-mediated immunity is likely to facilitate survival in mucosal environments such as the nasal passage and may contribute to systemic infections.

Enhanced major histocompatibility complex class I binding and immune responses through anchor modification of the non-canonical tumour-associated mucin 1-8 peptide.

Designing peptide-based vaccines for therapeutic applications in cancer immunotherapy requires detailed knowledge of the interactions between the antigenic peptide and major histocompatibility complex (MHC) in addition to that between the peptide-MHC complex and the T-cell receptor. Past efforts to immunize with high-affinity tumour-associated antigenic peptides have not been very immunogenic, which may be attributed to the lack of T cells to these peptides, having been deleted during thymic development. For this reason, low-to-medium affinity non-canonical peptides represent more suitable candidates. However, in addition to the difficulty in identifying such antigens, peptide binding to MHC, and hence its ability to induce a strong immune response, is limited. Therefore, to enhance binding to MHC and improve immune responses, anchor modifications of non-canonical tumour-associated peptides would be advantageous. In this study, the non-canonical tumour-associated peptide from MUC1, MUC1-8 (SAPDTRPA), was modified at the MHC anchor residues to SAPDFRPL (MUC1-8-5F8L) and showed enhanced binding to H-2Kb and improved immune responses. Furthermore, the crystal structure of MUC1-8-5F8L in complex with H-2Kb was determined and it revealed that binding of the peptide to MHC is similar to that of the canonical peptide OVA8 (SIINFEKL).

Three-dimensional structures of carbohydrate determinants of Lewis system antigens: implications for effective antibody targeting of cancer.

Lewis system carbohydrate antigens have been shown to be expressed at high levels in many cancers of epithelial cell origin, including those of colon, breast, lung, prostate and ovary. The type 1 (Le(a) and Le(b)) antigens are important histo-blood groups, while type 2 (Le(x) and Le(y)) antigens in healthy individuals are only expressed, at relatively low levels, by a few tissues, including some epithelial cells. Thus, the type 2 antigens are considered to be tumour-associated antigens and are promising targets for cancer treatment, including antibody-based immunotherapy. In this review, we discuss the conformational characteristics of the free and bound forms of Lewis oligosaccharides and the 3D structures of antibodies in complex with Le(y) and Le(x) antigens. Collectively, the structural studies have demonstrated that the Lewis determinants are rigid structures, which generally maintain the same conformation in the free and bound states. The rigid nature and similarities in shape of type 1 and 2 Lewis oligosaccharides appear to make them perfectly suited to driving a structurally convergent immune response (at least in the case of Le(y) specific antibodies) toward a highly specific recognition of individual carbohydrate determinants, which is a goal in the development of effective antibody-based cancer treatments.

Crystal structures of human antibodies: a detailed and unfinished tapestry of immunoglobulin gene products.

Sequencing of all human immunoglobulin (Ig) germline gene segments has recently been completed. However, our first glimpses of the recombined products of this combinatorial gene system were in the 1970s, in landmark publications, reporting the crystal structures of two human myeloma proteins, the Mcg lambda light chain dimer and the New IgG1(lambda) Fab. Although numerous crystal structures of murine and human antibodies have now been determined, only a relatively small proportion of the human germline genes have had their corresponding protein three-dimensional structures resolved. Therefore, further structural investigations are required before the inherent diversity of the antibody repertoire can be fully appreciated. We discuss the detailed structural information available for human antibodies with regard to their immune functions. Also discussed, is how the structural information is finding application in the 'humanization' of murine antibodies as part of their development as 'biopharmaceuticals' for the treatment of human disease.

Bovine IgM antibodies with exceptionally long complementarity-determining region 3 of the heavy chain share unique structural properties conferring restricted VH + Vlambda pairings.

Naturally occurring antibody repertoires of cattle (Bos taurus) include a group of IgMlambda antibodies with exceptionally long complementarity-determining region 3 of the heavy chain (CDR3H) segments, containing multiple Cys residues. These massive CDR3H segments will greatly influence the tertiary and quaternary structures of the bovine IgM combining sites. As an antibody's combining site is formed by both heavy and light chains, we have analyzed the nucleotide sequences and structural properties of the lambda-light chains that pair with micro -heavy chains containing exceptionally long CDR3H. There appears to be an exquisite selective pressure for the use of three V(lambda)1 genes (V(lambda)1x and two new V(lambda)1d and V(lambda)1e genes) in IgM with unusually long CDR3H. The V(lambda)1d and V(lambda)1e genes are similar to each other, but diverge from the other V(lambda)1 genes into two closely related subfamilies. The available bovine V(lambda) genes are classified into three V(lambda) gene families: V(lambda)1, V(lambda)2 and V(lambda)3 based on nucleotide similarity >/=80%. Further, analysis of total Ser content and positions of Ser residues in the sequences was found to be sufficient to classify the cattle V(lambda)1 subfamilies. Patterns of Ser residues differ for V(lambda) domains from ruminant species (e.g. cattle, sheep and goats) and other mammals (e.g. humans and mice). These 'Ser signatures' can be used to track divergent evolution in lambda-light chains. Interestingly, Ser90L in complementarity-determining region 3 of the light chain (CDR3L) occurred in all V(lambda) domains that pair with V(H) regions containing exceptionally long CDR3H. A structural role for Ser90L was revealed in homology models of V(lambda) domains, i.e. to hold the ascending polypeptide of CDR3L in a relatively tight space between the N-terminal segment and residues from CDR1L. The CDR3L of V(lambda) domains also occupied smaller volumes if paired to V(H) domains with extremely long CDR3H (>/=48 residues), and were more variable in their conformation and filled larger volumes if CDR3Hs were