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We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains., Competing Interests: Competing interest statement: G.D.Y. is a board member, employee, and shareholder of Regeneron Pharmaceuticals, Inc. Other authors are employees and shareholders of Regeneron Pharmaceuticals, Inc.

Although at first glance the diversity of the immunoglobulin repertoire appears random, there are a number of mechanisms that act to constrain diversity. For example, key mechanisms controlling the diversity of the third complementarity determining region of the immunoglobulin heavy chain (CDR-H3) include natural selection of germline diversity (D H ) gene segment sequence and somatic selection upon passage through successive B-cell developmental checkpoints. To test the role of D H gene segment sequence, we generated a panel of mice limited to the use of a single germline or frameshifted D H gene segment. Specific individual amino acids within core D H gene segment sequence heavily influenced the absolute numbers of developing and mature B-cell subsets, antibody production, epitope recognition, protection against pathogen challenge, and susceptibility to the production of autoreactive antibodies. At the tip of the antigen-binding loop (PDB position 101) in CDR-H3, both natural (germline) and somatic selection favored tyrosine while disfavoring the presence of hydrophobic amino acids. Enrichment for arginine in CDR-H3 appeared to broaden recognition of epitopes of varying hydrophobicity, but led to diminished binding intensity and an increased likelihood of generating potentially pathogenic dsDNA-binding autoreactive antibodies. The phenotype of altering the sequence of the D H was recessive for T-independent antibody production, but dominant for T-cell-dependent responses. Our work suggests that the antibody repertoire is structured, with the sequence of individual D H selected by evolution to preferentially generate an apparently preferred category of antigen-binding sites. The result of this structured approach appears to be a repertoire that has been adapted, or optimized, to produce protective antibodies for a wide range of pathogen epitopes while reducing the likelihood of generating autoreactive specificities., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Antibodies can rapidly evolve in specific response to antigens. Affinity maturation drives this evolution through cycles of mutation and selection leading to enhanced antibody specificity and affinity. Elucidating the biophysical mechanisms that underlie affinity maturation is fundamental to understanding B-cell immunity. An emergent hypothesis is that affinity maturation reduces the conformational flexibility of the antibody's antigen-binding paratope to minimize entropic losses incurred upon binding. In recent years, computational and experimental approaches have tested this hypothesis on a small number of antibodies, often observing a decrease in the flexibility of the complementarity determining region (CDR) loops that typically comprise the paratope and in particular the CDR-H3 loop, which contributes a plurality of antigen contacts. However, there were a few exceptions and previous studies were limited to a small handful of cases. Here, we determined the structural flexibility of the CDR-H3 loop for thousands of recent homology models of the human peripheral blood cell antibody repertoire using rigidity theory. We found no clear delineation in the flexibility of naïve and antigen-experienced antibodies. To account for possible sources of error, we additionally analyzed hundreds of human and mouse antibodies in the Protein Data Bank through both rigidity theory and B-factor analysis. By both metrics, we observed only a slight decrease in the CDR-H3 loop flexibility when comparing affinity matured antibodies to naïve antibodies, and the decrease was not as drastic as previously reported. Further analysis, incorporating molecular dynamics simulations, revealed a spectrum of changes in flexibility. Our results suggest that rigidification may be just one of many biophysical mechanisms for increasing affinity.

The VHHs are antigen-binding region/domain of camelid heavy chain antibodies (HCAb). They have many interesting biotechnological and biomedical properties due to their small size, high solubility and stability, and high affinity and specificity for their antigens. HCAb and classical IgGs are evolutionary related and share a common fold. VHHs are composed of regions considered as constant, called the frameworks (FRs) connected by Complementarity Determining Regions (CDRs), a highly variable region that provide interaction with the epitope. Actually, no systematic structural analyses had been performed on VHH structures despite a significant number of structures. This work is the first study to analyse the structural diversity of FRs of VHHs. Using a structural alphabet that allows approximating the local conformation, we show that each of the four FRs do not have a unique structure but exhibit many structural variant patterns. Moreover, no direct simple link between the local conformational change and amino acid composition can be detected. These results indicate that long-range interactions affect the local conformation of FRs and impact the building of structural models., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

In this present study, we engineered hypervariable loop 2 (HV2) of the IgNAR variable domain in a way that it solely facilitates antigen binding, potentially functioning as an autonomous paratope. For this, the surface-exposed loop corresponding to HV2 was diversified and antigen-specific variable domain of IgNAR antibody (vNAR) molecules were isolated by library screening using yeast surface display (YSD) as platform technology. An epithelial cell adhesion molecule (EpCAM)-specific vNAR was used as starting material, and nine residues in HV2 were randomized. Target-specific clones comprising a new HV2-mediated paratope were isolated against cluster of differentiation 3ε (CD3ε) and human Fcγ while retaining high affinity for EpCAM. Essentially, we demonstrate that a new paratope comprising moderate affinities against a given target molecule can be engineered into the vNAR scaffold that acts independent of the original antigen-binding site, composed of complementarity-determining region 3 (CDR3) and CDR1.

Broadly neutralizing antibodies (bNAbs) isolated from chronically HIV-1 infected individuals reveal important information regarding how antibodies target conserved determinants of the envelope glycoprotein (Env) spike such as the primary receptor CD4 binding site (CD4bs). Many CD4bs-directed bNAbs use the same heavy (H) chain variable (V) gene segment, VH1-2*02, suggesting that activation of B cells expressing this allele is linked to the generation of this type of Ab. Here, we identify the rhesus macaque VH1.23 gene segment to be the closest macaque orthologue to the human VH1-2 gene segment, with 92% homology to VH1-2*02. Of the three amino acids in the VH1-2*02 gene segment that define a motif for VRC01-like antibodies (W50, N58, flanking the HCDR2 region, and R71), the two identified macaque VH1.23 alleles described here encode two. We demonstrate that immunization with soluble Env trimers induced CD4bs-specific VH1.23-using Abs with restricted neutralization breadth. Through alanine scanning and structural studies of one such monoclonal Ab (MAb), GE356, we demonstrate that all three HCDRs are involved in neutralization. This contrasts to the highly potent CD4bs-directed VRC01 class of bNAb, which bind Env predominantly through the HCDR2. Also unlike VRC01, GE356 was minimally modified by somatic hypermutation, its light (L) chain CDRs were of average lengths and it displayed a binding footprint proximal to the trimer axis. These results illustrate that the Env trimer immunogen used here activates B cells encoding a VH1-2 gene segment orthologue, but that the resulting Abs interact distinctly differently with the HIV-1 Env spike compared to VRC01.

Mice transgenic for human Ig loci are an invaluable resource for the production of human Abs. However, such mice often do not yield human mAbs as effectively as conventional mice yield mouse mAbs. Suboptimal efficacy in delivery of human Abs might reflect imperfect interaction between the human membrane IgH chains and the mouse cellular signaling machinery. To obviate this problem, in this study we generated a humanized rat strain (OmniRat) carrying a chimeric human/rat IgH locus (comprising 22 human V(H)s, all human D and J(H) segments in natural configuration linked to the rat C(H) locus) together with fully human IgL loci (12 Vκs linked to Jκ-Cκ and 16 Vλs linked to Jλ-Cλ). The endogenous Ig loci were silenced using designer zinc finger nucleases. Breeding to homozygosity resulted in a novel transgenic rat line exclusively producing chimeric Abs with human idiotypes. B cell recovery was indistinguishable from wild-type animals, and human V(D)J transcripts were highly diverse. Following immunization, the OmniRat strain performed as efficiently as did normal rats in yielding high-affinity serum IgG. mAbs, comprising fully human variable regions with subnanomolar Ag affinity and carrying extensive somatic mutations, are readily obtainable, similarly to conventional mAbs from normal rats.

Anti-amyloid immunotherapy has been proposed as an appropriate therapeutic approach for Alzheimer's disease (AD). Significant efforts have been made towards the generation and assessment of antibody-based reagents capable of preventing and clearing amyloid aggregates as well as preventing their synaptotoxic effects. In this study, we selected a novel set of human anti-amyloid-beta peptide 1-42 (Abeta1-42) recombinant monoclonal antibodies in a single chain fragment variable (scFv) and a single-domain (VH) format. We demonstrated that these antibody fragments recognize in a specific manner amyloid-beta deposits in APP/Tg mouse brains, inhibit toxicity of oligomeric Abeta1-42 in neuroblastoma cell cultures in a concentration-dependent manner and reduced amyloid deposits in APP/Tg2576 mice after intracranial administration. These antibody fragments recognize epitopes in the middle/C-terminus region of Abeta, which makes them strong therapeutic candidates due to the fact that most of the Abeta species found in the brains of AD patients display extensive N-terminus truncations/modifications., (Copyright 2010 Elsevier B.V. All rights reserved.)

Probable germline gene sequences from thousands of aligned mature Ab sequences are inferred using simple computational matching to known V(D)J genes. Comparison of the germline to mature sequences in a structural region-dependent fashion allows insights into the methods that nature uses to mature Abs during the somatic hypermutation process. Four factors determine the residue type mutation patterns: biases in the germline, accessibility from single base permutations, location of mutation hotspots, and functional pressures during selection. Germline repertoires at positions that commonly contact the Ag are biased with tyrosine, serine, and tryptophan. These residue types have a high tendency to be present in mutation hotspot motifs, and their abundance is decreased during maturation by a net conversion to other types. The heavy use of tyrosines on mature Ab interfaces is thus a reflection of the germline composition rather than being due to selection during maturation. Potentially stabilizing changes such as increased proline usage and a small number of double cysteine mutations capable of forming disulfide bonds are ascribed to somatic hypermutation. Histidine is the only residue type for which usage increases in each of the interface, core, and surface regions. The net overall effect is a conversion from residue types that could provide nonspecific initial binding into a diversity of types that improve affinity and stability. Average mutation probabilities are approximately 4% for core residues, approximately 5% for surface residues, and approximately 12% for residues in common Ag-contacting positions, excepting the those coded by the D gene.

A central paradigm in immunology states that successful generation of high affinity antibodies necessitates an immense primary repertoire of antigen-combining sites. Much of the diversity of this repertoire is provided by varying one antigen binding loop, created by inserting randomly a D (diversity) gene out of a small pool between the V and J genes. It is therefore assumed that any particular D-encoded region surrounded by different V and J regions adopts a different conformation. We have solved the structure of two lysozyme-specific variable domains of heavy-chain antibodies isolated from two strictly unrelated dromedaries. These antibodies recombined identical D gene sequences to different V and J precursors with significant variance in their V(D)J junctions. Despite these large differences, the D-encoded loop segments adopt remarkably identical architectures, thus directing the antibodies toward identical epitopes. Furthermore, a striking convergent maturation process occurred in the V region, adapting both binders for their sub-nanomolar affinity association with lysozyme. Hence, on a structural level, humoral immunity may rely more on well developed maturation and selection systems than on the acquisition of large primary repertoires.

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., (Copyright 2004 John Wiley & Sons, Ltd.)

N-Linked carbohydrates are frequently found in the V region of Ig H chains and can have a positive or negative effect on Ag binding affinity. We have studied a murine anti-alpha(1-->6) dextran V(H) that contains a carbohydrate in complementarity-determining region 2 (CDR2). This carbohydrate remains high mannose rather than being processed to a complex form, as would be expected for glycans on exposed protein loops. We have shown that the glycan remained high mannose when murine-human chimeric Abs were produced in a variety of cell types. Also, when another carbohydrate was present in CDR1, CDR2, or CDR3 of the L chain, the V(H) CDR2 glycan remained high mannose. Importantly, we found that when the anti-dextran V(H) CDR2 replaced CDR2 of an anti-dansyl V(H), the glycosylation site was used, but H chains were withheld in the endoplasmic reticulum and did not traffic to the Golgi apparatus. These results suggest that inappropriate V region glycosylation could contribute to ineffective Ab production from expressed Ig genes. In some cases, a carbohydrate addition sequence generated by either V region rearrangement or somatic hypermutation may result in an Ab that cannot be properly folded and secreted.

A spontaneous, autoreactive autoantibody called SN5-18 (IgG2b, kappa) binds to a complex of H2A/H2B/dsDNA in chromatin, but erroneously appears to bind dsDNA when the Ab is used in a form that is not highly purified. Because of this finding, we evaluated the antigenic specificity of a prototypic anti-dsDNA Ab, 3H9/Vkappa4, now used widely in transgenic studies of tolerance and autoimmunity. We found that the purified mAb 3H9/Vkappa4 binds chromatin and specifically a complex of H2A/H2B/dsDNA, but not dsDNA in solid phase or in solution. When used in the form of culture supernatant or as a standard protein G preparation, mAb 3H9/Vkappa4 appears to bind dsDNA, apparently due to nuclear proteins in the preparation that assemble on target DNA. Because of the reported role of V(H)CDR3 Arg residues in dsDNA binding and the near identity of the SN5-18 sequence to other dsDNA-specific Ab, we tested the contributions of two V(H)CDR3 Arg residues in SN5-18 to chromatin specificity. We found that both these Arg residues at positions 104 and 106 were required for detectable chromatin binding. These results indicate a role for V(H)CDR3 Arg residues in chromatin specificity of lupus-derived autoantibodies. Further, they provide an explanation for a possible discrepancy in the form of tolerance observed in different anti-DNA Ig transgene models.

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.

We previously described T15H chain mutants that were impaired in assembly with L chain and in ability to be secreted from the cell. The unmutated T15L chain is unusual in that it is secretion-impaired in the absence of assembly with H chain. The T15L chain preferentially pairs with T15H in vivo, suggesting that if we introduced mutations that would allow secretion of free T15L chain, they might also lead to the secretion of the complex with the defective H chain. We mutated four positions in the germline T15L that had amino acids infrequently found in other kappa-chains. Mutation to the most frequently occurring amino acid at three of the four positions allowed secretion of free L chain, while the combination of two secretion-restoring mutations was synergistic. Coexpression of secretion-restored mutant L chains with the secretion-defective mutant H chains rescued secretion of the assembled H(2)L(2) complex, suggesting that during somatic hypermutation in vivo, deleterious mutations at the H chain may be compensated by mutations on the L chain. To our knowledge, this is the first example of mutations in IgL chains that are able to restore secretion-defective H chains to secretion competence in mammalian cells.

The monoclonal IgM cold agglutinins that bind to the I/i carbohydrate Ags on the surface of RBCs all have Ig H chains encoded by the V4-34 gene segment. This mandatory use indicates that distinctive amino acid sequences may be involved in recognition. Critical amino acids exist in framework region 1 (FR1) of V4-34-encoded Ig, and these generate a specific Id determinant which apparently lies close to the I binding site. However, I binding by Id-expressing Ig can be modulated by sequences in complementarity-determining region (CDR)(H)3. Examination of the crystal structure of an anti-I cold agglutinin has revealed a hydrophobic patch in FR1 involving residue W7 on beta-strand A and the AVY motif (residues 23-25) on beta-strand B. In this study we used mutagenesis to show that each of the strand components of the hydrophobic patch is required for binding the I carbohydrate Ag. In addition, the crystal structure reveals that amino acids in the carboxyl-terminal region of CDR(H)3 form a surface region adjacent to the hydrophobic patch. We propose that the I carbohydrate Ag interacts simultaneously with the entire hydrophobic patch in FR1 and with the outside surface of CDR(H)3. This interaction could leave most of the conventional binding site available for binding other Ags.

To better understand V gene usage, specificity, and clonal origins of IgE Abs in allergic reactions, we have constructed a combinatorial Ab library from the mRNA of an adult patient with atopic dermatitis. Sequence analysis of random clones revealed that 33% of clones used the IGHV6-1 H chain V gene segment, the only member of the V(H)6 gene family. IGHV6-1 is rarely used in the expressed adult repertoire; however, it is associated with fetal derived Abs. Features of the V(H)6 rearrangements included short complementarity-determining region 3, frequent use of IGHD7-27 D gene, and little nucleotide addition at the D-J junction. There was also a low level of mutation compared with V(H)1, V(H)3, and V(H)4 rearrangements. The library was expressed as phage-Fab fusions, and specific phage selected by panning on the egg allergen ovomucoid. Upon expression as soluble IgE Fabs, 12 clones demonstrated binding to ovomucoid, skim milk, and BSA by ELISA. Nucleotide sequencing demonstrated that the IGHV6-1 V gene segment encoded each of the 12 multiply reactive IgE Fabs. A cyclic peptide was designed from the complementarity-determining region 3 of several of these clones. The cyclic peptide bound both self and nonself Ags, including ovomucoid, human IgG, tetanus toxoid, and human and bovine von Willebrand factor. These results suggest that some IgE Abs may bind more than one Ag, which would have important implications for understanding the multiple sensitivities seen in conditions such as atopic dermatitis.

During an immune response, somatic mutations are introduced into the VH and VL regions of Ig chains. The consequences of somatic mutation in highly conserved residues are poorly understood. Ile51 is present in 91% of murine VH complementarity-determining region 2 sequences, and we demonstrate that single Ile51-->Arg or Lys substitutions in the PCG1-1 Ab are sufficient to severely reduce Ig secretion (1-3% of wild-type (WT) levels). Mutant H chains, expressed in the presence of excess L chain, associate with Ig binding protein (BiP) and GRP94 and fail to form HL and H2L assembly intermediates efficiently. The mutations do not irreversibly alter the VH domain as the small amount of mutant H chain, which assembles with L chain as H2L2, is secreted. The secreted mutant Ab binds phosphocholine-protein with avidity identical with that of WT Ab, suggesting that the combining site adopts a WT conformation. A computer-generated model of the PCG1-1 variable region fragment of Ig (Fv) indicates that Ile51 is buried between complementarity-determining region 2 and framework 3 and does not directly contact the L chain. Thus, the Ile51-->Arg or Ile51-->Lys mutations impair association with the PCG1-1 L chain via indirect interactions. These interactions are in part dependent on the nature of the L chain as the PCG1-1 VH single Ile51-->Arg or Ile51-->Lys mutants were partially rescued when expressed with the J558L lambda1 L chain. These results represent the first demonstration that single somatic mutations in V(H) residues can impair Ig secretion and suggest one reason for the conservation of Ile51 in so many Ig VH.

The emerging class of B cell superantigens includes HIV-1 gp120, which binds to many members of the V(H)3 Ig gene family. The present study addresses the structural features of V(H)3 antibodies conferring gp120 binding activity using a panel of recombinant full-length and Fab Ig proteins. Binding activity was fully conferred by the Fab portion of the Ig molecule. The V(H) region was the major determinant of binding; diverse light chains were permissive for gp120 binding. A series of recombinant V(H)3-V(H)1 chimeric molecules was created to analyze the contribution of different subregions of V(H)3 to gp120 binding. Hypervariable loop 1 (H1) substitution alone caused a 10-fold reduction in binding activity. The framework subregions (FR1, FR2 and FR3) and H2 also influenced binding, since substitutions of various combinations of these subregions conferred 10- to 100-fold binding reductions. We conclude that gp120 binding occurs through a non-conventional interaction involving multiple discontinuously arrayed residues spanning the V(H), and including roles in gp120 contact and favorable conformation of the V(H).

mAb Z22 is a highly selective IgG anti-Z-DNA Ab from an immunized C57BL/6 mouse. Previous studies showed that heavy chain CDR3 amino acids are critical for Z-DNA binding by the single chain variable fragment (scFv) comprising both V region heavy chain (VH) and V region light chain (VL) of mAb Z22 and that the VH domain alone binds Z-DNA with an affinity similar to that of whole variable fragment (Fv). To determine whether Z-DNA binding by VH alone and by Fv involves identical complementarity determining region residues, we tested effects of single or multiple amino acid substitutions in recombinant VH, scFv, and associated VH-VL heterodimers. Each recombinant product was a fusion protein with a B domain of Staphylococcal protein A (SPA). Z22VH-SPA alone was not highly selective; it bound strongly to other polynucleotides, particularly polypyrimidines, and ssDNA as well as to Z-DNA. In contrast, scFv-SPA or associated VH-VL dimers bound only to Z-DNA. VL-SPA domains bound weakly to Z-DNA; SPA alone did not bind. Introduction of multiple substitutions revealed that the third complementarity determining region of the heavy chain (CDR3H) was critical for both VH and scFv binding to Z-DNA. However, single substitutions that eliminated or markedly reduced Z-DNA binding by scFv instead caused a modest increase or no reduction in binding by VH alone. Association of VH-SPA with Z22VL-SPA restored both the effects of single substitutions and Z-DNA selectivity seen with Fv and intact Ab. Polypyrimidine and ssDNA binding by the isolated VH domain of immunization-induced anti-Z-DNA Ab resembles the activity of natural autoantibodies and suggests that VH-dependent binding to a ligand mimicked by polypyrimidines may play a role in B cell selection before immunization with Z-DNA.

The mouse monoclonal antibody (mAb) D12 specifically binds in the variable region (idiotype) of human V(H)3 encoded antibodies. We used mutational analysis to determine the subregions of a V(H)3 encoded antibody which effect the interaction with mAb D12. Recombinant antibodies composed of mutant heavy chains were produced using the baculovirus expression system. The results of this topographical study indicate that the combined conformations of FRI, CDR2 and FR3 are critical for mAb D12 binding. MAb D12 binding was not effected either by the heavy chain CDR3 sequence nor by the light chain. We previously demonstrated that structures within the same three subregions are required for the B cell superantigen Staphylococcal protein A (SPA) binding to V(H)3 encoded antibodies. Thus, some anti-idiotypic antibodies can interact with antibodies in a similar fashion to superantigens.

Size-diversity of Ig and T cell receptor antigen binding (CDR3) regions can be visualized by "CDR3 fingerprinting", and provides an estimate of B- or T-cell repertoire complexity. The method does not identify clonal diversity, however, which can only be determined by random sequencing of the CDR3s. In this study we demonstrate that a combination of fingerprinting and single strand conformation polymorphism (SSCP) analysis can be used for a rapid estimation of clonal diversity within mouse Ig antigen binding regions selected for size. This application may be useful in the analysis of clonal expansion within B- and T-cell repertoires.

The extent to which somatic mutation impairs the Ig complementarity-determining region (CDR) and framework region (FRW) structure/function is not clear. Previously, we found that the VH CDR2 of the murine T15 Ab is highly sensitive to mutation; 56% (26 of 46) of Abs mutated in vitro had reduced or no Ag binding capability, and 9% were secretion impaired. Here we test whether the T15 VH CDR2 structure is unique by mutating the VH CDR2 of the anti-PC-protein murine Ab, PCG1-1. PCG1-1 VH is encoded by the M141 gene and is unrelated in sequence or structure to that of T15 VH1. The majority (54%, 20 of 37) of PCG1-1 mutants carrying one to five mutations in VH CDR2 had reduced or abolished Ag binding, while 10% were secretion impaired. Taken together, mutational analysis of the VH1 and VH M141 genes demonstrates that impaired binding and secretion may be common outcomes of CDR2 somatic mutation. We also tested the tolerance of the VH FRW2 of T15 to mutation, expecting this sequence-conserved region to be highly sensitive to alterations. However, FRW2 accommodated many nonconservative changes, and only 12% (3 of 25) of secreted mutants had impaired Ag binding. Moreover, mutations in FRW2 caused secretion defects in 24% (8 of 33), a frequency twice that of VH CDR2 mutants. A total of 16 unique secretion mutants have now been identified. These findings suggest that B cell losses from somatic mutation may be extensive and due to varied causes not all related to Ag binding.

Staphylococcal protein A (SPA) is a B-cell superantigen which binds specifically to the variable region of human VH3 encoded antibodies. We undertook to identify the VH3 regions involved in the interaction with SPA by producing mutant antibodies in the baculovirus expression system. We had previously shown that a single amino acid change at position 57 in the CDR2 of a human SPA nonbinding VH3 encoded rheumatoid factor converted it to an SPA binder, implicating CDR2 in SPA binding. When regions of the mutated binder were exchanged with those from a mouse nonbinding antibody, the pattern of SPA binding indicated that residues in FR1, CDR2 and FR3 are involved in the interaction between VH3 encoded antibodies and SPA. In addition, all three regions are simultaneously required for SPA binding to occur. When any one of the three regions was altered, SPA binding was severely disrupted.

An important aspect of the study of antibody structure-function relationships involves analysis of natural or synthetic mutations of antigen-combining sites. The anti-hen egg lysozyme monoclonal antibody HyHEL-10 has been a focus for antibody structure-function studies. We have displayed on bacteriophage of a hybrid single chain Fv, containing the light chain variable region of HyHEL-10 and the heavy chain variable region of a structurally related but functionally distinct antibody, AS32. By using a combination of site-directed mutagenesis, complementary determining region grafting and molecular modeling, we have identified a number of contact and non-contact residues that are important in the affinity of HyHEL-10 for lysozyme. In particular, the heavy chain variable region framework residue at position 94 was shown to be an important determinant of high-affinity binding. The phage display approach eliminates the need for purification of antibodies and, when used in combination with polymerase chain reaction for variable region sequence mutagenesis, facilitates the rapid generation and characterization of mutant antibodies.

Mutations in the heavy chain complementarity determining region 2 (CDR2) of the phosphocholine-specific T15 Ab can have a dramatic effect on the ability of the Ab to bind Ag. A panel of multisite mutants that had lost detectable binding to phosphocholine-containing Ags was previously created by saturation mutagenesis of the CDR2 region of T15. Based on the predicted importance of amino acid changes represented in the multisite mutants, we have created single-site mutations, yielding a panel of Abs with which to test 17 of the 19 CDR2 residues. Of the 17 positions examined, only one, Arg52, is intolerant to change, yielding a nonbinder phenotype even with conservative amino acid replacement. Mutation at two other sites, Ala50 and Tyr55, can yield a nonbinder phenotype depending on the amino acid replacement. Single-site mutations of the remaining 14 positions allowed retention of binding ability. Thus, except for positions 50, 52, and 55, multiple mutations must be introduced into the CDR2 region to create a nonbinder phenotype. We provide a newly refined model of T15, illustrating the structure and the interactions of the CDR2 region. Our results imply that introduction of point mutations would not normally delete Ag-binding ability until two or more mutations had accumulated. This would minimize potentially harmful effects of somatic mutation on Ig V region genes and improve the chance of survival for an Ab such as T15, which in its unmutated form is already well suited to bind Ag.

Targeted disruption of the p50 subunit of NF-kappa B resulted in isotype class switch defects resembling those observed in mice in which the downstream IgH enhancer 3'alpha E(hs1,2) was deleted. We postulated that kappa B binding proteins may regulate class switching by interacting with 3'alpha E(hs1,2) or with other IgH 3' enhancers with which 3'alpha E(hs1,2) synergizes. kappa B binding sites were identified in 3'alpha E(hs1,2) and 3' alpha-hs4, the distal 3' IgH enhancer. A kappa B binding site within 3'alpha E(hs1,2) contributes to at least half the activity of the enhancer in plasma cells, while the same kappa B binding site participates in the complex repression of the enhancer in B cells. In the case of 3'alpha-hs4, a kappa B binding complex activates the enhancer in pre-B, B cells and plasma cells. Additional binding sites within 3'alpha-hs4 for factors known to regulate 3'alpha E(hs1,2), including Oct-1 and BSAP, were identified, and their contribution to 3'alpha-hs4 regulation during B cell development was assessed. Oct-1 positively regulates the enhancer in pre-B and B cells, while BSAP is a repressor in pre-B cells and an activator at the B cell stage. These studies identify kappa B binding proteins as key modulators of 3'alpha E(hs1,2) and 3'alpha-hs4, and suggest coregulation of the two enhancers by a common set of factors.

Essentially all cold agglutinins (CA) with red blood cell I/i specificity isolated from patients with CA disease stemming from lymphoproliferative disorders utilize the VH 4-34 (VH 4-21) gene segment. This near universality of the restricted use of a single gene segment is substantially greater than that demonstrated for other autoantibodies. The monoclonal antibody 9G4 exclusively binds VH 4-34 encoded antibodies and serves as a marker for the VH 4-34 gene segment. Previous studies form our laboratory localized the 9G4 reactive area to framework region 1 (FR1). In the present study, the relative roles of VH FR1, heavy (H) chain complementarity determining region 3 (CDRH 3) and the light (L) chain in I antigen binding were investigated. Mutants containing FR1 sequences from the other VH families, CDRH 3 exchanges, and combinatorial antibodies involving L chain interchanges were produced in the baculovirus system and tested in an I binding assay. The data indicate that FR1 of the VH 4-34 gene segment and the CDRH 3 are essential for the interaction between CA and the I antigen, with the CDRH 3 being fundamental in determining the fine specificity of antigen binding (I versus i). Mutants with substantially altered CDRH 1 and CDRH 2 regions bind I as long as the FR1 is VH 4-34 encoded and the CDRH 3 has a permissive sequence. Light chain swaps indicate that even though antigen binding is predominantly mediated by the H chain, the association with antigen can be abrogated by an incompatible L chain. The necessity for VH 4-34 FR1 explains the almost exclusive use of the VH 4-34 gene segment in cold agglutinins. We hypothesize that, as a general phenomenon, the H chain FR1 of many antibodies may be important in providing the contact required for the close association of antibody with antigen, while the CDRH 3 dictates the fine specificity and strenght of binding.

Antibodies can be made from repertoires of associated heavy and light chains displayed on the surface of bacteriophage, and are readily diversified by random point mutation or by chain shuffling. To make extensive variations around the "core" antigen binding contacts of a crystallographically solved mouse antibody NQ10/12.5 (gamma l, kappa), the NQ10 light chain was assembled in vitro with a repertoire of about 10(7) human heavy chains displayed on the surface of phage, and selected by binding to hapten. An antibody with a much improved affinity was isolated from the repertoire (K(a) = 10(9) M-1 compared with 10(8) M-1 for NQ10). The sequence of the human heavy chain (VH-IL) was highly related to NQ10. It conserved the same folds for the H1, H2 and H3 loops, six of the seven contact residues for hapten, and also a phOx binding motif (Asp-X-Gly-X-X) in the H3 loop. It appears that the new heavy chain partners for the NQ10 light chain often retain many critical antigen binding features found in the NQ10 heavy chain.

The carbohydrate-binding site in Fab fragments of an antibody specific for Salmonella serogroup B O-polysaccharide has been probed by site-directed mutagenesis using an Escherichia coli expression system. Of the six hypervariable loops, the CDR3 of the heavy chain was selected for exhaustive study because of its significant contribution to binding-site topography. A total of 90 mutants were produced and screened by an affinity electrophoresis/Western blotting method. Those of particular interest were further characterized by enzyme immunoassay, and on this basis seven of the mutant Fabs were selected for thermodynamic characterization by titration microcalorimetry. With regard to residues that hydrogen bond to ligand through backbone interactions, Gly102H could not be substituted, while several side chains could be introduced at Gly100H and Tyr103H with relatively little effect on antigen binding. There was, however, a preference for nonpolar side chains at position 103H. Substitution of His101H with carboxylate and amide side chains gave mutants with binding affinities approaching that of the wild type; complete side-chain removal by mutation to Gly was tolerated with a 10-fold reduction in binding constant. Analysis of binding by titration microcalorimetry revealed some dramatic thermodynamic changes hidden by the similarity of the binding constants. Similar effects were observed with residue changes in an Arg-Asp salt-bridge at the base of the loop. These results indicate that alterations to higher affinity anti-carbohydrate antibodies are characterized by an enthalpy-entropy compensation factor which allows for fundamental changes in the nature of the binding interactions but impedes engineering for increases in affinity.

We tested the value of a new library mutagenesis approach, called library enzymatic inverse PCR (LEIPCR), for expression-level enhancement of antibody Fv fragments produced in Escherichia coli. The production level of active, metal chelate-specific antibody from our constructs is limited by a low expression level of the second, heavy-chain cistron. To increase the production level, LEIPCR was applied to the wobble bases of the second cistron leader peptide. In LEIPCR mutagenesis, the entire plasmid is amplified using mutagenic primers with class-IIS restriction endonuclease (ENase) sites at their 5' ends. The PCR product is digested with the class-IIS ENase (here, BsaI; GGTCTCN[symbol: see text]NNNN[symbol: see text]), which removes its own recognition sequence, and the ends are self-ligated. Thus, LEIPCR can be used to make plasmid mutant libraries regardless of the nucleotide sequence, and independent of available ENase sites. The resulting library of 10(7) wobble mutants was screened for active Fv by a colony filter lift. A selected mutant was shown to produce between four- and elevenfold more active Fv than the wild type (wt), and fivefold more heavy chain. Mutations outside of the leader peptide were shown not to be involved. The mutated areas of the mRNAs of two different up-mutants may have less secondary structure than the wt. Thus, the sequence of the mRNA of the second leader peptide was limiting to the expression level of heavy-chain and active Fv.

A bifunctional molecule was genetically engineered which contained the secretory signal and four Fc-binding domains of Staphylococcus aureus protein A (FcA), fused to a single-chain Fv (scFv) derived from an immunoglobulin (Ig) G1 mouse monoclonal antibody (AS32) directed against the plant regulatory photoreceptor protein, phytochrome. The FcA::AS32scFv sequence was encoded in a single synthetic gene and expressed as a 60-kDa periplasmic protein in Escherichia coli. The bifunctionality of the fusion protein was established by its ability to bind to both IgG-agarose and phytochrome-sepharose. Growth of cultures, producing the FcA::AS32scFv, at 37 degrees C, resulted in a decrease in the periplasmic accumulation of the fusion protein, and an increased accumulation of an assumed degradation product which retained Fc-binding activity. Growth of cultures at lower temperatures favoured the accumulation of undegraded fusion protein. The recombinant fusion protein could be purified to homogeneity by a simple, rapid chromatography procedure.

A number of experiments on the shuffling of heavy and light chains from antibodies of defined specificity for the transition-state analogue hapten nitrophenyl phosphonamidate are described. The experiments report on the promiscuity of heavy and light chains in binding antigen and the feasibility of antibody redesign by this shuffling process. The concepts of incestuous and extraclonal promiscuous association are described. Shuffling opens the possibility of generating panels of antibodies with related specificity but of distinct idiotypic composition that may have significance in the use of human monoclonal antibodies in therapy.

In the course of studying the members of the T15 group of VH gene segments, some of which participate in the immune response to phosphorylcholine in the mouse, we identified a VH gene segment that contains three mutations preventing its expression. The mutations are an in-frame stop codon, a 4-base insertion which causes a termination codon to be shifted into the reading frame, and a modification of the recognition elements involved in the joining of VH and D gene segments during variable region formation. This pseudogene, which is 88-96% homologous to the other members of the T15 VH gene group, is probably of relatively recent origin and will presumably be deleted from the VH gene family eventually. We suggest that pseudogenes can only arise in multigene families and that the occurrence of pseudogenes will be a relatively frequent phenomenon in these families. Because the antibody gene families are made up of multiple gene elements, undergo two types of DNA rearrangements during differentiation, and employ several different RNA splicing mechanisms for expression, there are many different ways a particular antibody gene segment may become a pseudogene.

The organization and evolution of immunoglobulin variable region genes was studied by comparing human and mouse heavy chain variable region (VH) genes. We show that a VH gene subgroup constitutes a physically linked multigene family separated from another VH subgroup. We mapped the VHIII gene subgroup to be 3' to the VHII gene subgroup based on deletion of VH genes after V-D-J rearrangement. The results indicate that the human VHIII gene subgroup underwent a significant gene expansion as compared to the mouse VHIII subgroup. Amino acid sequence data indicate that human VHIII genes correspond to only a small subset of mouse VHIII genes. Human VHIII genes contain a shorter intron and are two codons shorter than most BALB/c mouse VHIII genes. The nature of nucleotide substitutions between VH genes within a species (human) is similar to that between genes of different species (human/mouse). Both contain approximately 50% silent substitutions.

Immunoglobulin variable (V) gene regions typify extensive multigenic families in terms of overall size, chromosomal arrangement and presence of large numbers of apparent pseudogenes. A unique mechanism of somatic reorganization involving recombination of VH, D and JH or VL and JL segments accompanies the differentiation of lymphoid cells and together with somatic mutation and other types of recombination accounts for V-region diversity. Although these processes have been well characterized in higher mammals, little is known concerning their origin and diversification during phylogenetic time. Previously, we described the blot-hybridization characteristics of murine VHIII probes with restriction enzyme-digested genomic DNA isolated from several phylogenetically critical species, including Caiman crocodylus, a modern representative of an ancient reptilian subclass. Here we have used a murine probe, S107V, to select homologous clones from a library of Caiman genomic DNA constructed in a lambda bacteriophage. The complete nucleotide sequence of a Caiman gene homologous to the murine VH gene and its adjacent 5' and 3' region is described. Comparison of the sequence with mammalian prototypes shows evidence of considerable organizational and structural homology extending outside the presumed VH-coding region and including elements believed to be involved in somatic recombination. Inferences about the evolution of this multigenic family can now be extended to the level of phylogenetic class.

Mouse immunoglobulin heavy-chain variable region (Ig VH) genes apparently arose from the approximately 600-base-pair-long (approximately 12 tandem repeats of the 48-base-pair-long primordial building block sequence TTC-AGC-AGC-CTG-ACT-GGA-TAT-GAC-CTG-GAG-TGG-ACT-TAC-TGC-GCA-AGA) that in the original reading frame specified the amino acid sequence Phe-Ser-Ser-Leu-Thr-Gly-Tyr-Asp-Leu-Glu-Trp-Thr-Tyr-Cys-Ala-Arg. The previously identified, shorter prototype building blocks merely represented particular portions of the above primordial sequence. Even today, the direct descendant in toto of this primordial sequence specifies the last one-sixth of each VH coding sequence: the 83rd to 98th amino acid residues. Furthermore, its four truncated derivatives specify the 4th to 14th, 17th to 23rd, 29th to 37th, and 38th to 48th amino acid residues. Accordingly, all three relatively invariant--therefore, conserved--framework regions (FW-1, FW-2, and FW-3) of VHs are specified by recognizable--therefore, conserved--descendants of the primordial sequence.

The multiplicity of heavy-chain variable-region (VH) genes in mouse and human DNA has been estimated using a mouse heavy-(H) chain cDNA clone. We found about 10 hybridization components in mouse DNA and about 20 components in human DNA. Cross-hybridization studies of variable region (V) genes indicate that these components represent the numbers of genes within the VH subgroups of each of these species. The arrangement and rearrangement of the H-chain gamma subclasses have been studied in order to assess possible mechanisms of the H-chain switch. Evidence has been found for rearrangement events involving the gamma 2a and gamma 2b constant-region (CH) genes in DNA from cells making IgG2a and IgG2b respectively. In addition we found that cells making IgG2a lack detectable genes for gamma1 and gamma 2b. Both sets of observations are discussed in relation to H-chain diversity and the switch.

Several genes for the variable region of immunoglobulin heavy chains (VH genes) have been isolated from human fetal liver DNA by using a cDNA plasmid probe containing a mouse VH sequence. The detectable VH genes are separated by 12-16 kilobases of DNA, and hybridization experiments show about 23 hybridizing VH genes in DNA of three different individuals. The complete nucleotide sequence of one of these human VH genes shows that it belongs to the human VHIII subgroup. The VH gene appears to contain an intervening sequence (104 bases in length) within a precursor sequence, between residues -4 and -5. The precursor sequence is itself 19 codons in length. The 3' end of the V gene seems to be at codon 93 or 94, and this is followed by the conserved sequences C-A-C-A-G-T-G and G-A-C-A-C-A-A-A-C-C. The presence of these sequences suggests that similar enzymatic mechanisms are involved in the integration of V genes in both heavy and light chains.

The genetic contro of the expression of an idiotype (Id-460) associated with the 2,4-dinitrophenyl (DNP)-binding BALB/c myeloma protein MOPC 460 was studied using congenic strains of mice. It was shown that the expression of high levels of Id-460 during secondary in vivo anti-DNP-ovalbumin responses was determined by genes governing immunoglobulin heavy-chain variable and kappa-light chain variable regions (V kappa). Appropriate alleles at both loci were required for the expression of Id-460. Genes in the major histocompatability complex and the X-linked immune deficiency gene found in strain CBA/N did not greatly affect Id-460 expression. The V kappa gene controlling Id-460 expression can be differentiated from Lyt-3, and it is the first instance in which expression of an idiotype subdivides the V kappa genes associated with the Lyt-3a allele. Although it is likely that the V kappa gene(s) involved are structural, the involvememt of a regulatory gene linked to the structural gene can not be excluded.

The cloned murine B-cell lymphoma line (BCL1) that expresses surface IgM and IgD is considered to be a model for the immunoglobulin gene expression of the mature virgin B cell. Of particular interest is the mechanism by which a single VH gene is shared by two CH genes. We examined the organization of the immunoglobulin heavy chain genes in BCL1 DNA. A single arrangement of CH genes was found with the expressed VHDJH gene complex just 5' to the Cmu gene. The complete DNA sequence of the VH gene was determined. No rearrangement occurred in the intervening DNA between the JH and C mu genes or between the C mu and C delta genes. We conclude that dual expression of mu and delta heavy chains using a single VH gene is accomplished by alternate processing of a primary transcript that encompasses the the VHDJH complex and both CH genes.

The sequences of four embryonic mouse immunoglobulin VH genes have been compared. All genes end at codon 98 and code for a hydrophobic signal peptide of 19 residues interrupted at codon -4 by an intron of 83 base pairs. Substitutions occur in all gene segments but at a significantly higher frequency in the hypervariable regions. The data suggest an evolutionary basis for the diversity of immunoglobulin genes. Divergence resulted also in a termination codon in two of the genes, suggesting that part of the V gene repertoire cannot be expressed unless some correction mechanism is available.

We have constructed a library of Balb/c mouse embryo DNA in the vector Charon 4A. The library was searched for sequences homologous to the VH region of a cloned cDNA of the UPC10 heavy chain mRNA. In this paper, we describe the structure and the partial nucleotide sequence of one of such clones (VH441). The nucleotide sequence of this germ-line gene indicates that it encodes amino-acids 1-98 of the X44 and J601 galactan-binding VH regions, but that it differs from the UPC10 VH segment by four single base changes. The VH gene appears to contain a 101 bases long intervening sequence within a precursor sequence identical to the precursor sequence of UPC10. The 3' non coding sequence of the V gene contains the two conserved sequences found in embryonic V DNA segments, CACAGTG and ACATGAACC, separated by 23 nucleotides and a sequence CACTGTG separated by 33 nucleotides from the first heptamer.

The polymorphic nature of the immunoglobulin VH genes was investigated by Southern blot analysis of liver DNA of sixteen different mouse strains and hybridization with VH probes. Differences in restriction enzyme pattern (REP) were observed and six different patterns of restriction fragments were found for the sixteen strains analyzed. No equivalent polymorphism was observed in another multigene family, the actins. The six patterns correlate with immunoglobin constant region allotypes (Igh-1). Experiments with Igh-1-congenic strains suggest that the VH REP is linked to immunoglobulin constant region haplotype. Mouse strains which share inherited idiotypes also share identical VH restriction pattern. This provides a structural basis for the genetic linkage between idiotypes and allotypes. It also indicates that different strains carry different VH gene repertoires, which may be the basis for the expression of different inherited idiotypes in various strains. We propose that a VH group in a set of linked genes that are coinherited as a cluster with the constant region genes and that VH and Ch can be regarded as an extended haplotype.

A putative diversity segment of immunoglobulin heavy-chain genes (D segments) has been identified 700 base pairs 5' to JH1 DNA on the germ-line genome of the mouse. This 10-base pair D segment is flanked by two sets of sequences related to (SEE FORMULAR IN TEXT) which are possible recognition sites for a recombinase. The spacer separating the heptamer and the nonamer is 12 base pairs long on both sides of the D segment. As the space separating the two signal sequences in VH DNAs and JH DNAs is 23 +/- 1 base pairs long, the two recombinations required for creation of a complete immunoglobulin VH gene, a VH--D joining and a D--JH joining, follow a 12/23-base pair spacer rule. Allelic exclusion is discussed with respect to D segments.