Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. III. A single VKII gene and one of several JK genes are joined by an invariant arginine to form the most common L chain V region

To characterize the L chain V region repertoire of IgG anti-Haemophilus influenzae type b capsular polysaccharide (Hib-PS) antibodies, clonal antibodies were purified from immune serum and internal amino acid sequences of VKII anti-Hib-PS L chains obtained. We examined VKII L chains because it is the most common VL family expressed in the anti-Hib-PS response. Comparison of VKII amino acid sequences, including the entire CDR2 and CDR3 regions, of five anti-Hib-PS clonal antibodies from four unrelated individuals revealed complete identity with the exception of a single CDR3 residue from one antibody. When the sequence of these antibodies was compared with known VKII genes and myeloma proteins, it was found to be identical to the human VKII gene, A2, whose genomic sequence is presented here. In addition, all five of the VKII anti-Hib-PS antibodies examined contain an arginine inserted at the V-J junction. Finally, in contrast to the extraordinary homology of the VKII-encoded residues, there is variability in the JK gene utilization by these antibodies. These results demonstrate that the most common L chain V region in IgG anti-Hib-PS antibodies is the product of a single germ-line gene. The invariant arginine insertion suggests that this residue has an important role in Ag binding.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type B polysaccharide. Demonstration of three types of V regions and their association with H and L chain isotypes

The antibody response to Haemophilus influenzae type b polysaccharide (Hib-PS) is pauciclonal but can vary between different individuals. To estimate the size of this antibody repertoire we examined the constant and V regions of human IgG anti-Hib antibodies from 14 individuals at the clonal level using various serologic and IEF methods. Examination of H chains showed that 11 of 14 individuals produced both IgG1 and IgG2 antibodies, two individuals produced only IgG2 and one individual produced only IgG1 antibody. All 14 individuals produced kappa-containing antibody clones and three persons also produced significant lambda-containing antibody clones. V region heterogeneity was examined by comparing cross-reactivity of anti-Hib-PS antibodies to the capsular polysaccharide of Escherichia coli K100 carbohydrate (K100 CHO). These studies showed that clones of IgG anti-Hib-PS antibodies cross-reactive with K100 CHO were present in 5 of 14 (36%) individuals and also revealed at least three types of V regions among these antibodies. The first type has no cross-reaction with K100 CHO and was found in 13 of the 14 individuals. The second type, found in three of 14 individuals, cross-reacts with K100 CHO and uses a lambda L chain V region. The third type, found in 2 of 14 individuals, cross-reacts with K100 CHO and uses a kappa L chain V region. Although the lambda type V region was found only in association with IgG2, the other two V region types associate with both IgG1 and IgG2. Thus, five IgG antibody clones are serologically discernable. An individual generally responds to Hib-PS by expressing several clones selected from these discernable antibody clones. Indeed, we can observe six individual response patterns among these 14 individuals and conclude that considerable variability in individual responses to Hib-PS can be achieved with very few V regions.     

Human immunoglobulin kappa light chain genes of subgroups II and III.

The first complete sequences of functionally rearranged VK genes (abbreviations ref. 1) of subgroups II and III are reported. The genes have been cloned from lymphoid cell lines synthesizing KII or KIII light chains as evidenced from immunochemical analyses with anti-VK subgroup-specific antisera. These data, together with the sequence of a KIV gene (described in the accompanying paper) and those of previously published KI genes make possible a comparison of genes representative of the four known V region subgroups of human K light chains. The VKII gene is distinguished from the VKI, VKIII, and VKIV genes by a much longer intron within the leader sequence: 426 bp vs ca. 120-220 bp. Blot hybridization experiments with human DNA digests using probes from the KII and KIII genes and from the respective upstream regions help to define subgroup specific probes and hybridization conditions.     

VH and VL gene usage by murine IgG antibodies that bind autologous insulin

To assess the recognition structures of antibodies that bind a self-Ag, we used mRNA analysis to identify the V region genes of IgG antibodies that bind autologous insulin. Four anti-insulin mAb from primary immunization of BALB/c mice use different combinations of H and L chain V region genes. Two VH genes are from the V-gam 3-2 and V-gam 3-8 families that are infrequently expressed in adult BALB/c mice, and two VH genes are members of the J558 family. Each anti-insulin antibody uses a different Vk gene family. Two antibodies express common Vk genes (Ox1 and Vk21C), whereas two other Vk genes are unusual in BALB/c mice. One Vk gene may represent a BALB/c equivalent of the VkOx2 subfamily and another is identical to a Vk used by anti-idiotypic antibodies from C57Bl/6 mice. When compared with known germ-line counterparts, all of the Vk sequences are close to germ-line configuration. In contrast, the germ-line counterparts for the anti-insulin VH genes are not known, however, they differ only in five to seven predicted amino acids from VH of other expressed antibodies. One antibody (mAb 123) differs in one amino acid in complementarity-determining regions 1 and 2 from the VH of the murine tumor BCL1, and another (mAb 126) employs an unmutated DFL16.1 germ-line D segment. These data suggest that antibodies binding autologous insulin use V gene components that are not extensively mutated, even when derived by immunization with heterologous insulin.     

Recognition of DNA by VH and Fv domains of an IgG anti-poly(dC) antibody with a singly mutated VH domain

Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. IV. The less frequently expressed VL are heterogeneous

We previously demonstrated that the human anti-Haemophilus influenzae type b polysaccharide (Hib-PS) VL repertoire is dominated by a product of the V kappa II gene, A2, and that V kappa II-A2 anti-Hib-PS antibodies have little or no somatic mutation in VL. To further study this VL repertoire, we studied non-A2 anti-Hib-PS antibodies that were identified either serologically or by amino-terminal amino acid sequence analysis. Of 15 non-A2 anti-Hib-PS antibodies from 12 vaccinated adults, we found four V lambda, five V kappa I, one non-A2 V kappa II, four V kappa III, and one V kappa IV antibodies. As expected, all but two of these subjects also produced V kappa II-A2 antibodies. Interestingly, one of these subjects lacks the A2 gene in the germ line. However, both subjects who did not produce detectable V kappa II antibody did produce normal amounts of total anti-Hib-PS antibody after vaccination. Candidate V kappa genes for the non-A2 antibodies were identified by comparison of up to 60 VL amino acid residues, including CDR1 and CDR2, with all sequenced V kappa genes. V kappa I antibodies appear to be products of three newly sequenced V kappa I genes, O8, O18, and L11, that are reported here. The O8 and O18 genes encode identical amino acid sequences. The non-A2 V kappa II antibody is a likely product of the A1 or A17 genes, the V kappa III antibodies are likely products of the A27 gene, and the V kappa IV antibody is a product of the single V kappa IV gene, B3. Unlike V kappa II-A2 antibodies, the V kappa I, V kappa III, and V kappa IV antibodies differed by one to five CDR residues from the germ line product of the candidate genes, suggesting the presence of somatic mutations. Thus, anti-Hib-PS antibodies can be divided into two types, the most frequently observed A2 antibodies with little or no somatic mutation and non-A2 antibodies that likely contain somatic mutations.     

Sequences of the VH and VL regions of murine monoclonal antibodies against 3-fucosyllactosamine

Many mAb that bind the carbohydrate antigenic determinant 3-fucosyl-lactosamine (3-FL), Gal beta 1-4[Fuc alpha-3]GlcNAc-R have been raised in BALB/c mice, and we are studying the structure and regulation of these antibodies. In this report, we present the first information about their amino acid sequences and the Ig gene segments used to encode them. V regions of the H and L chains of three anti-3-FL antibodies, PMN6, PMN29, and PM81, were sequenced by a combination of mRNA and amino acid sequencing. The L chain sequences of PMN6 and PM81 antibodies indicate that their VK and JK regions are encoded by VK24B and JK1 germ-line genes, respectively. The nucleotide and amino acid sequences of the H chains suggest that the three anti-3-FL antibodies are encoded by the VH441 gene segment of the X24 VH family, and this conclusion was supported by Southern filter hybridization with VH441 and JH3-JH4 probes. PMN29 has at least 11 amino acid substitutions, which is an unusually large amount of somatic mutation for an IgM antibody. Previous analyses of BALB/c genomic libraries with VHX24 and VH441 probes make it unlikely that this VH family contains additional germ-line genes, but this possibility cannot be excluded. All three antibodies use the DQ52 and JH4 gene segments. The single VH and VL gene segments used to encode the anti-3-FL antibodies is in contrast to the multiple VH and VL segments used by antibodies against other carbohydrate Ag such as alpha 1-6 dextran and group A streptococcal carbohydrate. VH441 also encodes the VH regions of antibodies against galactan and levan (beta 2-6 fructosan). The similarities among VH segments of antibodies against 3-FL, levan, and galactan, and the striking differences in their CDR3 sequences, suggest that CDR3 plays an important role in the formation of the Ag binding site. The use of a single VH segment from the smallest VH gene family by antibodies against at least three different carbohydrate determinants is noteworthy. It raises the possibility that the amino acid sequence encoded by VH441 has some general structural features that make it particularly well adapted for binding to carbohydrate sequences.     

Probing the human antibody repertoire to exogenous antigens. Characterization of the H and L chain V region gene segments from anti-hepatitis B virus antibodies.

Structural studies of human antibody V regions have been largely limited to those involving the fetal repertoire, autoantibodies, and malignant cell rearrangements, leaving the "normal" repertoire relatively unexplored. In this study we describe the nucleotide sequences of the H and L chain V regions of four antibodies specific for the surface Ag of the hepatitis B virus. Monoclonal cell lines were derived from healthy individuals who received standard immunizations with the serum-derived or recombinant hepatitis B virus vaccines by fusion of PBL to a heterohybridoma cell line, SPAZ-4. We utilized the polymerase chain reaction to amplify the H and L chain V regions for cloning and sequencing. The four antibodies express the following V region combinations: VHIII/V lambda V, VHIII/V kappa II, VHIV/V kappa I, VHV/V lambda III. When compared to germline genes with the closest sequence homology, all of the V regions appear to have undergone somatic mutation, ranging from 3.4 to 11.3% for the H chain, and 5.1 to 9.2% for the L chain. Analysis of the mutations shows them to be typical for an Ag-driven immune response.     

The immune response toward beta-adrenergic ligands and their receptors. VIII. Extensive diversity of VH and VL genes encoding anti-alprenolol antibodies

The V region genes (VH and VL) used in the immune response of BALB/c mice to alprenolol, a synthetic beta-adrenergic ligand, were examined by Southern blot and nucleotide sequence analyses. Fourteen anti-alprenolol hybridomas utilize 10 different combinations of six Vk, one V lambda, eight VH, three JK, one J lambda, and three JH genes. In addition to the combinatorial association, somatic mutations and junctional variation of assembled genes further contribute to diversity of the anti-alprenolol response. Although differing both in length and structure, the five H-chain third complementarity-determining region analyzed contain several acidic residues. Neither V gene utilization, nor H-chain third complementarity-determining-region structure can be simply correlated with affinity of the antibodies for the ligand. The anti-alprenolol V genes were compared with the corresponding sequences of unrelated antibodies. Antibody 37A4 shares a VH gene with anti-(Glu60Ala30Tyr10)n random terpolymer and anti-nitrophenyl antibodies, and a Vk gene with two anti-oxazolone antibodies. Antibodies 14C3 and 17C1 use the same germ-line VH and Vk genes as do anti-anti-idiotypic antibodies of the (Glu60Ala30Tyr10) system. These data demonstrate the genetic diversity of the antibody response to alprenolol, and illustrate the extensive flexibility of the immune system.     

Nucleotide sequences and three-dimensional modelling of the VH and VL domains of two human monoclonal antibodies specific for the D antigen of the human Rh-blood-group system.

The nucleotide sequences were determined for the VH and VL domains of two human IgG1 antibodies, Pag-1 and Fog-B, specific for the D antigen of the Rh-blood-group system. The VH-region genes of the two antibodies were derived from separate germ-line genes within the VH-IV gene family, but both antibodies used the same JH6 gene. The D-region genes differed from each other, and no similarity was found to known D regions. The light chain of Fog-B belongs to the V lambda-I subgroup and that of Pag-1 probably belongs to the V lambda-V subgroup; both light chains used the J2/3 gene. Three-dimensional models of the variable domains were made, based on those of known crystallographic structure. The surface contours at the combining sites are clearly different, consistent with the evidence that the antibodies recognize different but overlapping epitopes. Some details of the molecular modelling of hypervariable regions have been deposited as Supplementary Publication SUP 50155 (6 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.     

Different VL and VH germ-line genes are used to produce similar combining sites with specificity for alpha(1----6)dextrans

Monoclonal antibodies to alpha(1----6)dextrans produced in mice immunized with the T-independent antigens alpha(1----6)dextran or the stearylisomaltosyl oligosaccharides have been characterized immunochemically. To correlate the immunochemical properties of these monoclonal antibodies with their primary structure, we have sequenced the variable (V) regions of the light (L) and heavy (H) chains. Three V kappa germ-line genes belonging to two major gene families were used; differential J usages also contribute to diversity. Five different VH germ-line genes belonging to three major VH families were used. The VH genes were further modified by junctional diversity and differential J usage and possibly by somatic mutations. The effects of these modifications on the fine specificities of anti-alpha(1----6)dextrans are discussed. Thus far, six different combinations of VLJL-VH(D)JH chains that form groove-type combining sites specific for alpha(1----6)dextran have been found. We conclude that entirely different VL and VH can form combining sites specific for the internal linear sequence of alpha(1----6)dextran.     

Immunoglobulin genes of different subgroups are interdigitated within the VK locus

The variable regions of immunoglobulins are encoded by multigene families which are rearranged during B-cell differentiation. These families were classified in groups and subgroups based on their amino acid sequences. Genes belonging to a distinct subgroup are believed to occur in the genome within clusters. We are investigating the organization of human variable region genes of the kappa type (VK genes, ref. 1) in the germline and found now for the first time that VK sequences of three of the four different subgroups are interdigitated within the VK locus. We present evidence for the interspersion of two VKIII genes and a VKII pseudogene within an array of five VKI genes. All eight VK sequences are arranged in the same orientation. An evolutionary model for the generation of this 'mixed cluster' is discussed.     

Repertoire diversity of antibody response to bacterial antigens in aged mice. II. Phosphorylcholine-antibody in young and aged mice differ in both VH/VL gene repertoire and in specificity

Aging of mice is accompanied by both quantitative and qualitative changes in antibody responses to phosphorylcholine (PC), an immunodominant epitope of Streptococcus pneumoniae R36a strain (Pn). In order to study these changes at the molecular level, we generated PC-specific hybridomas from young (3 to 4 mo) and aged (20 to 24 mo) mice of different strains after primary immunization with S. pneumoniae R36a strain. These mAb were tested for Ig VH and VL gene family utilization, idiotopic repertoire, and cross-reactivity with unrelated Ag. Hybridomas from young mice (BALB/c, C57BL/6, and D1.LP) uniformly expressed the VH-S107 and V kappa-22 genes as well as most idiotopes of the T15 family, which were identified with different anti-T15 mAb. In contrast, the PC-reactive mAb from aged mice were quite heterogeneous: only 2 out of 13 utilized VHS107, 1 of 13 used VH7183, and 3 of 13 used VHJ558 gene family. Moreover, none of these mAb used L chain encoded by V kappa 22(0/13), but surprisingly they frequently expressed some of the T15 idiotope. In addition, the PC-binding mAb from aged mice showed broad cross-reactivity with various mouse and foreign proteins, whereas the mAb from young mice did not. These results demonstrate the genetic shift in antibody response of aging mice to PC, which is accompanied by a change in the antibody specificity. Interestingly, the qualitative repertoire change appears to be unrelated to the magnitude of antibody response, for the aged BALB/c mice maintain a very high reactivity to PC.     

Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure

Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.     

Two induced anti-Z-DNA monoclonal antibodies use VH gene segments related to those of anti-DNA autoantibodies

The cDNA for H and L chain V regions of two anti-Z-DNA mAb, Z22 and Z44, were cloned and sequenced. These are the first experimentally induced anti-nucleic acid antibody sequences available for comparison with autoantibody sequences. Z22 and Z44 are IgG2b and IgG2a antibodies from C57BL/6 mice. They recognize different facets of the Z-DNA structure. They both use VH10 family genes and share 95% sequence base sequence identity in the VH and leader sequences; however, they differ in the 5'-untranslated region of the VH mRNA, indicating they arise from different germline genes. Both use JH4 segments. They differ from each other very extensively in the CDR3 of both H and L chains. The most closely related H chains in the current GenBank/EMBL data base are two mouse IgG anti-DNA autoantibodies, one from an MRL-lpr/lpr mouse (MRL-DNA4) and one from an NZB/NZW mouse (BV04-01). Z22 and Z44 share 95% sequence identity with these antibodies in the VH segment. In addition, Z22 is identical to MRL-DNA4 at 91% of the positions in the 5'-untranslated region of the H chain mRNA. The two antibodies share 95% base sequence identity in the V kappa segment. The most closely related L chains, with 97 to 98% sequence identity, are the V kappa 10b germline gene for Z22 and the V kappa 10a germ line gene, which is associated with A/J anti-arsonate antibodies and BALB/c anti-ABO blood group substance antibodies, for Z44. Z22 and Z44 share several structural features (similarities in VH, JH, and V kappa) but differ very markedly in the L chain CDR1 and both H and L chain CDR3 sequences; these regions may determine the differences in their specific interactions with Z-DNA.     

Monoclonal antibodies to streptococcal group A carbohydrate. I. A dominant idiotypic determinant is located on Vk

In an effort to better define the antibody repertoire to streptococcal group A carbohydrate (GAC), somatic cell hybrids were prepared from A/J mice immunized with streptococcal vaccine. Most antibodies were IgG3K and IgMK, while 2 of 26 antibodies were lambda type. Each of the IgG3 antibodies had a distinct isoelectric point consistent with previous estimates of clonal repertoires of approximately 200. IEF analysis of the L chains, however, showed that about half of the antibodies produce a common L chain, called VK1GAC, previously identified in A/J anti-GAC serum antibodies. Additional support for the structural similarity of these L chains was gained by developing an idiotype antiserum to VK1GAC. All proteins with the common L chain spectrotype react strongly with anti-VK1GAC. Thus, it appears that anti-GAC antibodies are composed of H chains bearing a few VH regions pairing with a few L chains.     

Cloning and sequence analysis of the VH and VL regions of an anti-myelin/DNA antibody from a patient with peripheral neuropathy and chronic lymphocytic leukemia

We have cloned and determined the nucleotide sequence of the Ig VH and VL region genes of an IgM kappa mAb that binds to denatured DNA and myelin from a patient (POP) with chronic lymphocytic leukemia and peripheral neuropathy. Sequence analysis indicates that the V region of the kappa L chain gene (PopVK) has 99% homology to a V kappa IIIa germ-line gene and the V region of the mu H chain gene (PopVH) has 96% homology to the VH26 germ-line gene that is a member of the VH3 gene family. It is likely the V kappa and VH genes arose from these respective germ-line genes via somatic mutation or from closely related genes. V kappa III genes have frequently been used by other IgMk mAb especially those with rheumatoid factor activity, and the VH26 gene with no somatic mutation has been used by several anti-DNA antibodies, suggesting the possibility of preferential association of these or related germ-line genes with autoantibodies. The minor differences between the sequences of POP's VH and V kappa genes and sequences used by other autoantibodies, may be responsible for this antibody's crossreactivity with myelin and, as a result, the autoimmune neuropathy.     

Strain-dependent restricted VH and VL usage by anti-bacterial levan monoclonal antibodies.

The immune response to polysaccharides is highly regulated and has several distinguishing features, including restricted clonotype and isotype expression. The basis for this highly restricted response is not fully understood. To address these questions in a systematic manner, we have generated a panel of 102 mAb from CBA/CaHN (CBA/Ca) and BALB/cAnN (BALB/c) mice after one and two injections of bacterial levan (BL), a beta(2----6)-linked polyfructosan with beta(2----1)-linked fructose branch points (inulin determinant, In). This panel of mAb was examined for isotype, fine specificity, VH and VL region gene family usage and relationships between these parameters. After one or two injections of BL in both strains, mAb were IgM and IgG3. Fine specificity and VH/VL gene family usage differed markedly, however, between the two strains. Only 4% (2/51) of CBA/Ca mAb recognized the In determinant, whereas 77% (40/51) of BALB/c mAb recognized this epitope. In both strains, VH usage was restricted and certain families were overrepresented. In CBA/Ca mice, the overall response to BL was dominated by VHJ558 (45%, 23/51), the largest VH family, but VH36-60 (27%, 14/51) and VHJ606 (25%, 13/51) were also highly utilized and overrepresented. In BALB/c mice, the overall response to BL was dominated by VHJ606 (79%, 39/49 designated), a relatively small VH family. More importantly, after a single immunization with BL one particular VH/VL pair (VHJ606/ kappa 11) was used by 88% (36/41) of BALB/c mAb and was associated with In reactivity. In summary, BALB/c and CBA/Ca responses to BL differ in fine specificity and VH/VL usage.     

Contributions of a highly conserved VH/VL hydrogen bonding interaction to scFv folding stability and refolding efficiency.

The assembly of single-chain Fv (scFv) antibody fragments, consisting of an interconnected variable heavy chain (VH) and variable light chain (VL), is a cooperative process that requires coupled folding and domain association. We report here an initial investigation of VH/VL domain-domain assembly with a site-directed mutagenesis study that probes a highly conserved VH/VL hydrogen bonding interaction. Gln168 of the S5 scFv (Kabat VH 39) is absolutely conserved in 95% of all VH, and Gln44 (Kabat VL 38) is found in 94% of all kappa VL (Glx in 95% of all lambda VL). These side chains form two hydrogen bonds in head-to-tail alignment across the VH/VL interface. Double mutant cycles at Gln168 and Gln44 were constructed to first investigate their contribution to thermodynamic folding stability, second to investigate whether stability can be improved, and third to determine whether refolding efficiencies are affected by mutations at these positions. The results demonstrate that the Gln168-Gln44 interaction is not a key determinant of S5 scFv folding stability, as sequential modification to alanine has no significant effect on the free energy of folding. Several mutations that alter the glutamines to methionine or charged amino acids significantly increase the thermodynamic stability by increasing the m(g) associated with the unfolding isotherm. These effects are hypothesized to arise largely from an increase in the VH/VL association free energy that leads to tighter coupling between domain-domain association and folding. All of the mutants also display a reduced antigen binding affinity. Single and double methionine mutants also displayed significant increases in refolding efficiency of 2.4- to 3-fold over the native scFv, whereas the double alanine/methionine mutants displayed moderate 1.9- to 2.4-fold enhancement. The results suggest that reengineering the VH/VL interface could be useful in improving the stability of single-chain antibodies, as Ala/Met mutations at these conserved positions increase the free energy of folding by 46% while minimally perturbing binding affinity. They also could be useful in improving scFv recovery from inclusion bodies as the mutations increase the refolding efficiency by more than twofold.     

A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds.