Complete variable region sequences of five homologous high affinity anti-digoxin antibodies

High affinity murine A/J anti-digoxin monoclonal antibodies exhibit diversity in binding specificity for structurally related cardiac glycosides. They utilize several VH and VL genes. Among this diverse set, however, five antibodies share V region amino-terminal sequences that are remarkably homologous. The five antibodies were divided into three subsets based on different fine specificity-binding patterns. Therefore, complete V region sequences were determined by Edman degradation and by nucleotide sequence analysis. The VH region homology among the five antibodies was 84 to 100% and the VL region homology was 89 to 99%. The sequence data are consistent with the use of single (or closely related) VH and VL genes encoding the five antibodies. Four antibodies, derived from the same fusion (40-40, 40-120, 40-140, and 40-160), use identical D, JH2, and JK5 gene segments and identical junctions suggesting that they are clonally related. The fifth antibody (35-20) uses different D and JH1 gene segments but the same JK5 gene segment. All five antibodies share a cross-reactive idiotype. The three antibodies that exhibit the greatest degree of homology (40-40, 40-120, and 40-140) also share indistinguishable antigen-binding patterns as well as private idiotopes not present on the other two antibodies. Antibody 40-160, which has the next most homologous sequence, shares idiotopes with the first set but binds preferentially to different sites on the hapten, whereas antibody 35-20 has the most divergent sequence. In general, the degree of sequence homology among the five antibodies correlates with their hierarchical order based on hapten and idiotype fine specificity patterns.     

Structural modeling of sequence specificity by an autoantibody against single-stranded DNA

11F8 is a sequence-specific pathogenic anti-single-stranded (ss)DNA autoantibody isolated from a lupus prone mouse. Site-directed mutagenesis of 11F8 has shown that six binding site residues (R31VH, W33VH, L97VH, R98VH, Y100VH, and Y32VL) contribute 80% of the free energy for complex formation. Mutagenesis results along with intermolecular distances obtained from fluorescence resonance energy transfer were implemented here as restraints to model docking between 11F8 and the sequence-specific ssDNA. The model of the complex suggests that aromatic stacking and two sets of bidentate hydrogen bonds between binding site arginine residues (R31VH and R96VH) and loop nucleotides provide the molecular basis for high affinity and specificity. In part, 11F8 utilizes the same ssDNA binding motif of Y32VL, H91VL, and an aromatic residue in the third complementarity-determining region to recognize thymine-rich sequences as do two anti-ssDNA autoantibodies crystallized in complex with thymine. R31SVH is a dominant somatic mutation found in the J558 germline sequence that is implicated in 11F8 sequence specificity. A model of the mutant R31S11F8.ssDNA complex suggests that different interface contacts occur when serine replaces arginine 31 at the binding site. The modeled contacts between the R31S11F8 mutant and thymine are closely related to those observed in other anti-ssDNA binding antibodies, while we find additional contacts between 11F8 and ssDNA that involve amino acids not utilized by the other antibodies. These data-driven 11F8.ssDNA models provide testable hypotheses concerning interactions that mediate sequence specificity in 11F8 and the effects of somatic mutation on ssDNA recognition.     

Antibody engineering for the analysis of affinity maturation of an anti-hapten response.

The influence of structural variation, previously observed in a panel of V186.2 VH/V lambda 1-expressing anti-NP antibodies from the secondary response, on the affinity of these antibodies was examined by site-specific mutagenesis and recombinant antibody construction. A tryptophan----leucine exchange at position 33 in the VH segment of all but one of the high-affinity antibodies is the most frequently observed somatic mutation and by itself leads to a 10-fold higher affinity; all other somatic exchanges are irrelevant for affinity selection. In the single case of a high-affinity antibody without this common exchange, high affinity is mediated by a combination of mutations (including a one-codon deletion) in VH and the particular D-JH rearrangement carried by this antibody. The data indicate that the pattern of somatic diversification through hypermutation is shaped by affinity selection, but that only a single point mutation is available in the VH and the VL gene of lambda 1 chain-bearing anti-NP antibodies which by itself leads to an increase of hapten-binding affinity. Based on the analysis of two secondary response antibodies from which somatic mutations in VH and VL have been eliminated, it is also concluded that the recruitment of B cell clones into the pathway of hypermutation involves a mechanism which is not based upon affinity differences towards the antigen.     

Somatic evolution of diversity among anti-phosphocholine antibodies induced with Proteus morganii

The variable region sequences of light and heavy chains (VL and VH) were determined for 11 hybridoma antibodies produced in response to the PC moiety on Proteus morganii. These hybridomas were derived from two separate fusions, one obtained from mice early in a secondary response and the other from late in a secondary response. All of these antibodies possessed a cross-reactive idiotype found on anti-PC antibodies in the M603 family, and exhibited preferential specificity for PC in the context of P. morganii. We found that all of the antibodies were derived from a single VH/VL pair. VH was encoded by V1, DFL16.1 and JH1, and VL was encoded by a consensus VK8 gene and JK5. Antibodies differed from each other by somatic point mutations that occurred at a high rate. The mutations in VL were approximately one-third as abundant as those in VH and were randomly distributed throughout the molecule. Mutations in VH were concentrated in CDR 2 and 3 and had a replacement to silent ratio that was three to six times greater than predicted from random accumulation. Based on the sequence data, a single genealogic tree with multiple branches could accommodate all the hybrids from a fusion. We concluded that in both examples the anti-PC response arose by somatic mutation and stepwise selection from a single precursor. Antigen binding studies with these 11 hybridomas and a 12th that had no mutations revealed that the acquisition of preferential specificity for antigen was dependent on somatic mutation of germline genes. Additional binding studies demonstrated that continued selection during clonal expansion was probably antigen driven. An unexpected finding was five independently selected antibodies from one fusion that had identically mutated VH and VL sequences. We suggest that the hypermutation mechanism is not a continuously active process during clonal expansion and that it is regulated, probably during the mid to late phase of the primary response.     

Clustered H chain somatic mutations shared by anti-p-azophenylarsonate antibodies confer enhanced affinity and ablate the cross-reactive idiotype

A cluster of four consecutive CDR2 somatic mutations are shared by the VH regions of two independently isolated hybridoma antibodies with specificity for p-azophenylarsonate (Ars). The mutations appear to be derived by a series of independent events. To assess the influence of these shared somatic mutations on antibody affinity for Ars and on idiotypy, we introduced them, via site-directed mutagenesis, into the V region of an anti-Ars antibody that was otherwise unmutated and we eliminated them from the mutated context of one of the two antibodies in which they were originally found. Results of affinity measurements by fluorescence quenching and of idiotypic binding assays performed on these engineered mutants demonstrated that the shared mutations increased affinity for Ars and eliminated the predominant Id associated with strain A anti-Ars antibodies and four of five idiotypes defined by anti-idiotypic mAb. These results support the interpretation that a strong affinity-based selection pressure has favored the clonal expansion of B cells with receptors containing these mutations despite the loss of a predominant Id. Thus, in producing antibodies containing V regions conferring high affinity for Ag, the combined processes of somatic mutation and clonal selection have generated a common structural solution through parallel repeats.     

Mutational analysis of avidity and fine specificity of anti-levan antibodies

Using the polyfructose, bacterial levan, as a model polysaccharide, we analyzed how V regions affect binding in anti-polysaccharide mAbs. Previously, panels of mAb were constructed from bacterial levan-immunized BALB/c and CBA/Ca mice. The BALB/c mAb were mostly germline VHJ606:Vkappa11, and a subset contained presumed somatic mutations in the complementarity-determining regions (CDRs) that correlated with increases in avidity for the beta(2-->1) inulin linkage of levan. The CBA/Ca mAb were more heterogeneous in V gene usage, but a subset of inulin-nonreactive mAb were VHJ606:Vlambda and had VH sequence differences in the CDRs from the VHJ606 regions of the BALB/c mAb. In this report, VHJ606 Abs containing various combinations of specifically mutated H and L chains were produced by engineered transfectants and tested for inulin avidity and levan binding. Two presumed somatic mutations seen in CDRs of the BALB/c hybridomas were shown to directly cause marked increases in avidity for inulin (VH N53H, 9-fold; VL N53I, 20-fold; together, 46-fold) but not for beta(2-->6) levan. Exchange of either positions 50 or 53 in VH or the H3 loop between the BALB/c and CBA/Ca mAb resulted in either fine specificity shift or total loss of bacterial levan binding. Three-dimensional models of the V regions suggested that residues that affect binding to inulin alone are near the edge of the CDR surface, while residues involved with binding both forms of levan and affecting fine specificity are in the VH:VL junctional area.     

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.     

Complete heavy and light chain variable region sequence of anti-arsonate monoclonal antibodies from BALB/c and A/J mice sharing the 36-60 idiotype are highly homologous

Structural and serologic studies on murine A/J monoclonal anti-arsonate antibodies resulted in the identification of a second idiotype family (Id36-60) in addition to the predominant idiotype family (IdCR). Id36-60, unlike IdCR, is a dominant idiotype in the BALB/c strain but is a "minor" idiotype in the A/J strain. The complete heavy and light chain variable region (VH and VL) amino acid sequences of a representative Id36-60 hybridoma protein from both the A/J and BALB/c strains have been determined. There are only four amino acid sequence differences between the VH of antibody 36-60 (A/J) and antibody 1210.7 (BALB/c). Two of these differences arise from single nucleotide changes in which the A/J and BALB/c Id36-60 VH germline gene sequences differ. The two other differences are the result of somatic mutation in hybridoma protein 36-60. In addition, Id36-60 heavy chains employ the same D and JH3 segments in both strains. The entire Vk2 VL of 36-60 and 1210.7 differ by only two amino acids, suggesting that like the heavy chains, they are derived from highly homologous VL genes. The same Jk segment is used in both antibodies. A comparison of the amino acid sequence data from Id36-60-bearing hybridomas suggests that a heavy chain amino acid difference accounts for the diminished arsonate binding by the 1210.7 hybridoma protein. Because the 1210.7 heavy chain is the unmutated product of the BALB/c VH gene, somatic mutation in VH may be required to enhance Ars affinity in this system.     

A V kappa-J kappa junctional change in an antidigoxin recombinant antibody destroys digoxin-binding activity

A set of high affinity antidigoxin antibodies were previously identified with high homologous V kappa 1A L chain sequences but were associated with two entirely different VH regions and two dramatically different specificities for digoxin analogs. Antibodies 40-20, 40-60, 40-90, and 40-100 displayed similar binding specificities but differed from that of antibody 26-10. In a previous study using somatic cell fusion for Ig chain recombination we demonstrated that a recombinant antibody consisting of the H chain of antibody 26-10 and the L chain of antibody 40-20 retained digoxin binding and the 26-10 Id, but displayed a binding specificity pattern dominated by the 26-10 H chain donor. In the present study we produced three additional chain recombinant antibodies that contain the 26-10 H chain recombined with each of the L chains of antibodies 40-60, 40-90, and 40-100. All four recombinants expressed the 26-10 Id indistinguishably from the 26-10 antibody. Two of the recombinants (using the 40-60 and 40-90 L chains) bind digoxin; however, the recombinant using the 40-100 L chain failed to bind digoxin. Complete sequence analyses of the 40-20, 40-60, 40-90, and 40-100 VH and VL regions were performed. Antibodies 40-90 and 40-100 have identical VH region sequences but differed only in their L chains at position 96 (proline/leucine). This single difference at the VK-JK junction abolished digoxin binding in the context of one H chain (26-10), but does not cause a significant change in binding in association with the "normal" parental chains 40-90 and 40-100. Thus, structurally closely related VL regions can recombine with different VH regions to form digoxin binding sites of different specificity; in one binding site the identity of a L chain junctional residue is critical whereas in the second binding site that residue is unimportant. Molecular modeling studies revealed major differences between calculated binding site structures for 26-10 when leucine is substituted for proline at position 96 in the 26-10 VL region.     

Immunoglobulin chain recombination among antidigoxin antibodies by hybridoma-hybridoma fusion

Conditions necessary for in vitro chain recombination of high affinity (10(9) to 10(12) M-1) antidigoxin monoclonal antibodies resulted in decreased affinity for both intact "native" and chain recombinant molecules. Chain recombination by somatic cell fusion was used instead to study the effects on antigen specificity and idiotypy of recombinants in which an homologous light (L) chain substituted for the parental L chain. The antidigoxin antibody 26-10 utilizes a VL sequence highly homologous to that of antibody 40-20, an antidigoxin antibody which uses a different VH gene than does 26-10 and lacks significant reactivity with an anti-26-10 idiotypic serum. The drug-marked antidigoxin cell line 26-10 (gamma 2a, kappa) and a drug-marked light chain producing variant of antidigoxin hybridoma 45-20 (lambda 1) which lacks both digoxin binding and idiotypy were fused. The fusion progeny (gamma 2a, kappa, lambda 1) which binds digoxin and is idiotype-positive, was selected for kappa loss (resulting in loss of digoxin and idiotype binding) and then fused with a heavy (H) chain loss variant of antidigoxin hybridoma 40-20 (kappa, digoxin nonbinding, idiotype negative). The resultant cell line CR-57 (gamma 2a, kappa, lambda) secretes antibodies which assemble the 26-10 H chain with both the 40-20 kappa-chain and the 45-20 lambda 1-chain. The affinity purified recombinant species consisting of 26-10 H chain and 40-20 kappa-chain expresses complete 26-10 idiotypic determinants. However, this recombinant antibody binds digoxin with decreased affinity and altered specificity relative to native 26-10. The binding specificity pattern nonetheless is most similar to the H chain donor. Amino acid and nucleotide sequence analyses of the respective light chains demonstrate six variable region differences between them, two of which are in complementarity-determining regions and the remainder in the framework. Hybridoma-hybridoma fusion provides an alternative to in vitro chain recombination for studying the contribution of chain combinational diversity to antibody diversity, antigen binding, and idiotypy.     

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

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

Genetics of the phosphocholine-specific antibody response to Streptococcus pneumoniae. Germ-line but not mutated T15 antibodies are dominantly selected

The role that somatic mutations play in the phosphocholine-specific, antibody response to Streptococcus pneumoniae was examined by studying sets of hybridomas from different individual mice. As expected most of the cell lines were from the T15 anti-phosphocholine family and were not encoded by the v1 gene of the T15 VH family and V kappa 22. A minority of antibodies were from the M603 (v1/V kappa 8) and M511 (v1/V kappa 24) families. Three additional antibodies were encoded by the v11 gene of the T15 family; two were paired with a V lambda and the other with a V kappa 1 gene. In vitro binding studies showed that T15- and M603-like antibodies had the highest affinity for S. pneumoniae. Complete sequencing of the VH and VL mRNA from 25 of the hybridomas revealed somatic mutations in 11 of the antibodies. A total of 17 independently derived T15 positive cell lines were studied in detail, six of these were mutated. These mutations were scattered throughout the V regions and the replacement to silent ratio was typical of that for framework regions. Statistical evaluation of the placement of mutations showed that there was a slight but significantly decreased frequency of mutations in complementarity determining regions. Comparisons of mutated and unmutated T15-related antibodies showed that mutations caused a decrease in binding to S. pneumoniae in every case. These results argue that the optimal specificity for this molecular form of phosphocholine is encoded in the germline and that Ag-driven events favor selection of B cells expressing these germ-line encoded antibodies.     

Heavy and light chain variable single domains of an anti-DNA binding antibody hydrolyze both double- and single-stranded DNAs without sequence specificity

Anti-DNA antibodies (Abs) are of biomedical interest because they are associated with autoimmune diseases in human and mice. Previously we isolated an anti-DNA monoclonal Ab 3D8 from an autoimmune-prone MRL-lpr/lpr mouse. Here we have characterized DNA binding kinetics and hydrolyzing activities of the recombinant single chain variable fragment (scFv) and the single variable domains of heavy chain (VH) and light chain (VL) using various single-stranded (ss) and double-stranded (ds) DNA substrates. All the Abs bound to both ds- and ssDNAs without significant preferential sequence specificity showing scFv higher affinities (KD = approximately 17-74 nm) than VH (KD = approximately 2.4-8.4 microm) and VL (KD = approximately 3.2-72 microm), and efficiently hydrolyzed both ds- and ssDNAs without sequence specificity in a Mg2+-dependent manner, except for the poor activity of 3D8 scFv for ss-(dT)40. Elucidated crystal structure-based His to Ala mutations on the complementarity determining regions of VH (His-H35 --> Ala) and/or VL (His-L94 --> Ala) of 3D8 scFv significantly inhibited the catalytic activities, indicating that the His residues are involved in the catalytic mechanism of 3D8 scFv. However, the DNA hydrolyzing activities of single domain VH and VL were not affected by the mutations, indicative of their different catalytic mechanisms from that of 3D8 scFv. Our results demonstrate single domain Abs with DNase activities for the first time, which might provide new insights into substrate recognition and catalytic mechanisms of anti-DNA Abs.     

The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin

Previously we reported that the variable heavy chain region (VH) of a human beta2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (VL) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4VH and paired VL in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of VH and VL sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived VL sequences were expressed with IS4VH and the VH of an anti-dsDNA antibody, B3. Six variants of IS4VH, containing different patterns of arginine residues in CDR3, were paired with B3VL and IS4VL. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.     

Patterns of mutations and selection in antibodies to the phosphocholine-specific determinant in Proteus morganii

The contribution of somatic mutation to the generation of an antibody response was investigated by using the phosphocholine (PC) determinant in the bacterium Proteus morganii as the model Ag. The response to this determinant is restricted to a single VH/VL pair and apparently is derived from only one or two precursors per mouse. In this study we examined hybridoma antibodies from nine individual mice which produced representatives of 12 different clones. We found that all antibodies reactive with the PC Ag of P. morganii contained somatic mutations; the number ranged from 2 to 20. Two clusters of mutations were observed, one in complementarity-determining residue (CDR) 2 and the other in CDR 3 of VH. Examination of a three-dimensional model of M603, an antibody with the same V region composition as the anti-PC antibodies under study, showed that these clusters occupied an area of the binding site which presumably interacts with carrier elements of the PC epitope in P. morganii. A high incidence of recurring mutations were found in both clusters, and one of these was invariant, leading to an Asn for Asp substitution at 95. Ag binding studies with these antibodies and an additional one, which was unmutated except for the invariant substitution at 95, showed that: 1) antibodies having only the 95Asn mutation failed to bind the PC Ag of P. morganii, 2) addition of a second recurring mutation, at 52a (CDR 2), was sufficient to create strong binding to the P. morganii Ag, and 3) accumulation of mutations was directly correlated with increased binding activity for Ag. These results show that somatic mutations play a critical, if not essential, role in generating specificity for this PC Ag, and that Ag, and most likely a carrier element of the epitope, is a primary force in the continued selection and expansion of Ag-reactive B cells.     

Antibody engineering: the use of Escherichia coli as an expression host.

The hypervariable loops of an antibody molecule are supported on the relatively conserved beta-sheeted frameworks of the heavy- and light-chain variable domains (designated VH and VL domains, respectively). Residues within and flanking these loops interact with antigen and confer the specificity and affinity of antigen binding on the immunoglobulin molecule. Thus, the isolation and expression of VH and VL domain genes are of particular interest both for analysis of the determinants of antibody specificity and for generation of fragments with binding affinities for use in therapy and diagnosis. The PCR can now be used to isolate diverse repertoires of antibody VH and VL domain genes from antibody-producing cells from different species, including humans and mice. The genes can be expressed as either secreted or surface-bound Fv or Fab fragments, using Escherichia coli expression systems, and the desired antigen-binding specificity screened for or, preferably, selected. The use of E. coli as an expression host allows the required antigen-binding specificity to be isolated in clonal form in a matter of days. The VH and VL domain genes can also be hypermutated and higher-affinity variants isolated by screening or selection. Thus, the use of this technology should allow the isolation of novel binding specificities or specificities that are difficult to generate by hybridoma technology. It will also facilitate the isolation of human-derived Fv/Fab fragments that may be less immunogenic in therapy. This approach therefore has almost unlimited potential in the generation of therapeutics with binding specificities to order. The fragments can be used either alone or linked to effector functions in the form of antibody-constant domains or toxins. The new technology could prove to be a method of choice for the rapid and convenient production of designer antibodies.     

Mutation of a single conserved residue in VH complementarity-determining region 2 results in a severe Ig secretion defect

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.     

Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme.

To experimentally examine the functional roles of somatically derived structural variation in the lysozyme-binding mAb HyHEL-10, we have introduced three different point mutations and one insertion at two different sites in HyHEL-10 by site-directed mutagenesis and expression of the mutant antibodies. Mutation of Asp----Ala at position 101 of the H chain returns a somatically mutated residue to its germline sequence for HyHEL-10, and reduces affinity for chicken lysozyme by approximately 9000-fold. Lengthening the third H chain hypervariable region by two amino acids reduces affinity by about 2000-fold. Two mutations, Asp----Thr at position 101 in the H chain and Lys----Thr at position 49 in the L chain, model somatic differences found in another structurally related but functionally distinguishable mAb and minimally decrease affinity for chicken lysozyme. The H chain mutation Asp101VVH----Thr has little effect on affinity for other avian lysozymes but does alter relative fine specificity for these lysozymes. The L chain mutation Lys49VK----Thr increases affinity for duck lysozyme by approximately fivefold. Neither of the positions mutated, 101 in the H chain nor 49 in the L chain, nor the residues near the insertion contact lysozyme in the x-ray structure of the HyHEL-10 F(ab)-HEL complex. The results suggest that these mutations, which model observed somatic mutations, produce functional variation by indirect or long-range effects.     

Effects on interaction kinetics of mutations at the VH-VL interface of Fabs depend on the structural context

The influence of framework residues belonging to VH and VL modules of antibody molecules on antigen binding remains poorly understood. To investigate the functional role of such residues, we have performed semi-conservative amino acid replacements at the VH-VL interface. This work was carried out with (i) variants of the same antibody and (ii) with antibodies of different specificities (Fab fragments 145P and 1F1h), in order to check if functional effects are additive and/or similar for the two antibodies. Interaction kinetics of Fab mutants with peptide and protein antigens were measured using a BIACORE instrument. The substitutions introduced at the VH-VL interface had no significant effects on k(a) but showed small, significant effects on k(d). Mutations in the VH module affected k(d) not only for the two different antibodies but also for variants of the same antibody. These effects varied both in direction and in magnitude. In the VL module, the double mutation F(L37)L-Q(L38)L, alone or in combination with other mutations, consistently decreased k(d) about two-fold in Fab 145P. Other mutations in the VL module had no effect on k(d) in 145P, but always decreased k(d) in 1F1h. Moreover, in both systems, small-magnitude non-additive effects on k(d) were observed, but affinity variations seemed to be limited by a threshold. When comparing functional effects in antibodies of different specificity, no general rules could be established. In addition, no clear relationship could be pointed out between the nature of the amino acid change and the observed functional effect. Our results show that binding kinetics are affected by alteration of framework residues remote from the binding site, although these effects are unpredictable for most of the studied changes.     

Structural characterization of H chain-associated idiotopes of anti-p-azophenylarsonate monoclonal antibodies

The majority of antibodies directed against p-azophenylarsonate (Ars) protein conjugates elicited during secondary immune responses of A/J mice bear a heritable cross-reactive Id (CRIa or IdCR) which corresponds to the utilization of a unique combination of variable region gene segments that can differ by somatic mutations. One such monoclonal anti-Ars antibody, 44-10, bears IdCR as defined by rabbit antisera but does not react with two anti-idiotypic mAb, 5Ci and AD8, which react with all primary (unmutated) IdCR+ antibodies and some secondary response IdCR+ antibodies. We therefore determined the complete sequence of antibody 44-10, which differs from the germline encoded (unmutated) IdCR+ antibody 36-65 at four positions in the H chain V region (VH): position 55 in the second complementarity determining region, 100 and 107 (D-gene junctions) and 110 (in JH2). The 44-10 L chain is unmutated. Sequence analyses of five other secondary immune response anti-Ars IdCR+ antibodies chosen on the basis of sharing one or more of the amino acid substitutions found in 44-10, were correlated with idiotypic expression of this set of antibodies. The results suggest that the mutation at VH position 55 (Asn----Lys) is responsible for loss of the 5Ci idiotope. To substantiate this hypothesis, oligonucleotide-directed mutagenesis of the germline encoded (unmutated) IdCR+ antibody was used to produce two mutants, one with VH Lys 55 and the other containing residues at positions 100, 107 and 110 identical to those found in 44-10. Id binding studies on these mutants confirm that 5Ci idiotope loss is due to conformational changes resulting from a mutation at VH position 55. This mutation also results in loss of the AD8 idiotope in the structural context of antibody 44-10.