VL Families Research Articles

Effect of VH-VL Families in Pertuzumab and Trastuzumab Recombinant Production, Her2 and FcγIIA Binding.

Many therapeutic antibodies are humanized from animal sources. In the humanization process, complementarity determining region grafting is tedious and highly prone to failure. With seven known VH families, and up to six known κ VL families, there are choices aplenty. However, the functions of these families remain largely enigmatic. To study the role of these V-region families, we made 84 recombinant combinations of the various VH and VL family whole IgG1 variants of both Trastuzumab and Pertuzumab. We managed to purify 66 of these to investigate the biophysical characteristics: recombinant protein production, and both Her2 and FcγIIA binding. Our findings revealed combinations that showed improved recombinant antibody production and both antigen and receptor binding kinetics. These findings show the need to rethink antibodies as a whole protein, relooking of the functions of the antibody domains, and the need to include immunoglobulin receptor investigations for effective antibody therapeutics development.

Molecular Characterization of Murine Monoclonal Antibody Variable Regions Specific for Hepatitis B Surface Antigen.

Hepatitis B virus (HBV) surface antigen (HBsAg) induces a vigorous neutralizing antibody response, which causes effective protection against HBV infection. Little is known about the profile of variable region genes of immunoglobuline heavy (VH) and light (VL) chains rearranged in anti-HBs antibodies, and also the possible association of this profile with specificity pattern of these antibodies to mutant forms of HBsAg. The present study determined the nucleotide sequence of VH and VL genes of mouse monoclonal antibodies (MAbs) generated against HBsAg. Hybridoma clones secreting anti-HBsAg MAbs were developed from hyperimmunized Balb/c mice. VH and VL gene sequences of all MAbs were determined by amplifying the genes using a panel of VH and VL family specific primers by reverse transcription polymerase chain reaction. The reactivity pattern of anti-HBs MAbs with different mutant forms of HBsAg was evaluated by enzyme-linked immunosorbent assay, and then the profile of antigen specificity and its association to VH/VL family expression was analyzed. Twenty-three murine hybridomas producing anti-HBs MAbs were generated. Nucleotide sequence analysis revealed that heavy chains of these MAbs were encoded by IGHV genes from the HV1 (52%), HV6 (22%), HV5 (17%), and HV3 (9%) families in combination with IGHJ2 (57%), HJ1 (26%), and HJ4 (17%). Besides, 56% of MAbs used IGHD1 genes in their VDJ rearrangements. Concerning the IGKV gene, 26% and 22% of clones used KV4 and KV10 gene families, while the rest of the clones used KV8, KV6, KV1, KV12, and KV14 gene families. Besides, the IGKJ2 gene was the most represented KJ gene (43%). No association was found between the specificity pattern of MAbs to mutant forms of HBsAg with their preferential V, D, and J genes usage for most of MAbs. The data suggest that heavy chains of anti-HBs MAbs preferentially use genes derived from the IGHV1, IGHV6, IGHJ2, and IGHD1 families. In contrast to heavy chains, which predominantly use four families of IGHV genes, light chains use more diverse IGKV gene families.

The immunogenicity of humanized and fully human antibodies

Monoclonal antibodies represent an attractive therapeutic tool as they are highly specific for their targets, convey effector functions and enjoy robust manufacturing procedures. Humanization of murine monoclonal antibodies has vastly improved their in vivo tolerability. Humanization, the replacement of mouse constant regions and V framework regions for human sequences, results in a significantly less immunogenic product. However, some humanized and even fully human sequence-derived antibody molecules still carry immunological risk. To more fully understand the immunologic potential of humanized and human antibodies, we analyzed CD4+ helper T cell epitopes in a set of eight humanized antibodies. The antibodies studied represented a number of different VH and VL family members carrying unique CDR regions. In spite of these differences, CD4+ T cell epitopes were found only in CDR-sequence containing regions. We were able to incorporate up to two amino acid modifications in a single epitope that reduced the immunogenic potential while retaining full biologic function. We propose that immunogenicity will always be present in some antibody molecules due to the nature of the antigen-specific combining sites. A consequence of this result is modifications to reduce immunogenicity will be centered on the affinity-determining regions. Modifications to CDR regions can be designed that reduce the immunogenic potential while maintaining the bioactivity of the antibody molecule.

Unusually Similar Patterns of Antibody V Segment Diversity in Distantly Related Marsupials

A pattern of coevolution between the V gene segments of Ig H and L chains has been noted previously by several investigators. Species with restricted germline V(H) diversity tend to have limited germline V(L) diversity, whereas species with high levels of germline V(H) diversity have more diverse V(L) gene segments. Evidence for a limited pool of V(H) but diverse V(L) gene segments in a South American opossum, Monodelphis domestica, is consistent with this marsupial being an exception to the pattern. To determine whether M. domestica is unique or the norm for marsupials, the V(H) and V(L) of an Australian possum, Trichosurus vulpecula, were characterized. The Ig repertoire in T. vulpecula is also derived from a restricted V(H) pool but a diverse V(L) pool. The V(L) gene segments of T. vulpecula are highly complex and contain lineages that predate the separation of marsupials and placental mammals. Thus, neither marsupial follows a pattern of coevolution of V(H) and V(L) gene segments observed in other mammals. Rather, marsupial V(H) and V(L) complexity appears to be evolving divergently, retaining diversity in V(L) perhaps to compensate for limited V(H) diversity. There is a high degree of similarity between the V(H) and V(L) in M. domestica and T. vulpecula, with the majority of V(L) families being shared between both species. All marsupial V(H) sequences isolated so far form a common clade of closely related sequences, and in contrast to the V(L) genes, the V(H) likely underwent a major loss of diversity early in marsupial evolution.

One-step cloning of anti tissue transglutaminase scFv from subjects with celiac disease

Celiac disease is characterized by intestinal mucosal injury and malabsorption precipitated by dietary exposure to gluten of some cereals with a prominent role being played by gliadins, specific antigenic determinants found in wheat gluten. Patients suffering from celiac disease have serum antibodies recognizing gliadin, as well as the endomysial autoantigen tissue transglutaminase. Phage display antibody libraries have revealed ectopic production of anti-transglutaminase antibodies by intestinal lymphocytes with a biased use of the VH5 antibody gene family. Here we report a study on the pairing of VH and VL families in the antibodies to transglutaminase. Our results led to the construction of small phage display antibody libraries based on the amplification of the two genes in the VH5 family from intestinal lymphocytes. This method can be used for the rapid characterization of the anti-transglutaminase response in a potentially large number of subjects including asymptomatic patients whose serum antibodies may be undetectable.

Characterisation of a fourth immunoglobulin light chain isotype in the common carp

Three isotypes of immunoglobulin (Ig) light (L) chain, designated L1A, L1B, and L3, have been characterised in the common carp ( Cyprinus carpio L.) to date. In this paper the molecular cloning of a fourth IgL isotype in carp, designated L2, is described. A reverse transcription–polymerase chain reaction (RT–PCR) method including 5′- and 3′-RACE was used to isolate carp L2 cDNA clones. The VL sequences could be divided into two distinct VL families, designated VL2-1 and VL2-2, most similar to rainbow trout (68% similarity) and zebrafish (78%) VL2 amino acid sequences, respectively. The CL amino acid sequences showed the highest similarity to zebrafish L2 (80%), and contained the characteristic cysteines necessary for intradomain or interchain disulphide bridges as did the VL sequences. Neither the VL nor CL sequences demonstrated such a high similarity to the other carp IgL isotypes, L1A, L1B, and L3. For JL segments, sequence variations appeared to be confined to a few positions. In the course of 5′- and 3′-RACE, cDNA clones containing recombination signal sequence (RSS), representatives of IgL sterile transcripts, were obtained. Southern blot analyses suggested that the locus of carp L2 has a cluster-like organisation. Phylogenetic analyses showed that both carp VL2 and CL2 amino acid sequences highly clustered with other teleost L2 sequences.

The antibody site in Atlantic salmon; phage display and modeling of scFv with anti-hapten binding ability.

Cloning and sequencing the cDNA of around 50 VH (VDJ) and 15 VL genes in Atlantic salmon demonstrated nine VH families (above 80% identity within each family) and one dominating but relatively diverse VL family in this species. The highest variability of the VH was seen in the CDR3, but CDR2 also expressed a modest variability. The ‘whole’ antibody repertoire was expressed as single chain Fv (scFv) in a phage display library by combining 12 VH and two VL specific primers (FR1/μ1 and FR1/CL, respectively). The PCR products (VH and VL) were ligated (with a G-rich spacer) into the λ Surf-Zap (Stratagene) vector and expressed as a surface fusion protein on the M13 phage. Anti-TNP and anti-FITC specific scFv clones were isolated by panning using hapten-coated magnetic beads and the coding DNA sequenced. The specificities of the anti-TNP and anti-FITC clones were similar to mouse monoclonal antibodies. 3D-models of the active sites (CDRs) of the anti-TNP and anti-FITC clones suggest hapten-interacting structures of the salmon antibody site similar to mammalian antibodies.

Molecular structure of eight human autoreactive monoclonal antibodies.

The heavy (H) and light (L) chain V-region sequences of eight human autoreactive immunoglobulin M (IgM) monoclonal antibodies (mAbs: BY-4, BY-7, BY-12, IRM-3, IRM-7, IRM-8, IRM-10 and CDC-1) were determined at the cDNA level. All VH and VL families were identified. Four different VH families were represented, VH3 being the most common as five of the mAbs (BY-7, BY-12, IRM-3, IRM-8 and CDC-1) used genetic elements of this family, whereas VH1, VH2 and VH4 were only present in IRM-7, BY-4 and IRM-10, respectively. BY-4, BY-7, BY-12, IRM-7 and IRM-10 reacted with a variety of self as well as non-self antigens, thus exhibiting polyreactive behaviour. Comparison of the gene segments utilized by these mAbs with their germline counterparts revealed that the gene segments were close to germline configuration. The length of H-CDR3 was found to be relatively long (27-60 nucleotides) among the polyreactive mAbs and the presence of Tyr and Trp residues in this region seems to be of vital importance for polyreactivity. We have analysed the utilization of gene elements and the presence of amino acid residues in regions particularly important for antigen binding, such as CDR. Common molecular features relating to the function of the mAbs are discussed.

Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3′ to 5′ exonuclease activity

Degenerate primers were designed for PCR amplification of unknown mouse immunoglobulin (Ig) light (L) and heavy (H) chain variable (V) genes. Each subgroup of mouse Ig gene sequences [Kabat, E.A., Wu, T.T., Perry, H.H., Gottesman, K.S., Foeller, C., 1991. Sequences of Proteins of Immunological Interest, 5th edn. US Department of Health and Human Services, Public Health Service, NIH.] was analyzed, and highly degenerate primers in the framework one (FR1) region were designed. A single highly degenerate FR1 primer sufficed for the amplification of light chains; for heavy chains, a series of FR1 primers was used. At the same time, we assessed the effect of 3′ to 5′ exonuclease activity of DNA polymerase on the utilization of these degenerate primers. Using Taq polymerase, which lacks 3′ to 5′ exonuclease activity, we successfully amplified the Ig VL and VH genes expressed in more than a hundred monoclonal hybridoma cell lines reactive against a phosphonamidate hapten. Sequence analysis of the cloned VL and VH genes, 52 of each, showed that they are derived from multiple germline families (10 of the 17 VL families and 9 of the 14 VH families) as recently defined [Martinez, C., Lefranc, M., 1998. The mouse ( Mus musculus) immunoglobulin kappa variable (IGKV) genes and joining (IGKJ) segments. Exp. Clin. Immunogenet. 15, 184.]. The universality of our primers was also demonstrated by successful amplification of other mouse hybridoma cell lines that are specific to different antigens.

A second immunoglobulin light chain isotype in the rainbow trout.

A novel immunoglobulin (Ig) light chain isotype, termed IgL2, has been isolated from trout lymphoid tissues both by reverse transcription - polymerase chain reaction (PCR) and screening of cDNA libraries. The CL domain of the new isotype shares only 29% residues with a recently cloned trout IgL isotype, termed IgL1, which has some similarities to Ckappa and Clambda isotype domains of several vertebrate species. Using anchored PCR, a VL element rearranged to CL2 was isolated. It is a member of a new VL family (VL2) of which four members were sequenced. These differ in the sequence of CDR1 and CDR2 but are remarkably similar in CDR3, i. e., at the junction between VL and JL segments. VL elements are rearranged to novel JL elements which differ from those described for VL1-CL1 rearrangements. Two cDNA clones contained JL-CL2 segments but no VL segments. The JL segments were preceded by typical rearrangements signal sequences [RSS, nonamer-23 base pair (bp) spacer-heptamer]. Further upstream of RSS were located two to three near identical 53 bp repeats, each of which included a 16 bp sequence similar to KI and KII sequences located at similar places in human and mouse Jk1 genes. These sequences are believed to act as binding sites for the protein KLP, which could be a transcriptional factor involved in the synthesis of germline Jk transcripts. Their phylogenic conservation in vertebrates suggests that they have an important role in B-cell differentiation. Remarkably, an RNA species of about 0.7 kilobase is the predominant IgL mRNA in trout spleen and coincides in size with JLCL2 transcripts. Genomic DNA blot analysis indicates that the trout L2 locus has a cluster-like organization similar to the trout L1 locus and the IgL locus of several teleost fish. A phylogenic analysis of VL2 and CL2 corroborates their low similarity to other vertebrate IgL chains and suggests an ancient diversification of the IgL locus.

A third Ig light chain gene isotype in Xenopus laevis consists of six distinct VL families and is related to mammalian lambda genes.

Xenopus laevis is a unique model for studying the ontogenetic development of immune functions. A short primer PCR amplification method was employed to amplify fragments from Xenopus genomic DNA that are related to Ig light chains and TCR. One fragment was identified that appeared to represent a novel type of light chain and was used as a probe to recover the corresponding cDNA from a spleen cDNA library. We designate these light chains type III. Using an iterative screening procedure, six families of VL genes, two distinct JL and two distinct CL sequences, were identified. In a comparison of phylogenetically diverse light chains, the type III genes align with higher vertebrate lambda genes. Southern blot hybridization analyses with genomic DNA from different animals showed the VL and CL sequences to be both diverse and polymorphic. Intrafamily sequence comparisons of VL genes revealed additional diversity. Collectively, these studies confirm the existence of a third type of light chain gene in Xenopus, establish a high degree of genetic variation in the sequences encoding the light chain V regions, and provide the most significant evidence to date for the presence of a lambda-like light chain gene at the phylogenetic level of the amphibians.

Idiotype manipulation for autoimmune diseases: where are we going?

In the past few years, there has been considerable progress in the identification and characterization of B and T cell idiotypes associated with autoimmunity1,2. The identification of human VH and VL families and the more widely available techniques for sequencing the genes encoding these regions is beginning to help elucidate the structural basis of idiotypy. Awareness of these structures has stimulated experiments designed to suppress, specifically, aberrant immune responses. Such therapy is an attractive prospect but its clinical utility remains unproved. While the concept of the idiotype network as originally defined by Jerne2 has been modified since it was first proposed over 20 years ago, we share the view of Coutinho3 that its manipulation may well contribute solutions to autoimmune disease. In this article, we will outline some of the more recently described links between idiotypes and certain autoimmune conditions and examine the possibilities for the management of these disorders by modulation w...

B-Cell superantigens: Definition and potential impact on the immune response

Superantigens have been extremely helpful tools in exploring fundamental questions in immunobiology including mechanisms of cell activation, tolerance, and autoimmunity. Until recently, attention has been focused exclusive on T-cell superantigens. However, new data suggest that there are superantigens that directly activate B cells. By definition, these agents (1) stimulate a high frequency of B cells, (2) target B cells that have restricted usage of VH or VL family genes, and (3) bind to immunoglobulins outside the sites that bind conventional antigens. A candidate B-cell superantigen that has received considerable attention in this laboratory is staphylococcal protein A. This agent is best known to the immunologist because of its ability to bind to the Fc fragment of IgG. This binding has been localized to two alpha-helical structures on each of four or five homologous regions that comprise the extracellular domain of protein A. However, it is now clear that protein A contains a second site that binds to determinants on the Fab regions of certain immunoglobulins independently of their heavy-chain isotype. In man this so-called alternative site appears to bind only to immunoglobulins that utilize heavy-chain genes of the VH3 subfamily. In the mouse this type of binding is restricted to immunoglobulins using heavy chains belonging to the S107 and J606 VH families. In this review, we examine the growing list of microbial products that dominate B-cell superantigenic properties. Using staphylococcal protein A as a model for a B-cell superantigen, we consider the potential impact of this novel class of antigens on the immune response. We focus on the ability of B-cell superantigens to influence the expression of the B-cell repertoire. In addition, we consider the hypothesis that the interaction of a B-cell superantigen with its reactive serum immunoglobulins activates the classical complement cascade and thus represents a powerful stimulant of tissue inflammation.

Sequence analysis and antigen binding characteristics of Ig SCID Ig+ mice.

SCID mice as they age may develop a limited number of functional B and T cell clones. VH and VL gene sequences of hybridoma antibodies derived from the spleens of five adult SCID Ig+ mice revealed nine clonally unrelated, i.e. derived from different precursor lymphocytes, groups of Ig. Of these, four used the VH7183 family, one Q52, three J558 and one VH3609. VH7183 was seen in excess over J558 despite a 5-fold larger number of J558 family members in the mouse genome. Preferential use of D proximal VH genes along with reduced N region addition in the heavy chain VDJ joins of these antibodies suggest derivation from a population of lymphocytes with characteristics of the perinatal repertoire. All of the hybridomas produced kappa light chains of several different VL family groups. Sequences of 23 VH and 22 VL genes of hybridomas derived from one mouse (no. 45) showed that they were all clonally related, differing only in somatic mutations. Autoreactivity of these antibodies to cellular antigens [including (U1)RNP-specific A protein and SmB] was analyzed, and shown to correlate with the geneology and specific mutations within members of the clone. The geneological tree derived from sequence data permitted a study of the microevolution of a clone of B cells and showed that as it developed there was a tendency toward increased strength of self-reactive binding as well as alterations in specificity toward these autoantigens. Analysis of replacement:silent mutation ratios within members of this clone reflected limited affinity maturation in mouse 45 and none for another mouse, no. 58. These data are consistent with the progressive differentiation of B cells in an immunodeficient state in the absence of clonal competition with B cells of similar antigen specificities as they populate the spleen.

Variable Region Gene Analysis of Pathologic Human Autoantibodies to the Related i and I Red Blood Cell Antigens

Abstract To investigate the molecular basis of the autoimmune response to the related i and I carbohydrate antigens, we studied cold agglutinins (CA) from B-cell clones and from the peripheral circulation of patients with lymphoproliferative syndromes. Sequence analyses of expressed variable region genes indicate that both anti-i and anti-I specificities from B- cell clones from two patients are encoded by the VH4.21 or a very closely related VH4 heavy chain gene, whereas the expressed light chain genes differed. The anti-i-secreting B-cells express unmutated germline- encoded VH4.21 and VKI gene sequences. The VH region gene encoding anti- I has the closest homology (97%) to the VH4.21 germline gene and differs at the protein level by only three amino acids. In contrast, while the VL region gene encoding anti-I is most homologous (96%) to the VKIII, kv328 germline gene, there are seven amino acid differences due to nonrandom replacement mutations, which suggests a role for antigen-mediated selection in the anti-I response of this individual. These studies were extended by a structural survey of 20 additional serum CA using antipeptide antibodies specific for determinants in VH and VL regions. All anti-I and anti-i CA were shown to express VH4 heavy chains, and 14 of 17 CA expressed a previously described VH4 second hypervariable region determinant, termed VH4-HV2a. We also found that 13 of 14 anti-I CA used VKIII light chains, while the anti-i CA used light chains from at least three VL families. Taken together, the data show that anti-i and anti-I CA probably both derive from the VH4.21 gene (or a closely related gene). Furthermore, the restricted VH and different VL gene use in anti-i and anti-I CA may reflect the close structural relationship of the i and I antigens.

Variable region gene analysis of pathologic human autoantibodies to the related i and I red blood cell antigens

To investigate the molecular basis of the autoimmune response to the related i and I carbohydrate antigens, we studied cold agglutinins (CA) from B-cell clones and from the peripheral circulation of patients with lymphoproliferative syndromes. Sequence analyses of expressed variable region genes indicate that both anti-i and anti-I specificities from B-cell clones from two patients are encoded by the VH4.21 or a very closely related VH4 heavy chain gene, whereas the expressed light chain genes differed. The anti-i-secreting B-cells express unmutated germline-encoded VH4.21 and VKI gene sequences. The VH region gene encoding anti-I has the closest homology (97%) to the VH4.21 germline gene and differs at the protein level by only three amino acids. In contrast, while the VL region gene encoding anti-I is most homologous (96%) to the VKIII, kv328 germline gene, there are seven amino acid differences due to nonrandom replacement mutations, which suggests a role for antigen-mediated selection in the anti-I response of this individual. These studies were extended by a structural survey of 20 additional serum CA using antipeptide antibodies specific for determinants in VH and VL regions. All anti-I and anti-i CA were shown to express VH4 heavy chains, and 14 of 17 CA expressed a previously described VH4 second hypervariable region determinant, termed VH4-HV2a. We also found that 13 of 14 anti-I CA used VKIII light chains, while the anti-i CA used light chains from at least three VL families. Taken together, the data show that anti-i and anti-I CA probably both derive from the VH4.21 gene (or a closely related gene). Furthermore, the restricted VH and different VL gene use in anti-i and anti-I CA may reflect the close structural relationship of the i and I antigens.

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. II. IgG antibodies contain VH genes from a single VH family and VL genes from at least four VL families.

To define the V gene family repertoire of human IgG anti-Haemophilus influenzae type b polysaccharide antibodies, we purified six IgG1 and nine IgG2 anti-Hib-PS antibodies to monoclonality from immune serum of six individuals and performed N-terminal amino acid sequence analysis. Of the 15 clonal antibodies we examined, all H chain V regions were of the VHIII family. In contrast, the L chains of these antibodies were clearly from at least four different VL families; VKI, VKII, VKIII, and V lambda. Interestingly. VL family expression correlated with the cross-reactivity of these antibodies to the capsular carbohydrate of Escherichia coli K100. VKII antibodies did not cross-react, whereas antibodies expressing V lambda, VKI, or VKIII generally cross-reacted. We conclude that L chain V regions are very important contributors to the limited heterogeneity in this antibody repertoire.

Molecular characterization of antibodies bearing Id-460. I. The structure of two highly homologous VH genes used to produce idiotype positive immunoglobulins.

Abstract The heavy chain immunoglobulin genes encoding a variety of antibodies specific for DNP or Pasteurella pneumotropica and bearing the dominant idiotype of MOPC 460, Id-460, were cloned and sequenced. The VH genes encoding the M460 and D35 (DNP binding) antibodies were found to be homologous but not identical to the VH gene encoding the LB8 (P. pneumotropica binding) monoclonal antibody. Two of at least eight genes in the VH460 cross-hybridizing gene family can encode Id-460 positive antibodies. The VH460 gene family overlaps with the gene family described by VH36-60 and more completely describes this germ-line VH gene family. We have previously demonstrated a genetic requirement for VK1 expression in order to observe the expression of Id-460 in anti-DNP antibody responses. Southern blot analysis of these monoclonal antibody-producing cells demonstrates, as with the cross-hybridizing VH, that two cross-hybridizing VK1 genes (VK1A and VK1C) can be used to encode Id-460-positive immunoglobulins. We demonstrate that immunoglobulin idiotype determinant expression can be the result of the expression of nonidentical but highly homologous genes in the VH460 cross-hybridizing VH gene family and also in the VK1 cross-hybridizing VL family.