Kappa Chain Loci Research Articles

Resolving haplotype variation and complex genetic architecture in the human immunoglobulin kappa chain locus in individuals of diverse ancestry

Immunoglobulins (IGs), critical components of the human immune system, are composed of heavy and light protein chains encoded at three genomic loci. The IG Kappa (IGK) chain locus consists of two large, inverted segmental duplications. The complexity of the IG loci has hindered use of standard high-throughput methods for characterizing genetic variation within these regions. To overcome these limitations, we use long-read sequencing to create haplotype-resolved IGK assemblies in an ancestrally diverse cohort (n = 36), representing the first comprehensive description of IGK haplotype variation. We identify extensive locus polymorphism, including novel single nucleotide variants (SNVs) and novel structural variants harboring functional IGKV genes. Among 47 functional IGKV genes, we identify 145 alleles, 67 of which were not previously curated. We report inter-population differences in allele frequencies for 10 IGKV genes, including alleles unique to specific populations within this dataset. We identify haplotypes carrying signatures of gene conversion that associate with SNV enrichment in the IGK distal region, and a haplotype with an inversion spanning the proximal and distal regions. These data provide a critical resource of curated genomic reference information from diverse ancestries, laying a foundation for advancing our understanding of population-level genetic variation in the IGK locus.

Abstract P2-19-01: Characterization of fully human internalizing anti-HER2 monoclonal antibodies that compete with Trastuzumab for binding to HER2

Abstract The human epidermal growth factor receptor (HER) family of receptors plays a significant role in the pathogenesis of several human cancers. Among them, HER2 is overexpressed in several cancers. In particular, overexpression with or without gene amplification occurs in 15-30% of breast cancers and this has both prognostic and predictive implications. HER2 overexpression and amplification is associated with shorter disease-free and overall survivals and with resistance to certain hormonal agents as well as increased risk of metastasis to the brain. Two types of treatment targeting HER2 biological activity have been developed: anti-Her2 monoclonal antibodies and small molecule tyrosine kinase inhibitors such as lapatinib and neratinib targeting HER2 and EGFR and Afatinib targeting all HER family members. Trastuzumab is a humanized anti-HER2 monoclonal antibody that binds to domain IV of HER2 extracellular segment and blocks its signaling. It is the first therapeutic antibody targeting Her2 overexpression approved by the FDA. It is administered in combination with standard of care chemotherapy. More recently, since it was shown that Trastuzumab was an internalizing antibody, two antibody drug conjugates using trastuzumab have been approved and used in the standard of care: Ado-Trastuzumab-Emtansine (Kadcyla) consisting of trastuzumab conjugated to the drug mertansine DM1 and fam-trastuzumab-deruxtecan-nhki (Enhertu). Enhertu is another antibody drug conjugate using Trastuzumab to deliver a topoisomerase inhibitor deruxtecan. Our laboratory has been focused in developing fully human internalizing anti-Her2 antibodies that compete or not with trastuzumab for binding to Her2. These antibodies have been developed by immunizing fully human mice with recombinant Her2 protein. Human Ab producing Tc mice (TC-mAb mice) stably maintain a mouse-derived engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig knockout background. After production of hybridoma secreting fully human immunoglobulins, the screening process included inhibition of binding of trastuzumab to Her2 by enzyme linked immunoassay and by Octet epitope binning as well as internalization assay. Several internalizing antibodies with Kd ranging from 10-9 M to 10-12 M were selected. They were stratified by their ability to compete or not with trastuzumab. They were further characterized for their ability to deliver a cytotoxic payload in Her2 overexpressing cells as well as for their efficacy to inhibit proliferation, signaling and migration of Her2 overexpressing breast cancer cells. Data related to these antibodies will be presented here. In conclusion, the use of fully human TC mice in our laboratory represents an attractive approach to develop fully human monoclonal antibodies to high value cancer targets that can by-pass the need for humanization and affinity maturation of antibodies. Citation Format: Ginette Serrero, Jianping Dong, Binbin Yue, Udaya Yerramalla, Jun Hayashi. Characterization of fully human internalizing anti-HER2 monoclonal antibodies that compete with Trastuzumab for binding to HER2 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-19-01.

Efficient human-like antibody repertoire and hybridoma production in trans-chromosomic mice carrying megabase-sized human immunoglobulin loci

Trans-chromosomic (Tc) mice carrying mini-chromosomes with megabase-sized human immunoglobulin (Ig) loci have contributed to the development of fully human therapeutic monoclonal antibodies, but mitotic instability of human mini-chromosomes in mice may limit the efficiency of hybridoma production. Here, we establish human antibody-producing Tc mice (TC-mAb mice) that stably maintain a mouse-derived, engineered chromosome containing the entire human Ig heavy and kappa chain loci in a mouse Ig-knockout background. Comprehensive, high-throughput DNA sequencing shows that the human Ig repertoire, including variable gene usage, is well recapitulated in TC-mAb mice. Despite slightly altered B cell development and a delayed immune response, TC-mAb mice have more subsets of antigen-specific plasmablast and plasma cells than wild-type mice, leading to efficient hybridoma production. Our results thus suggest that TC-mAb mice offer a valuable platform for obtaining fully human therapeutic antibodies, and a useful model for elucidating the regulation of human Ig repertoire formation.

Allelic 'choice' governs somatic hypermutation in vivo at the immunoglobulin κ-chain locus

Monoallelic demethylation and rearrangement control allelic exclusion of the immunoglobulin kappa-chain locus (Igk locus) in B cells. Here, through the introduction of pre-rearranged Igk genes into their physiological position, the critical rearrangement step was bypassed, thereby generating mice producing B cells simultaneously expressing two different immunoglobulin-kappa light chains. Such 'double-expressing' B cells still underwent monoallelic demethylation at the Igk locus, and the demethylated allele was the 'preferred' substrate for somatic hypermutation in each cell. However, methylation itself did not directly inhibit the activation-induced cytidine-deaminase reaction in vitro. Thus, it seems that the epigenetic mechanisms that initially bring about monoallelic variable-(diversity)-joining rearrangement continue to be involved in the control of antibody diversity at later stages of B cell development.

Differential accessibility at the kappa chain locus plays a role in allelic exclusion.

Gene rearrangement in the immune system is always preceded by DNA demethylation and increased chromatin accessibility. Using a model system in which rearrangement of the endogenous immunoglobulin kappa locus is prevented, we demonstrate that these epigenetic and chromatin changes actually occur on one allele with a higher probability than the other. It may be this process that, together with feedback inhibition, serves as the basis for allelic exclusion.

The endogenous retrovirus Mtv-8 on mouse chromosome 6 maps near several kappa light chain markers.

Endogenous retroviruses are known to affect expression of cellular genes in the vicinity of their integration sites. The endogenous mouse mammary tumor provirus (Mtv-8) previously has been reported to reside on mouse chromosome 6 near the immunoglobulin kappa chain locus. Using pairs of mouse strains on the BALB/c (Mtv-8 positive) and C58 (Mtv-8 negative) backgrounds which are congenic for chromosome 6 genetic markers, we have confirmed the chromosome assignment of this provirus. Moreover, we have analyzed the N1 progeny of a (B6 X C58) X C58 backcross to determine the segregation of the Mtv-8 provirus with respect to polymorphisms in the Igk-VSer and Igk-J loci. The results with congenic and backcross mice together with results of others suggest that Mtv-8 is located approximately 0.52 cM from several closely linked kappa markers on chromosome 6.

The active immunoglobulin kappa chain gene is packaged by non-ubiquitin-conjugated nucleosomes.

To elucidate the molecular features of active chromatin, we have mapped, by two-dimensional electrophoresis, the protein composition of nucleosomes that package the immunoglobulin kappa chain gene of mouse plasmacytoma cells. Nucleoprotein particles that possess the active kappa chain gene comigrate with bulk mononucleosomes that contain high mobility group proteins HMG-14 or -17 but lack histone H1. High electrophoretic resolution of the underlying core particles, after removal of ubiquitin by isopeptidase treatment, reveals that these nucleosomes are nonubiquitinated, even though they coincidently migrate with bulk ubiquitinated particles. This distinctive electrophoretic behavior may be correlated with the presence of histone H2A.X. Nucleosomes exhibiting these unusual properties appear to span at least 10 kilobases, in both transcribed and nontranscribed regions, suggesting that mechanisms independent of transcription exist to initiate, maintain, and propagate a common chromatin phenotype over long distances along the kappa chain locus.

Variant (6 ; 15) translocation in a murine plasmacytoma occurs near an immunoglobulin kappa gene but far from the myc oncogene.

Chromosome translocations in B-lymphoid tumours are providing intriguing insights and puzzles regarding the role of immunoglobulin genes in the activation of the myc oncogene (reviewed in refs 1, 2). The 15 ; 12 translocations found in most murine plasmacytomas and the analogous 8 ; 14 translocation in human Burkitt's lymphomas involve scissions of murine chromosome 15 (human chromosome 8) near the 5' end of the c-myc gene and subsequent fusion near an immunoglobulin heavy-chain gene. The less well characterized 'variant' translocations found in about 15% of such tumours also involve the myc-bearing chromosome band, but exchange occurs with a chromosome bearing an immunoglobulin light-chain locus--in mice, the kappa-chain locus bearing chromosome 6 (refs 3-5) and, in man, chromosome 2 (or 22), at the same band at which the kappa (or lambda) locus lies (reviewed in ref. 1). The Burkitt variant translocations involve scissions 3' of c-myc; one 8 ; 22 translocation placed the C lambda locus just 3' of c-myc, but usually the chromosome 8 breakpoint is a greater, but unknown, distance away from c-myc, more than 20 kilobases (kb) in one 8 ; 2 translocation involving the C kappa gene. Little is known about the murine 6 ; 15 translocations, although a C kappa gene cloned from one plasmacytoma (PC7183) is linked, via chromosome 12 sequences, to an unidentified region of chromosome 15 (ref. 11). We describe here the chromosome fusion region from plasmacytoma ABPC4, which displays the typical reciprocal 6;15 translocations. We find that the chromosome 6 breakpoint is near C kappa but, unlike those in the heavy-chain locus, not at a position where immunoglobulin genes normally recombine. Moreover, the chromosome 15 sequences involved in the ABPC4 translocation are not derived from the vicinity of c-myc.

Murine T cell receptor beta chain is encoded on chromosome 6.

Southern blot analysis of somatic cell hybrid lines indicates that the beta chain of the T cell receptor for antigen maps to chromosome 6 of the mouse. An experiment testing hybridization of the constant region of this gene to DNA from a hybrid cell line containing a translocation of chromosome 6 supports the localization of this gene to the proximal (centromeric) one-third of chromosome 6, in the same general region as the immunoglobulin kappa chain locus. This may be another indication of the shared evolutionary origins of the genes encoding both T and B cell antigen recognition.

Recombination between kappa chain genetic markers in the mouse.

In this study we report the first instance of recombination between kappa chain genetic markers in the mouse. The recombination frequency, 0.45% (95% limits, 0.12-1.61), is similar to that previously found for recombination between the kappa chain locus and the Lyt-2,3 locus (0.3%, 95% limits, 0.05-1.6), but is relatively low in comparison with that found at the heavy chain locus (0.41-5.4%). Lyt-2,3-typing of the recombinants permits a partial ordering of the kappa chain and Lyt-2,3 loci as (Lyt-2,3, Igk-Ef1) - Igk-Ef2. Light chains controlled by the two kappa markers include the Vk-(ser) subgroup (controlled by Igk-Ef1) and Vk-1 (controlled by Igk-Ef2). One of the recombinants has been recovered in a homozygous state ("NAK") and should be suitable for Vk gene mapping studies.

The translocated c-myc oncogene of Burkitt lymphoma is differentially regulated in lymphoblastoid vs plasma cells.

We have shown that the c-myc oncogene translocates from its normal position on band q24 of chromosome 8 to the heavy chain locus on chromosome 14 in Burkitt lymphomas with the t(8;14) chromosome translocation, while the c-myc oncogene remains on chromosome 8 and either the lambda or the kappa chain locus translocates to a region distal to the c-myc oncogene in Burkitt lymphoma with the t(8;22) and t(2;8) translocations, respectively (Dalla Favera 1982, 1983; Erikson 1983, 1983a; Croce 1983). Independently of whether it is structurally rearranged, the c-myc oncogene involved in the translocation is transcriptionally active, while the c-myc oncogene on the normal chromosome 8 is transcriptionally silent (Erikson 1983, 1983a; Croce 1983; Nishikura 1983; ar-Rushdi 1983). We have also shown that while the c-myc oncogene involved in the translocation escapes normal transcriptional control, the untranslocated c-myc oncogene on normal chromosome 8 is repressed in plasma cells (Erikson 1983; 1983a; Croce 1983; Nishikura 1983; ar-Rushdi 1983). To determine whether the activation of the translocated c-myc oncogene depends on the differentiated state of the B cells harboring the c-myc oncogene involved in the translocation, we have hybridized different human Burkitt lymphoma cells with human lymphoblastoid cells and with mouse plasmacytoma cells and examined the hybrids for the expression of the normal and of the involved c-myc oncogene.KeywordsBurkitt LymphomaLymphoblastoid Cell LineDaudi CellST486 CellHuman Lymphoblastoid CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

An immunoglobulin V gene polymorphism in the rat.

Anti-phenyloxazolone (phOx) antibodies of different AVN rats (primary response) share several isoelectric focusing bands. These bands were not shared by antibodies of some other rat strains, including DA. An anti-idiotype reagent was prepared (in rabbits) that bound radioactive anti-phOx antibodies of AVN rats but not normal AVN immunoglobulin. This binding was strongly inhibited by AVN anti-phOx antisera, but not by AVN anti-BOC-p-azobenzene arsonate-tyrosine antisera or DA anti-phOx antisera. Anti-phOx antisera of (AVN X DA)F1 rats were also strongly inhibitory indicating the presence of the idiotype (Ox-r1). Antisera of backcross rats (AVN X DA) X DA either resembled F1 hybrid sera (31 rats) or DA sera (23 rats). The data suggest that the presence of idiotype Ox-r1 is controlled by one gene, or genes linked to each other. The gene(s) is not linked to the Ig kappa chain locus. It may be a V gene of the Ig H chain.