Human Kappa Chains 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.

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

Abstract 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 IG loci has hindered effective use of standard high-throughput methods for characterizing genetic variation within these regions. To overcome these limitations, we leverage 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 a common novel ∼24.7 Kbp structural variant harboring a functional IGKV gene. Among 47 functional IGKV genes, we identify 141 alleles, 64 (45.4%) of which were not previously curated. We report inter-population differences in allele frequencies for 14 IGKV genes, including alleles unique to specific populations within this dataset. In addition, we identify haplotypes carrying signatures of gene conversion that associate with enrichment of SNVs in the IGK distal region. Finally, we identify extensive polymorphism in the IG Lambda (IGL) locus, including copy number variation for genes encoding IGLC and IGLJ genes. These data lay a foundation for advancing our understanding of how genetic variation in IG loci impacts expressed antibody repertoires in human populations.

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.

A highly efficient modified human serum albumin signal peptide to secrete proteins in cells derived from different mammalian species

Signal peptides (SPs) are key elements in the production of recombinant proteins; however, little information is available concerning different SP in mammalian cells other than CHO. In order to study the efficiency of different SPs to direct the traffic along the secretory pathway of the green fluorescence protein (GFP) and a scFv-Fc fusion protein; CHO-K1, HEK293 and NS0 cell lines were transfected in a transient and stable way. SP of human azurocidin (AZ), modified human albumin (mSA), modified Cricetulus griseus Ig kappa chain V III region MOPC 63 like (mIgκ C) and modified human Ig kappa chain V III region VG (mIgκ H) were evaluated. The efficiency of SPs to translocate a propeptide across the ER membrane was evaluated by fluorescence microscopy and flow cytometry for the GFP inside the secretory pathway, and by antigen-specific indirect ELISA for the scFv-Fc outside the cell. The mSA SP was successful in directing the secretion of the active proteins in these different types of mammalian cells, regardless of the transgene copy number. The goal of this work was to demonstrate that a modified version of SA SP might be used in different mammalian cells employing the same expression vector.

Theraputic Monoclonal Antibody Interference In Immunofixation Electrophoresis

AbstractAbstract 4996The application of monoclonal antibodies represents an increasingly powerful weapon in the arsenal against hematologic diseases. With the advent of new therapies come previously unrecognized laboratory interference that if not properly identified could have clinical repercussions. We encountered two patients, neither of whom had a history of plasma cell dyscrasia, who underwent treatment with rituximab for immune thrombocytopenic purpura (ITP) and were discovered to have a monoclonal IgG kappa band on serum protein electrophoresis (SPEP) followed by immunofixation (IFX). Both patients had severe, steroid-resistant ITP and received rituximab on an empiric basis, at a weekly dose of 375 mg/m2 IV. In Patient #1, an evaluation for secondary causes of thrombocytopenia included SPEP and IFX. The IFX yielded a faint, atypical IgG kappa band that migrated to the far cathodal zone of the gel. No band was seen on SPEP alone. Since the patient was treated with rituximab the day before these studies were performed, we considered the possibility that the IgG kappa band was due to circulating rituximab. Rituximab is a chimeric antibody composed of human IgG1 kappa chain constant regions and heavy- and light-chain variable regions from a murine anti-CD20 antibody. The elimination half-life of this therapeutic antibody increases with subsequent doses and is approximately 60 hours for the first dose and 174 hours for the fourth dose (range 26 to 442 hours). An aliquot of rituximab was obtained from pharmacy and analyzed by SPEP/IFX, showing an IgG kappa monoclonal protein band identical to the M-protein in the patient's serum. Repeat assessment of serum from this patient obtained immediately prior to her fourth treatment with rituximab demonstrated that the IgG kappa paraprotein was less intense on IFX compared to the first IFX. Several hours after the fourth rituximab infusion was completed, a more intense band corresponding to the IgG kappa paraprotein was detected again on IFX. In Patient #2, who also received rituximab for steroid-resistant ITP, a similar circulating rituximab-related protein was detected on IFX performed one hour after his fourth rituximab infusion. IFX performed on a sample obtained immediately before treatment did not demonstrate a paraprotein, and testing one week after treatment showed that the band had resolved. We believe that awareness of the presence of a faint monoclonal protein by SPEP/IFX following recent administration of rituximab is important to avoid unnecessary further evaluation for a pathologic monoclonal gammopathy. Alternatively, if suspicion exists, SPEP/IFX should be conducted prior to the initiation of treatment with rituximab or after the treatment course is completed in order to avoid uncertainty. Furthermore, it is important to be cognizant of this finding in patients on maintenance rituximab for an indolent lymphoma and an associated paraprotein who may undergo interval monitoring of the monoclonal gammopathy. It is possible that a similar phenomenon may be seen with other therapeutic monoclonal antibodies with a dosing regimen and half-life that is similar to rituximab, such as cetuximab, a monoclonal antibody directed against the epidermal growth factor receptor. Finally, the presence of a characteristic band on immunofixation may allow for the qualitative assessment of the presence of a therapeutic monoclonal antibody in the serum. Further studies are necessary to determine the sensitivity and specificity of such a laboratory test. Disclosures:Off Label Use: Rituximab is an anti-CD20 chimeric antibody and is used for the treatment of immune thrombocytopenic purpura.

A Novel Therapeutic Target in Inflammatory Uveitis: Transglutaminase 2 Inhibitor

PurposeOur goal was to investigate the effects of inhibition of transglutaminase 2 (TGase 2) on endotoxin-induced uveitis (EIU)MethodsEIU was induced in female Lewis rats by single footpad injections of 200 µg of lipopolysaccharide (LPS). TGase 2 inhibitors were administered intraperitoneally 30 minutes before and at the time of LPS administration. Rats were sacrificed 24 hours after injection, and the effects of the TGase 2 inhibitors were evaluated by the number of intraocular inflammatory cells present on histologic sections and by measuring the TGase 2 activity and TGase products in the aqueous humor (AqH). TGase 2 substrates were also assayed in AqH from uveitis patients.ResultsClinical indications of EIU, the number of cells present on histologic sections, and TGase 2 activity in AqH increased in a time-dependent manner, peaking 24 hours after LPS injection. Inflammation in EIU was significantly reversed by treatment with TGase inhibitors. A 23-kDa cross-linked TGase substrate was identified in the AqH from EIU rats and uveitis patients. MALDI-TOF analysis showed that this substrate in uveitis patients was human Ig kappa chain C region.ConclusionsTGase 2 activity and its catalytic product were increased in the AqH of EIU rats. TGase 2 inhibition attenuated the degree of inflammation in EIU. Safe and stable TGase inhibitors may have great potential for the treatment of inflammatory uveitis.

Enhancement of Antibody-dependent Cellular Cytotoxicity by Tandem Fc Multimerization

Monoclonal antibodies are being used as therapeutics for a number of cancers, such as leukemia, breast and colon cancers, and a lot of monoclonal antibodies specific for tumor-related antigens have been on clinical trials. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms by which antibodies exert anti-tumor effects. ADCC occurs through interaction between the Fc domains of IgG antibodies bound to target cells and Fcgamma receptors on the surface of effector cells. In our study, a chimeric antibody, designated M-Ab, was constructed with the V regions from mouse anti-CD20 mAb 1F5 and the C regions from human IgG1 and kappa chain. Two or three Fc domains were tandemly repeated downstream of the C-terminus of the M-Ab to give D0-Ab (Fc dimer Ab without a linker), T0-Ab (Fc trimer Ab without a linker), and T3-Ab (Fc trimer Ab with a (GGGGS)(3) linker in front of the second and third hinge regions). Here, we show that Fc tandem repeat antibodies bind to all the low-affinity Fcgamma receptors with very potent avidities and have greatly enhanced ADCC activity. T3-Ab is about 100 times more potent than the parental 1F5 chimeric antibody in terms of both Fcgamma receptor binding and exerted ADCC activity at a 50-100 times less concentration as compared with the parental antibody. Thus, Fc tandem repeat antibodies are anticipated to be candidates for anti-tumor therapeutics and useful tools to elucidate the biological roles of Fcgamma receptors.

Antibody reactivity against animal retroviruses in multiple sclerosis

A total of 27 MS patients and 82 controls were studied for presence of antibodies reacting with maedi-visna virus and bovine leukemia virus by means of 2 ELISA techniques. Furthermore, the differentiated antibody response against BLV antigens was evaluated by immunoblotting. People with elevated activities against MVV were detected in the MS group as well as controls. Elevated antibody activity against BLV as determined by biotinylated protein A and peroxidase-labelled streptavidin tended to occur more frequently in MS patients than in controls. No such difference was found, however, when peroxidase-conjugated antibodies against human kappa chain were used for detection of antibodies reacting with BLV. Immunoblotting revealed no difference between the MS and the control group when anti-kappa antibodies were used to detect antibody binding to BLV antigens. The protein A method revealed significantly decreased antibody activity against a BLV antigen with molecular weight of about 29 kD in MS compared with controls, whereas a significant rise in number of MS patients responding to a component of about 53 kD was observed. The differences between the 2 detection systems could be due to unspecific reactions. The present data indicate that MS patients exhibit an inhomogeneous and, in some cases elevated, antibody activity against BLV.

The reported germline repertoire of human immunoglobulin kappa chain genes is relatively complete and accurate

We describe a bioinformatic analysis of germline and rearranged immunoglobulin kappa chain (IGK) gene sequences, performed in order to assess the completeness and reliability of the reported IGK repertoire. In contrast to the reported heavy-chain gene repertoire, which includes many dubious sequences, only five IGK variable gene (IGKV) alleles appear to have been reported in error. There was, however, insufficient evidence to justify removing these IGKV genes from the germline repertoire. Bioinformatic analysis of apparent mismatches between reported germline genes and 1,863 expressed IGK sequences suggested the existence of two unreported IGKV polymorphisms. Genomic screening of 12 individuals led to the confirmation of both of these polymorphisms, IGKV1-16*02 and IGKV2-30*02. We also show that in contrast to the heavy chain, the IGK repertoire is dominated by sequences that use just a handful of kappa variable (IGKV) and junction (IGKJ) gene pairs. There is also little modification of IGKV and IGKJ genes by the processes of exonuclease removal and N nucleotide addition. The expressed IGK repertoire therefore lacks diversity and the junction region is particularly constrained. Remarkably, the analysis of a dataset of 435 relatively unmutated rearranged kappa genes showed that ten amino acid sequences account for almost 10% of the rearrangements, with identical sequences being derived from as many as seven independent sources. Such dominant sequences are likely to have important roles in the operation of the humoral immune response.

Molecular basis for the cross-reactive idiotypes on human anti-IgG autoantibodies (rheumatoid factors).

High titres of anti-IgG autoantibodies (rheumatoid factors, RF) are characteristic of patients with rheumatoid arthritis, Sjögren's syndrome, and mixed cryoglobulinaemia, and may contribute to immune complex formation and tissue damage. The monoclonal RFs from cryoglobulinaemia patients frequently display cross-reactive idiotypes. The genetic basis for the cross-reactive idiotypes on RF autoantibodies has not been determined. To clarify structural and genetic relationships among RFs from unrelated subjects, a series of anti-peptide antibodies have been generated that define primary sequence-dependent idiotypes on RF heavy and light chains. Multiple monoclonal and polyclonal RFs from unrelated individuals have been probed by Western blotting with the anti-idiotypic reagents. The results show that sequences in the kappa light chain variable region represent a common structural element among RF autoantibodies. This hypothesis is confirmed by the cloning and sequencing of the conserved germline variable region gene which encodes human RF kappa chains.

Generation and characterization of a high‐affinity chimaeric antibody against hepatitis B surface antigen

Antibody against HBsAg (hepatitis B surface antigen) is advocated for the passive immunotherapy in certain cases of hepatitis B infections. A recombinant monoclonal antibody against HBsAg would offer several advantages over the currently used polyclonal human hepatitis B immunoglobulin. 5S is a mouse monoclonal antibody that binds to HBsAg with very high affinity. However, this mouse antibody cannot be used for therapeutic purposes, as it may elicit antimouse immune responses. Chimaerization, by replacing mouse constant domains with human counterparts, can reduce the immunogenicity of this molecule. We have cloned the V(H) (heavy-chain variable region) and V(L) (light-chain variable region) genes of this mouse antibody, and fused them with C(H)1 (heavy-chain constant domain 1) of human IgG1 and C(L) (light-chain constant domain) of human kappa chain respectively. These chimaeric genes were cloned into a mammalian expression vector (pFab-CMV), which has a modular cassette coding for part of the hinge, C(H)2 and C(H)3 of human IgG1. The recombinant construct was transfected in CHO (Chinese-hamster ovary) cells to generate a stable transfectoma. The resulting transfectoma was maintained in a serum-free medium and the full-length chimaeric anti-HBsAg antibody was purified from the culture supernatant. The yield of the purified chimaeric antibody was moderate ( approximately 5.5 mg/l). We further characterized the chimaeric antibody using several in vitro techniques. It was observed that the chimaeric molecule was glycosylated and expressed in the expected heterodimeric form. This chimaeric antibody has very high affinity and specificity, similar to that of the original mouse monoclonal antibody.

High affinity mouse-human chimeric Fab against Hepatitis B surface antigen

Passive immunotherapy using antibody against hepatitis B surface antigen (HBsAg) has been advocated in certain cases of Hepatitis B infection. We had earlier reported on the cloning and expression of a high affinity scFv derived from a mouse monoclonal (5S) against HBsAg. However this mouse antibody cannot be used for therapeutic purposes as it may elicit anti-mouse immune responses. Chimerization by replacing mouse constant domains with human ones can reduce the immunogenicity of this antibody. We cloned the V(H) and V(L) genes of this mouse antibody, and fused them with CH1 domain of human IgG1 and C(L) domain of human kappa chain respectively. These chimeric genes were cloned into a phagemid vector. After initial screening using the phage display system, the chimeric Fab was expressed in soluble form in E. coli. The chimeric Fab was purified from the bacterial periplasmic extract. We characterized the chimeric Fab using several in vitro techniques and it was observed that the chimeric molecule retained the high affinity and specificity of the original mouse monoclonal. This chimeric antibody fragment was further expressed in different strains of E. coli to increase the yield. We have generated a mouse-human chimeric Fab against HBsAg without any significant loss in binding and epitope specificity. This chimeric Fab fragment can be further modified to generate a full-length chimeric antibody for therapeutic uses.

Identification of a type of human IgG-like protein in shrimp Penaeus vannamei by mass spectrometry

Recently, infectious diseases have delayed the growth of shrimp aquaculture. Interest has been focused on immune molecules and defense mechanisms to reduce these diseases in shrimp aquaculture. In invertebrates, various immunoglobulin superfamily (IgSF) molecules have been characterized in body tissues and fluids, which play a significant role in innate defense. In the current study, we found that a protein in shrimp serum, referred as an IgG-like protein, could be reacted with goat anti-human IgG, specifically. The IgG-like protein was purified from the serum of shrimp Penaeus vannamei by affinity chromatography using CNBr-activated sepharose 4B. The purified protein was subsequently analysed using one-dimensional sodium sulphate-polyacrylamide gel electrophoresis (1-DE), two-dimensional sodium sulphate-polyacrylamide gel electrophoresis (2-DE) and immunoblotting. Furthermore, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and quadrupole time-of-flight (Q-TOF) tandem mass spectrometry methods were used for peptide mass fingerprint (PMF) and sequencing of the protein, respectively. Sequence information and PMF queried against the NCBI database confirmed the identity of the protein as hemocyanin. Conserved domain search showed that there was an Ig-like conserved domain of 252 amino acid residues in the C-terminus of arthropoda hemocyanins. In addition, four and one conserved regions were found between hemocyanin and human Ig heavy chain and Ig kappa chain, respectively. These results indicate that in addition to copper-binding domains hemocyanin has an Ig-like conserved domain, which would confer some new functions to multifunctional respiratory pigment of crustaceans.

Diversification of exogenous genes in vivo in Neurospora.

We have adapted the meiotic recombination hotspot cog of Neurospora crassa for shuffling exogenous DNA, providing a means of generating novel genes in situ from sequences introduced into chromosomes. Genes to be diversified are inserted between the his-3 locus and cog. Diversification crosses are heterozygous both for alleles of the exogenous DNA and for auxotrophic alleles of his-3. Progeny selected for ability to grow without histidine supplementation are enriched for exchange events within the exogenous DNA. Exchange events initiated by cog can propagate past DNA sequences mismatched for more than 370 bp and complete exchanges in patches of matched sequence as short as 24 bp, parameters that make the system suited for use in the directed evolution of genes for protein engineering. Here we demonstrate the system by shuffling human immunoglobulin kappa chain genes and also endoglucanase genes derived from different species of fungi.

Chimeric Human/Murine Monoclonal IgM Antibodies to HIV‐1 Nef Antigen Expressed on Chronically Infected Cells

Human IgM antibody (Ab) to gangliosides induced cytolysis of HIV-1-infected cells by homologous human complement. We expected that any human IgM Ab reactive with HIV-1 infected cells could cause complement-mediated cytolysis. The trans-chromosome mouse (TC mouse) contains human chromosomes harboring genes responsible for immunoglobulin production. Spleen cells from TC mice immunized with recombinant Nef were fused with mouse myeloma cells to generate hybridomas, and we selected those that produced human mu-chain-positive Abs reactive with Nef fixed on an ELISA plate. However, the L-chain of the monoclonal Abs (mAbs) were murine lambda in type and were chimeric, and we could not succeed in obtaining mAb with human mu- and human kappa-chains. The chimeric mAbs reacted with the HIV-1 infected cells as seen with flow cytometric analysis, and the surface expression of Nef was also detectable on chronically infected OM10.1 cells which had no detectable gp120. However, although the reaction of the chimeric IgM mAb with HIV-1-infected MOLT4 cells induced C3 deposition on cell surfaces on incubation with fresh human serum, the cells remained unlysed, as determined by 51Cr release assay. The amount of Nef antigen on the cells might not have been high enough to overcome the function of HRF20 (CD59) that restricts formation of membrane attack complexes of homologous complement. However, combination of anti-Nef IgM mAb with other IgM mAbs reactive with the surface of HIV-1-infected cells may induce a synergistic effect in complement mediated cytolysis.

Human kappa-like expression in the axial organ of the sea star Asterias rubens (Echinoderma).

The axial organ of sea star Asterias rubens is a primitive immune organ which in certain conditions produces an antibody-like factor (ALF). This A.L.F. presents homologies with the human kappa chain. The ALF could be composed of many kappa chains devoid of any disulfide bond. The ontogeny of kappa expression was studied. Just after metamorphosis of the sea star, certain mesodermic cells which become axial organ cells express human kappa-like activity.

An efficient route to the production of an IgG-like bispecific antibody.

Production of IgG-form bispecific antibody (BsAb-IgG) by co-expressing two antibodies in transfected cells is often inefficient owing to the unwanted pairing between the component heavy and light chains. We have developed an efficient method for the production of a novel IgG-like BsAb by using the natural dimerization mechanism between IgG heavy and light chains. Two single-chain Fv (scFv) of different specificity are fused to the constant domain of human kappa chain (C(L)) and the first constant domain of human heavy chain (C(H1)), to form two polypeptides, (scFv)(1)-C(L) and (scFv)(2)-C(H1)-C(H2)-C(H3), respectively. Co-expression of the two polypeptides in mammalian cells results in the formation of a covalently linked IgG-like hetero-tetramer, Bs(scFv)(4)-IgG, with dual specificity. Our approach yields a homogeneous bispecific IgG-like antibody product with each molecule containing four antigen binding sites, two for each of its target antigens. A Bs(scFv)(4)-IgG was prepared using two scFv antibodies each directed against a different epitope of a vascular endothelial growth factor receptor, the kinase insert domain-containing receptor (KDR). The Bs(scFv)(4)-IgG is capable of simultaneously binding to the two epitopes on the receptor. Further, the Bs(scFv)(4)-IgG also retains the antigen-binding efficacy and biological activity of its component antibodies.

Improved detection of Beet necrotic yellow vein virus in a DAS ELISA by means of antibody single chain fragments (scFv) which were selected to protease-stable epitopes from phage display libraries.

The detection of Beet necrotic yellow vein virus (BNYVV) in stored sugar beets by means of monoclonal antibodies or antibody single chain fragments (scFv) often poses problems, because the immunodominant C-terminal epitope of the viral coat protein is readily lost due to proteolysis. Clones which produce scFv specific for protease-stable BNYVV epitopes were selected from two naive phage display libraries. Fusion proteins of the scFv with a human IgG kappa chain (expressed from the newly designed vector pCL) or with alkaline phosphatase,respectively, allow the ELISA detection of BNYVV even in stored sugar beets with a sensitivity which was comparable or often higher than that achieved with polyclonal antibodies.

A very limited number of keywords (main patterns) describes all sequences of the human variable heavy (VH) and kappa (Vkappa) domains.

Sequences of the variable heavy (VH) and kappa (Vkappa) domains of Ig structures were divided into 21 fragments that correspond to strands, loops, or parts of these structural units of the variable domains. Amino acid sequences of fragments (termed "words") were collected from the 1,172 human heavy and 668 human kappa chains available in the Kabat database. Statistical analysis of words of 17 fragments was performed (fragments that comprise the complementary determining regions' fragments will not be discussed in this paper). The number of different words (those with different residues in at least one position) ranged, for various fragments, from 11 to 75 in the kappa chains, and from 23 to 189 in the heavy chains. The main result of this study is that very few keywords, or main patterns of words, were necessary to describe over 90% of the sequences (no more than two keywords per fragment in the kappa and no more than five per fragment in the heavy chains). No identical keywords were found for different fragments of the variable domains. Keywords of aligned fragments of the VH and Vkappa domains were different in all but two instances. Thus, knowing the keywords, one can determine whether any given small part of a sequence belongs to a heavy or kappa chain and predict its precise localization in the sequence. In addition, by using all of the keywords obtained through analysis of the Kabat database, it was possible to describe completely the sequences of the human VH and Vkappa germ-line segments.