Mouse Kappa Light Chain Research Articles

Engineering and expression of a chimeric transferrin receptor monoclonal antibody for blood–brain barrier delivery in the mouse

Protein therapeutics may be delivered across the blood-brain barrier (BBB) by genetic fusion to a BBB molecular Trojan horse. The latter is an endogenous peptide or a peptidomimetic monoclonal antibody (MAb) against a BBB receptor, such as the insulin receptor or the transferrin receptor (TfR). Fusion proteins have been engineered with the MAb against the human insulin receptor (HIR). However, the HIRMAb is not active against the rodent insulin receptor, and cannot be used for drug delivery across the mouse BBB. The rat 8D3 MAb against the mouse TfR is active as a drug delivery system in the mouse, and the present studies describe the cloning and sequencing of the variable region of the heavy chain (VH) and light chain (VL) of the rat 8D3 TfRMAb. The VH and VL were fused to the constant region of mouse IgG1 heavy chain and mouse kappa light chain, respectively, to produce a new chimeric TfRMAb. The chimeric TfRMAb was expressed in COS cells following dual transfection with the heavy and light chain expression plasmids, and was purified by protein G affinity chromatography. The affinity of the chimeric TfRMAb for the murine TfR was equal to the 8D3 MAb using a radio-receptor assay and mouse fibroblasts. The chimeric TfRMAb was radio-labeled and injected into mice for a pharmacokinetics study of the clearance of the chimeric TfRMAb. The chimeric TfRMAb was rapidly taken up by mouse brain in vivo at a level comparable to the rat 8D3 MAb. In summary, these studies describe the genetic engineering, expression, and validation of a chimeric TfRMAb with high activity for the mouse TfR, which can be used in future engineering of therapeutic fusion proteins for BBB drug delivery in the mouse.

Rabbit Immune Repertoires as Sources for Therapeutic Monoclonal Antibodies: The Impact of Kappa Allotype-correlated Variation in Cysteine Content on Antibody Libraries Selected by Phage Display

The rabbit immune repertoire has long been a rich source of diagnostic polyclonal antibodies. Now it also holds great promise as a source of therapeutic monoclonal antibodies. On the basis of phage display technology, we recently reported the first humanization of a rabbit monoclonal antibody. The allotypic diversity of rabbit immunoglobulins prompted us to compare different rabbit immune repertoires for the generation and humanization of monoclonal antibodies that bind with strong affinity to antigens involved in tumor angiogenesis. In particular, we evaluated the diversity of unselected and selected chimeric rabbit/human Fab libraries that were derived from different kappa light chain allotypes. Most rabbit light chains have an extra disulfide bridge that links the variable and constant domains in addition to the two intrachain disulfide bridges shared with mouse and human kappa light chains. Here we evaluate the impact of this increased disulfide bridge complexity on the generation and selection of chimeric rabbit/human Fab libraries. We demonstrate that rabbits with mutant bas and wild-type parental b9 allotypes are excellent sources for therapeutic monoclonal antibodies. Featured among the selected clones with b9 allotype is a rabbit/human Fab that binds with a dissociation constant of 1 nM to both human and mouse Tie-2, which will facilitate its evaluation in mouse models of human cancer. Examination of 228 new rabbit antibody sequences allowed for a comprehensive comparison of the LCDR3 and HCDR3 length diversity in rabbits. This study revealed that rabbits exhibit an HCDR3 length distribution more closely related to human antibodies than mouse antibodies.

A method of estimating the numbers of human and mouse immunoglobulin V-genes.

Mutations in immunoglobulin V-genes can be due to gene multiplication, allelic variations, mutations induced by antigens or somatic mutations, etc., and various combinations of these. Since the number of different mouse lambda light V-gene nucleotide sequences is relatively small, a pairwise comparison between these sequences can provide a rough idea as to the contributions of the above mechanisms to the number of nucleotide differences between sequences. A plot of occurrences against the number of differences suggests that differences between one to five can be attributed to somatic mutations. Six to 12 differences can be allelic. Thirteen to 17 may be due to allelic variations together with somatic mutations. Differences > 17 appear to be derived from gene multiplication. Although these numbers are most likely somewhat different in humans, they can nevertheless provide a rough guide to sort out the effect of gene multiplication. Estimations of human heavy, kappa and lambda light chain immunoglobulin V-genes are in reasonably good agreement with recent experimental studies. For mouse kappa light and heavy chains, our estimations can provide some insight to future analyses by direct sequencing of these gene segments.

Chimaeric monoclonal antibodies encoded by the human VH26 gene from naïve transgenic mice display a wide range of antigen-binding specificities.

To elucidate the molecular basis for the ability of antibodies encoded by the human VH26 heavy-chain variable region gene to react with diverse antigens, we have generated 34 hybridomas secreting chimaeric monoclonal antibodies (human mu heavy chain/mouse light chains) from transgenic mice. The transgenic mice carry an immunoglobulin minilocus containing the human VH26 gene, human DH and JH gene segments, and genes encoding the human C mu region. The minilocus in these animals undergoes functional rearrangement resulting in the production of chimaeric antibodies in which human mu heavy chains utilizing the VH26 gene are paired with mouse kappa or lambda light chains. The hybridomas described in this study were generated from naïve animals and were selected solely on the basis of human mu-chain expression. The antibodies described have covalently attached mouse light chains and are multimeric in structure. The binding properties of the antibodies were examined using a panel of both self- and foreign antigens using enzyme-linked immunosorbent assays, agglutination or radio-immunoprecipitation assays and immunofluorescence. Chimaeric immunoglobulins from 21 of the 34 hybridoma clones (61.7%) reacted with one or more antigens, of which 13 (38.2%) reacted with more than two antigens. These studies demonstrate that the VH26 gene, in combination with human DH and JH gene segments, and mouse light-chain genes, is able to encode antibodies with a wide range of ligand-binding specificities. These findings have important implications in the context of the possible origins of autoantibodies encoded by VH26 which may play a role in the pathogenesis of a number of autoimmune conditions.

Generation of a mouse strain that produces immunoglobulin kappa chains with human constant regions

Humanized antibodies are highly efficient as immunotherapeutic reagents and have many advantages over rodent antibodies. A mouse strain was generated by gene targeting to replace the mouse kappa light chain constant (C) region gene with the human C kappa gene. Mice homozygous for the replacement mutation (C kappa R) produced normal concentrations of serum antibodies, most of which carry chimeric kappa light chains, and mounted normal immune responses to hapten-protein conjugates. This technology provides a feasible option for the generation of high-affinity humanized antibodies by means of the powerful somatic hypermutation-selection mechanism.

The structure of the variable regions of mouse monoclonal antibodies to hepatitis B virus core antigen.

From a panel of monoclonal antibodies (MoAbs) directed against E. coli-derived native and denatured hepatitis B virus (HBV) core antigen we have selected a set of specific MoAbs which recognize different linear antigenic determinants: MoAb C1-5--cl epitope; MoAb 14K8--less immunogenic N-terminal region; and MoAbs 13C9, 10F10 and 14E11, 14G3--the immunodominant region between amino acids 134 and 140. We have applied the polymerase chain reaction technique to clone Ig VH and VL region genes, and appropriate full-length cDNA clones were obtained and characterized by nucleotide sequence analysis. Among the six heavy chain variable region sequences examined, three VH families were represented. Two of them belong to the 7183 (MoAb C1-5) and 3609 (14B8) families respectively and four, having only two amino acid changes in the CDR2 region, to the J558 family. These four probably are derived from a single expanded B-cell clone. The light chain sequences indicate that their VL are encoded by V kappa 21, V kappa 19 and V kappa 3 germline genes. Unlike VH genes, light chain genes are closely related to known representatives of mouse kappa light chain families and are employed also by MoAbs raised against other antigens.

Light chain heterogeneity in the amphibian Xenopus

Three subpopulations of light chains in Xenopus can be distinguished by monoclonal antibodies as well as by electrophoretic mobility on SDS-PAGE, peptide map and cell surface distribution. Analysis of these proteins from LPS-stimulated lymphocytes culture supernatants by two-dimensional gel electrophoresis showed a heterogeneity comparable to that observed for mouse kappa light chains. However, evidence from the selective expression of light chain subpopulations, as well as highly restricted light chain representation in anti-DNP antibodies, supports earlier findings that an antibody response in Xenopus is greatly limited in heterogeneity.

Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family‐specific oligonucleotide probes

In recent work, the polymerase chain reaction (PCR) has been used to amplify rearranged mouse and human immunoglobulin heavy and kappa light chain variable (V) genes. Here we have optimized the design of the PCR primers for human V genes and used them to amplify cDNA from human peripheral blood lymphocytes. Cloning and sequencing revealed a diverse repertoire of V genes, and the presence of members of each human V gene family. After alignment of the sequences, we identified a region conserved within V gene families, but differing between families, and used this to design family-specific oligonucleotide probes.

Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase

The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP2.Ag) and splenocytes from a rat immunized with whole mouse immunoglobulin (Ig)G. The hybrid-hybridoma generated from this fusion expresses and secretes rat Igs of the IgG1 and IgG2a subclasses, as determined by radial immunodiffusion. In competitive binding solid-phase enzymatic assays, McC10 was found to cross-react with all four mouse IgG subclasses as well as mouse kappa light chains. In contrast, in this type of assay, McC10 did not appear to recognize mouse IgA, IgM or lambda light chains. However, IgM-bearing kappa light chains were recognized by immunocytochemistry. Epitope specificity of this bi-specific antibody was more clearly determined on immunoblots where McC10 was found to exclusively recognize mouse kappa light chains and display no cross-reactivity with mouse Ig heavy chains nor with kappa light chains from rat or rabbit. In addition, McC10 was used successfully in two-step immunocytochemistry (ICC) for the localization of enkephalin, nerve growth factor (NGF) receptor and paired helical filament-immunoreactive sites in rat brain, rat skin and human brain, respectively, using mouse IgG's and IgM's as primary antibodies. McC10 compared favourably with peroxidase-anti-peroxidase (PAP) ICC with respect to sensitivity but was markedly superior with respect to specificity when used in fixed human brain or rat skin.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide sequence of a new BALB/c mouse kappa light chain variable region gene

Nucleotide sequence of a new BALB/c mouse kappa light chain variable region gene

Determinant differences between the rabbit and mouse immunoglobulinxenhancers impair the activity of the rabbit enhancer in mouse myeloma cells

Enhancer activity of the rabbit immunoglobulin kappa light chain gene intron conserved region (KICR) was examined in mouse myeloma cells using transient expression experiments. Compared to the homologous region of the mouse kappa light chain gene, the rabbit KICR shows nearly no stimulatory effect on expression of the indicator gene, cat. Experiments with mouse-rabbit chimeric KICRs indicated that differences in the region around the NF-kappa B binding site are responsible for the impaired activity of the rabbit KICR whereas mouse sequences covering the kappa E2 and kappa E3 motifs can be replaced by the equivalent rabbit fragment without affecting enhancer function. Creation of a perfect mouse NF-kappa B target sequence in the rabbit gene only partially restores enhancer activity. Furthermore, mouse and rabbit DNA fragments encompassing the NF-kappa B target sequence behave in an identical manner in an electrophoretic mobility shift assay. The results indicate species-related functional differences in the immunoglobulin kappa light chain gene enhancer and suggest that although the NF-kappa B binding site plays a crucial role in enhancer activity surrounding gene elements are also necessary for full enhancer effect.

Protein-Binding Sites in Ig Gene Enhancers Determine Transcriptional Activity and Inducibility

Individual protein-binding sites within the mouse immunoglobulin heavy chain and kappa light chain gene enhancers were altered, making it possible to examine the functional role of the sites during transcription. The E motifs, which bind factors that are present in many if not all cells, mostly behave as transcriptional activating sites. The only known heavy chain enhancer site that binds a lymphocyte-specific factor, the "octamer" site, plays a critical role in transcription but only in a truncated form of the enhancer. In the full enhancer, no one site is crucial because of an apparent functional redundancy. The site in the kappa enhancer that binds a factor specific to mature B cells, kappa B, was crucial to the constitutive activity of the enhancer in B cells. This factor is also inducible in pre-B cells, and the site was necessary for inducibility of the kappa enhancer. Thus, the sites defined by protein binding are important for the functional activity of immunoglobulin enhancers, with the sites that bind proteins restricted in their cellular distribution playing the most important roles.

Rearrangement and expression of rabbit immunoglobulin kappa light chain gene in transgenic mice.

To determine whether a foreign unrearranged immunoglobulin gene can be functionally rearranged and expressed in vivo, a rabbit b9 kappa light chain gene construct containing a single germ-line kappa chain variable (V) region gene (V kappa), the five kappa chain joining (J) segments (J kappa), and the kappa chain constant (C) region germ-line gene (C kappa) was introduced into fertilized mouse eggs. Eleven transgenic mice carrying 1-30 copies of the rabbit kappa gene construct were obtained. Rearrangement of the transgene due to V kappa-J kappa recombination was observed in the spleen of all the mice lines analyzed. Only the J kappa 1 and J kappa 2 segments, which have canonical sequences for rearrangement and high-level expression, are utilized in assembly of the rabbit gene. V kappa-J kappa 1 and V kappa-J kappa 2 joining was also observed in the thymus but not in nonlymphoid tissue. Polyadenylylated rabbit kappa chain transcripts of 1.2 kilobases were found in the spleen of the transgenic mice. The level of transcription was low despite a high level of rearrangement. Three transgenic mice lines secreted kappa light chains encoded by the foreign rabbit gene. Serum rabbit kappa chains were associated with mouse mu and gamma 1 heavy chains. However, hybrid antibody molecules containing both rabbit and mouse kappa light chains were also found in the serum of these animals. These results suggest that, although the rabbit kappa chain gene construct contains the necessary sequences for gene assembly, sequences implicated in stage- and tissue-specific regulation of kappa chain gene rearrangement are either missing or not recognized by mouse lymphoid cells.

Chromosomal loop anchorage sites appear to be evolutionarily conserved

We have previously identified a class of DNA sequence elements, termed matrix association regions (MARs), which specifically bind to nuclear matrices in vitro and are believed to be at the bases of chromosomal loops in vivo [1]. Here we demonstrate that nuclear matrices prepared from the yeast Saccharomyces cerevisiae will specifically bind an MAR sequence derived from the mouse kappa light chain immunoglobulin gene. This suggests that both MAR sequences and their binding sites have been strongly evolutionarily conserved.

Genetic polymorphism at the ? chain locus in mice: Comparisons of restriction enzyme hybridization fragments of variable and constant region genes

Variable (V kappa) and constant (C kappa) region genes of the mouse kappa light chain have been compared in inbred strains and in geographically isolated or genetically separated populations of mice by Southern blot analysis of endonuclease-restricted germline DNA. In most cases, the C kappa gene is found on a single restriction fragment while the V kappa genes of the V kappa 19 and V kappa 21 groups are each found on several (6-18) fragments. The restriction fragment (RF) patterns of V kappa 19 and V kappa 21 groups are both polymorphic when compared among inbred mouse strains. Southern blot patterns of V kappa 21 and V kappa 19 of inbred strains are also found among some geographically isolated populations of mice, suggesting that inbred strains acquired kappa loci from different subspecies. Some populations of geographical isolates show V kappa 21, V kappa 19, and C kappa contexts similar to inbred mice while more distantly related species within the genus Mus and laboratory rats show no apparent similarity in context to inbred strains. Variable region genes determining the RF patterns of V kappa 19 and V kappa 21 appear to be linked to each other and to the C kappa and Lyt-3 loci.

Different joining region J elements of the murine kappa immunoglobulin light chain locus are used at markedly different frequencies.

In order to assess whether DNA rearrangement occurs with equal frequency at each of the several J (joining region) elements in the mouse kappa light chain locus, we set out to determine the relative frequency of usage of J kappa segments in populations of B lymphocytes unperturbed by antigenic selection or cloning. To obtain such a population, we exposed a suspension of spleen cells to a mixture of mitogens capable of activating most B cells independently of their specificity for antigen. We estimated the relative usage of the J kappa elements in unspliced kappa chain gene transcripts in total and poly(A)-containing nuclear RNA, using an S1 nuclease protection assay, and in mature kappa chain mRNA, using a specifically primed cDNA hybridization assay. Both types of assay reveal a marked difference in the frequency of J kappa elements and indicate that their relative usage is: J1 approximately J2 much greater than J4 approximately J3. Comparison of the 5' flanking regions of the mouse J kappa elements, including the conserved putative recombination target sequences, shows no obvious differences consistent with the variation in recombinational efficiency, so we conclude that, although the consensus heptamer and nonamer signals may be sufficient to identify a recombination site, the probability that that site will be used depends also on other determinants. A review of published data suggests a nonequivalence of usage also among human J kappa elements and between mouse J lambda I and J lambda III loci. Extending the comparison to include these sets of sequences indicates that one of the determinants of frequency of J use may be the proximity of the consensus heptamer to a T-G dinucleotide within the J coding sequence, perhaps revealing an Escherichia coli gyrase-like substrate preference within a recombination enzyme.

Expression of a VHC kappa chimaeric protein in mouse myeloma cells.

The heavy (H) and light (L) chains of antibodies consist of variable (V) and constant (C) regions. The V regions of the heavy and light chains form the antibody combining site. To determine whether a V region could be functional when joined to a polypeptide other than its own C region, we constructed a chimaeric gene encoding the V region of a mouse heavy chain and the C region of a mouse kappa light chain ( VHC kappa). The heavy-chain gene is derived from an A/J mouse hybridoma cell line 36-65 whose antibody product (gamma 1, kappa) is specific for the hapten azophenylarsonate. We report here that, when introduced into a mouse myeloma cell line, the chimaeric gene is expressed and a protein of the expected molecular weight is secreted into the medium. As light chains tend to dimerize we expected that the VHC kappa protein might associate with light chain from the cell line 36-65 to form an antibody-binding molecule. Affinity binding experiments and Ka determination indicate that this is the case. Dimers of this type offer a novel and interesting alternative to existing antibody-binding molecules.

Intracisternal A-particle genes as movable elements in the mouse genome.

We analyzed two functionally defective mouse kappa light chain gene variants previously shown to contain novel insertions of repetitive DNA in their intervening sequences [Hawley, R. G., Shulman, M. J., Murialdo, H., Gibson, D. M. & Hozumi, N. (1982) Proc. Natl. Acad. Sci. USA 79, 7425-7429]. Heteroduplex analysis of the cloned genes shows that the insertions consist of intracisternal A-particle (IAP) genetic elements. Each insertion includes an IAP 5' long terminal repeat (LTR) sequence and extends to a characteristic IAP internal BamHI site where the IAP sequence is interrupted because the mutant genes were cloned from complete BamHI digests of the cellular DNAs. Restriction enzyme mapping indicates that the 5' LTR boundaries of the inserted IAP elements correspond closely to the previously determined rearrangement sites in the mutant genes. The IAP insertions in the two mutants can be distinguished by restriction-site differences and by the fact that one of them contains a deletion that is absent in the other. Nucleotide sequence data are presented for the LTRs of one full-length IAP gene copy randomly selected from a mouse genomic DNA library. These LTRs show many features typical of known integrated retroviral terminal repeat units, and the entire gene is bracketed by short direct repeats within the adjacent cellular DNA. Thus, the findings show that IAP genetic elements can appear in new locations in mouse cellular DNA and suggest that this may occur through a process of proviral insertion.

Use of monoclonal anti-mouse immunoglobulin to detect mouse antibodies.

Rat monoclonal antibodies specific for mouse kappa light chains and mouse gamma heavy chains have been generated. These rat monoclonal antibodies have been biosynthetically labelled with 35S methionine. The free label was dialyzed from the medium and, without further purification, the medium containing the radioactive monoclonal antibody was used in a radioimmunoassay to screen the sera of the immunized animals and hybridomas for specific mouse antibodies of the IgG class.

Amino acid sequence of the variable region of M149 mouse myeloma light chain: Comparison with the nucleotide sequence of K2 and K3 clones

The complete amino acid sequence of the kappa chain variable region from M 149, a myeloma urinary protein of BALB/c origin, has been determined. This sequence is compared with sequences of kappa chains from myeloma proteins that belong to the same group. The M 149 structure is also compared with the amino acid sequence derived from the nucleotide sequences of two EcoRI fragments (K2 and K3) of mouse DNA. These DNA fragments encode variable gene sequences closely related to mouse kappa light chain M149. Comparison of the protein sequence with the nucleotide sequence reveals differences in only 13–18 residues, which are clustered both in complementarity-determining regions and framework regions. When the probe derived from M149 tumour m-RNA was hybridized with mouse genome DNA, 6–8 different bands were revealed (Seidman et al., 1978 b). Furthermore, the amino acid sequences corresponding to the K2 and K3 fragments have not yet been found among kappa light chains whose structures have been determined. These results therefore, raise the possibility that a set of genes (6–8), some of which remain silent, might exist in the mouse genome corresponding to each of the many groups identified among kappa light chains. Recent work has shown that light chain genes are encoded in three segments, a major variable region sequence, a short J sequence and the constant region sequence. Five J segments have been identified for all BALB/c mouse kappa chains by means of nucleotide sequencing of embryonic DNA (Max et al., 1979; Sakano et al., 1979). The J2 segment of embryonic DNA corresponds to the amino acid sequence, of M 149 protein covering residues 96–108.