We have created mouse-human antibody molecules of defined antigen-binding specificity by taking the variable region genes of a mouse antibody-producing myeloma cell line with known antigen-binding specificity and joining them to human immunoglobulin constant region genes using recombinant DNA techniques. Chimeric genes were constructed that utilized the rearranged and expressed antigen-binding variable region exons from the myeloma cell line S107, which produces an IgA (kappa) anti-phosphocholine antibody. The heavy chain variable region exon was joined to human IgG1 or IgG2 heavy chain constant region genes, and the light chain variable region exon from the same myeloma was joined to the human kappa light chain gene. These genes were transfected into mouse myeloma cell lines, generating transformed cells that produce chimeric mouse-human IgG (kappa) or IgG (kappa) anti-phosphocholine antibodies. The transformed cell lines remained tumorigenic in mice and the chimeric molecules were present in the ascitic fluids and sera of tumor-bearing mice.

https://www.pnas.org/content/pnas/81/21/6851.full.pdf

Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains

To design and direct at will the specificity of T cells in a non-major histocompatibility complex (MHC)-restricted manner, we have generated and expressed chimeric T-cell receptor (TcR) genes composed of the TcR constant (C) domains fused to the antibody's variable (V) domains. Genomic expression vectors have been constructed containing the rearranged gene segments coding for the V region domains of the heavy (VH) and light (VL) chains of an anti-2,4,6-trinitrophenyl (TNP) antibody (SP6) spliced to either one of the C-region gene segments of the alpha or beta TcR chains. Following transfection into a cytotoxic T-cell hybridoma, expression of a functional TcR was detected. The chimeric TcR exhibited the idiotope of the Sp6 anti-TNP antibody and endowed the T cells with a non-MHC-restricted response to the hapten TNP. The transfectants specifically killed and produced interleukin 2 in response to TNP-bearing target cells across strain and species barriers. Moreover, such transfectants responded to immobilized TNP-protein conjugates, bypassing the need for cellular processing and presentation. In the particular system employed, both the TNP-binding site and the Sp6 idiotope reside almost exclusively in the VH chain region. Hence, introduction into T cells of TcR genes containing only the VHSp6 fused to either the C alpha or C beta was sufficient for the expression of a functional surface receptor. Apparently, the VHC alpha or VHC beta chimeric chains can pair with the endogenous beta or alpha chains of the recipient T cell to form a functional alpha beta heterodimeric receptor. Thus, this chimeric receptor provides the T cell with an antibody-like specificity and is able to effectively transmit the signal for T-cell activation and execution of its effector function.

https://www.pnas.org/content/pnas/86/24/10024.full.pdf

Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity

Immunoglobulins are the major secretory products of the adaptive immune system. Each is characterized by a distinctive set of glycoforms that reflects the wide variation in the number, type, and location of their oligosaccharides. In a given physiological state, glycoform populations are reproducible; therefore, disease-associated alterations provide diagnostic biomarkers (e.g., for rheumatoid arthritis) and contribute to disease pathogenesis. The oligosaccharides provide important recognition epitopes that engage with lectins, endowing the immunoglobulins with an expanded functional repertoire. The sugars play specific structural roles, maintaining and modulating effector functions that are physiologically relevant and can be manipulated to optimize the properties of therapeutic antibodies. New molecular models of all the immunoglobulins are included to provide a basis for informed and critical discussion. The models were constructed by combining glycan sequencing data with oligosaccharide linkage and dynamics information from the Glycobiology Institute experimental database and protein structural data from "The Protein Data Bank."

The Impact of Glycosylation on the Biological Function and Structure of Human Immunoglobulins

We have designed a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable domains by the polymerase chain reaction. The primers incorporate restriction sites that allow the cDNA of the variable domains to be force-cloned for sequencing and expression. Here we have applied the technique to clone and sequence the variable domains of five hybridoma antibodies and to express a mouse-human chimeric antibody that binds to the human mammary carcinoma line MCF-7. The technique should also lead to the cloning of antigen-binding specificities directly from immunoglobulin genes.

https://www.pnas.org/content/pnas/86/10/3833.full.pdf

Cloning immunoglobulin variable domains for expression by the polymerase chain reaction.

Mouse cell lines of different lineages have been established which constitutively secrete large quantities of recombinant mouse interleukins (mIL2, mIL3, mIL4 or mIL5). An existing bovine papilloma virus-based expression vector, pBV-1MTHA, was modified to allow transformed X63Ag8-653 myeloma cells, NIH 3T3 fibroblasts and C127 mammary tumor cells to stably carry multiple copies of the vector, to express the inserted cDNA encoding a single interleukin constitutively, and to secrete the interleukin in high quantities. Cell lines transformed with mIL2 cDNA stably carried 30-100 copies of the plasmid per cell and constitutively secreted biologically active mIL2 in quantities similar to those produced by murine EL4 thymoma cells or rat spleen cells stimulated with mitogens. Deletion of the 3' untranslated region containing AT-rich sequences from the mIL2 cDNA resulted in a 100-fold increase in the constitutive production and secretion of mIL2 by the transformants. Addition of a heavy metal further increased the production 2 to 6-fold. Cells transformed with 3'-deleted mIL3 cDNA constitutively secreted 300-1000 times higher activities of mIL3 than the myelomonocytic leukemia line WEHI3. mIL4 produced by the similar transformants induced [3H]thymidine uptake of a T cell line, a mast cell line and B leukemia cells, and enhanced the production of IgG1 by B cells. IL4 titers were 150 times higher than those produced by the concanavalin A-stimulated T cell line 2.19. mIL5 was secreted by similar transformants at 10-fold higher titers than those produced by concanavalin A-stimulated 2.19 T cells, as judged by the proliferation and maturation of B cell leukemia BCL1. The expression vectors should be useful in establishing eukaryotic cell lines producing proteins from full length cDNA clones at higher rates. The established cell lines secreting IL2, 3, 4 or 5 at high rate should be useful sources for these interleukins in the investigation of their function in the immune system.

Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors

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.

https://www.pnas.org/content/pnas/80/7/1992.full.pdf

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

The kappa light chain genes from two mutant hybridoma cell lines defective in kappa light chain synthesis were isolated and compared to the wild-type kappa light chain gene. In each case, the mutant kappa light chain genes were found to contain repetitive DNA elements in their intervening sequences that were not present in the intervening sequences of the wild-type kappa light chain gene. These elements were found to be related to the genes of intracisternal A particles. These results suggest that the decreased production of kappa light chain in the mutant cell lines is due to the presence of the intracisternal A particle-related genes.

https://www.pnas.org/content/pnas/79/23/7425.full.pdf

Mutant immunoglobulin genes have repetitive DNA elements inserted into their intervening sequences

The rearranged immunoglobulin heavy (mu) and light (kappa) chain genes cloned from the Sp6 hybridoma cell line producing immunoglobulin M specific for the hapten 2,4, 6-trinitrophenyl were inserted into the transfer vector pSV2-neo and introduced into various plasmacytoma and hybridoma cell lines. The transfer of the mu and kappa genes resulted in the production of pentameric, hapten-specific, functional IgM.

http://www.pnas.org/content/80/20/6351.full.pdf

Functional immunoglobulin M production after transfection of cloned immunoglobulin heavy and light chain genes into lymphoid cells

Previous studies have discerned two forms of polymeric mouse IgM: moderately cytolytic (complement-activating) pentamer, which contains J chain, and highly cytolytic hexamer, which lacks J chain. To investigate the relationships among polymeric structure, J chain content, and cytolytic activity, we produced IgM in J chain-deficient and J chain-proficient mouse hybridoma cell lines. Both hexamer and pentamer were produced in the absence as well as the presence of J chain. Hexameric IgM activated (guinea pig) complement approximately 100-fold more efficiently than did J chain-deficient pentamer, which, in turn, was more active than J chain-containing pentamer. These results are consistent with the hypothesis that J chain-containing pentamer cannot activate complement. We also analyzed the structure of IgM-S337, in which the mu-chain bears the C337S substitution. Like normal IgM, IgM-S337 was formed as a hexamer and as both J chain deficient- and J chain-containing pentamers. Unlike normal IgM, IgM-S337 dissociated in SDS into various subunits. For IgM-S337 pentamer, the predominant subunits migrated as mu2kappa2 and mu4kappa4, and the subunit distribution was unaltered by J chain. However, J chain was found only in the mu2kappa2 species, suggesting that some arrangement of inter-mu bonds directs incorporation of J chain. IgM-S337 hexamer also dissociated to mu2kappa2 and mu4kappa4, but also yielded several species migrating much more slowly in SDS-PAGE than wild-type mu12kappa12. To account for these forms, we propose that each mu-chain can interact with three other mu-chains and that some hexameric molecules contain two catenated mu6kappa6 circles.

Structural and Functional Analysis of J Chain-Deficient IgM

We report here the occurrence of homologous recombination between transferred and chromosomal immunoglobulin genes. Specifically, we have corrected a chromosomal immunoglobulin gene mutation by transferring pSV2neo vectors encoding the constant region of the immunoglobulin mu heavy chain to mutant hybridoma cells that bear a 2-base-pair deletion in the third constant region exon of their chromosomal mu gene. After DNA transfer, we detected G418-resistant transformants that produce normal IgM. Analysis of the DNA structure of the mu gene in these transformants indicates that in four of five cases the mu gene has been restored as a result of the integration of a single copy of the transfer vector by a reciprocal homologous recombination event; the fifth case seems to have resulted from gene conversion or double crossover. These results suggest that this technology might be adapted for mapping immunoglobulin gene mutations by marker rescue and for more convenient engineering of specifically altered immunoglobulin.

https://www.pnas.org/content/pnas/85/17/6432.full.pdf

Marc J. Shulman

Papers

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

Mutant immunoglobulin genes have repetitive DNA elements inserted into their intervening sequences

Functional immunoglobulin M production after transfection of cloned immunoglobulin heavy and light chain genes into lymphoid cells

Structural and Functional Analysis of J Chain-Deficient IgM

Homologous recombination can restore normal immunoglobulin production in a mutant hybridoma cell line.