Biomedical subjects
Publications and source records attributed to M D Scharff.
Somatic cell mutants with deletions in the immunoglobulin constant region were isolated from an IgG(2b) Ar-binding hybridoma. Rat anti-mouse immunoglobulins were used to identify the sites of the deletions. The mutant monoclonal antibodies differ from the parental molecule in their assembly states and are defective in various immunoglobulin activities, making them potentially more useful reagents.
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The S107 mouse myeloma cell line synthesizes an IgA antibody that binds the hapten phosphocholine and is similar if not identical in its heavy and light chain variable region sequence to the predominant antibody produced by BALB/c mice in response to immunization with phosphocholine. This cell line frequently and spontaneously generates somatic variants producing immunoglobulins with decreased ability to bind antigen. One such variant, S 107.U1, is described here. This variant has a decreased ability to bind phosphocholine when it is attached to a carrier, although its affinity for free hapten is the same as that of the parent. This decrease in antigen binding is associated with a single amino acid substitution at the fifth residue in the JH segment.
S107, a phosphocholine-binding myeloma protein, has been cloned in soft agar, and an antigen-binding variant has been isolated and characterized. The variant does not bind phosphocholine attached to carrier or as free hapten in solution but does retain antigenic determinants (idiotypes) of the parent. Chain recombination experiments suggest that the defect in binding is entirely in the heavy chain. Amino acid sequence analysis showed a single substitution--glutamic acid to alanine at position 35--in the first hypervariable or complementarity-determining region. In terms of the three-dimensional model of the phosphocholine-binding site, glutamic acid-35 provides a hydrogen bond to tyrosine-94 of the light chain that appears to be critical for stability of this portion of the binding site. The removal of this bond and the presence of the smaller alanine side chain is thus consistent with the loss in binding activity. These results suggest that small numbers of substitutions in antibodies, such as those presumably introduced by somatic mutation, may in some situations be effective in altering antigen-binding specificity.
Whereas mouse myelomas that secrete IgA half-molecules have been shown to arise in vivo, their origin has not been definitely established. We show that somatic variants secreting phenotypically similar molecules can arise directly from the normal IgA-secreting myelomas S107 and W3082. In addition to being improperly assembled, the variant proteins have distinct carboxy-terminal deletions and an aberrant heavy-light chain disulfide bond. For at least one of the variants, variable region serology and affinity for hapten are both unaffected by these changes. Southern and Northern blot analyses indicate normal size DNA restriction fragments and mRNA, suggesting premature termination as the mechanism of deletion. These results are discussed in relation to possible mutational hot spots and long-range interdomain interactions.
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An 18-kilobase DNA fragment containing the sequence coding for both the variable and constant regions of the S107 mouse immunoglobulin light chain was cloned from total cellular DNA. The complete nucleotide sequence of the kappa-chain variable-region gene is reported. Determination of the amino acid sequence encoded by the DNA is found to be identical to the protein sequence of the T15 light chain through residue 88. Direct sequence analysis confirmed that the J1 joining segment is used in the recombination event producing the active kappa light chain gene.
A somatic cell genetic technique has recently been developed that makes it possible to obtain very large amounts of homogeneous antibodies and to replenish the supply of the exact same antibodies whenever they are needed. This hybridoma technology has already contributed to major scientific advances and will surely improve the diagnosis and treatment of many diseases. Because the technology itself is relatively simple and inexpensive, it has captured the attention of basic scientists, clinicians, and industrial managers and investors.
We have cloned two rearranged kappa immunoglobulin genes from the mouse myeloma cell line S107, and find that both are expressed. One gene, designated S107A, encodes the secreted kappa chain that participates in phosphocholine binding and expression of the T-15 idiotype. The other gene, designated S107B, as described here, contains an unusual junction between a V region unrelated to that of S107A and a different J region. The V-J junction preserves the triplet reading frame, but 6 nucleotides have been deleted at the recombination site. Nucleotide sequence analysis of the germline V-region precursor of S107B in comparison with other germline kappa-variable sequences reveals an "extra" 2 nucleotides in S107B between codon 95 and the palindromic heptanucleotide CACAGTG previously implicated in V-J recombination; this difference may be relevant to the 6 nucleotide deletion. Both S107A and S107B genes are expressed in the S107 cell as protein products, but unlike the S107A kappa chain, the S107B protein product is not secreted into the medium. The expression of these two kappa genes in the S107 cell has implications for theories of allelic exclusion.
Hybridomas, the progeny of fusion between antibody-secreting spleen cells and myeloma cells, produce large quantities of a single species of antibody. They are an ideal source of serologic reagents since they can be grown in tissue culture, frozen, stored, and recovered when needed. Impending applications include production of monoclonal antibodies to a wide variety of serum components and pathogens.
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.
Serologic reagents have played an important role in the diagnosis, treatment, and epidemiology of infectious diseases. A new technology has been developed for generating homogeneous antibodies that can be produced in large amounts and are available indefinitely. This technique promises to increase the reliability and sensitivity of immunoassays and may even provide antibodies that can be used safely in vivo.
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Cultured mouse myeloma cells frequently generate variants in immunoglobulin expression and structure. In this paper the authors will review the phenotypes of the variants which have been identified and discuss the mechanisms which may be responsible for the somatic instability of the immunoglobulin genes in mouse myeloma cells.
Mouse myeloma cell lines synthesize large amounts of immunoglobulin on their membrane-associated polyribosomes. Variants which no longer synthesize immunoglobulins have been studied and shown to have the same number of tightly bound membrane-associated polyribosomes as the parental cell lines. These polyribosomes are still active in the synthesis of nonimmunoglobulin proteins.
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Monoclonal IgG1 anti-SRBC has been used to study the binding of monomeric and aggregated IgG1 to Fc receptors on mouse macrophages. Aggregated IgG1 was found to bind to Fc receptors on two macrophage cell lines and on primary macrophages. It competes for binding with IgG2b and not IgG2a. Like the binding of IgG2b, the binding of IgG1 is unaltered at 4 degrees C and is insensitive to trypsin or cytochalasin B. Antigen-bound IgG1, like IgG2b and IgG2a, mediates phagocytosis. Variant macrophage cell lines selected for the loss of phagocytosis through the IgG2b receptor no longer phagocytize IgG1 bearing SRBC. Monomeric IgG1 did not bind to either macrophage lines or primary macrophages. We conclude from these experiments that antigen-activated IgG1 binds to the same receptor as IgG2b.