Tumor-specific genetically engineered murine/human chimeric monoclonal antibody.

Murine variable and human constant region exons were fused to produce "chimeric" immunoglobulin gamma and kappa genes. These constructs were cotransfected into murine myeloma cells which then produced and secreted intact, functional antibody. Cells secreting the chimeric antibody were introduced into mice. The engineered immunoglobulin was subsequently harvested from ascites fluid and was purified by affinity chromatography. Its immunological properties were compared to those of the parental murine monoclonal (B6.2), which exhibits specificity for human breast, lung, and colon carcinoma cells. Competitive binding, immunofluorescent cell staining, and analysis of immunoprecipitated antigen gave similar results for the chimeric and murine B6.2. The biodistribution of chimeric and murine B6.2 after injection into mice bearing human tumors was found to be identical. These results suggest that murine/human chimeric antibodies may be viable clinical replacements for murine monoclonals with the potential for better immunological tolerance and pharmacological efficacy.

Selection by genetic complementation and characterization of the gene coding for the yeast porphobilinogen deaminase.

The porphobilinogen deaminase (PBG-D) gene of Saccharomyces cerevisiae has been isolated by genetic complementation of a mutant GL7 (alpha hem 3) strain, previously shown to be defective in this haembiosynthetic enzyme [Gollub, Liu, Dayan, Adlersberg & Sprinson (1977) J. Biol. Chem. 252, 2846-2854]. The gene was selected from a yeast wild-type genomic DNA library ligated into the shuttle vector YEp13. The complementing gene restored growth of the hem 3 (PBG-D) mutant strain on media in the absence of exogeneous haem or fatty acid and sterol supplements. The recombinant plasmid was retained in the Hem+ transformant provided that selective pressure for plasmid-dependent growth was maintained. Transformation of the mutant strain (hem 3) restored the PBG-D activity to levels up to 10-fold those of the parental strain. The mutant strain GL7 does not show any measurable enzymic activity. Analysis of the plasmid designated YEpPBG-D (containing the PBG-D gene) by hybrid-selected translation revealed that it contained the coding information for a single protein of apparent Mr 43,000. The coding region was localized on an 1.5 kb endonuclease-EcoRI fragment (E4), within the 5.5 kb genomic insert in YEpPBG-D.

Detection of Corynebacterium kutscheri in animal tissues by DNA-DNA hybridization.

Corynebacterium kutscheri, a pathogen of experimental rats and mice, is difficult to detect in the early stages of infection. A diagnostic assay was developed to reveal the organism in tissues of asymptomatic, infected animals. Three fragments of DNA specific to C. kutscheri were isolated, radiolabeled with 32P, and used in DNA-DNA hybridization assays with blots of impression smears of animal tissues on membrane filters. The C. kutscheri probes did not cross-hybridize with 15 DNA isolates from nonpathogenic corynebacteria. The method described is more rapid and more specific than conventional immunological and culture procedures used to detect C. kutscheri; the organism was detected in tissues of animals infected for only 1 week.

A genetically engineered murine/human chimeric antibody retains specificity for human tumor-associated antigen.

Chimeric immunoglobulin genes were constructed by fusing murine variable region exons to human constant region exons. The ultimate goal was to produce an antibody capable of escaping surveillance by the human immune system while retaining the tumor specificity of a murine monoclonal. The murine variable regions were isolated from the functionally expressed kappa and gamma 1 immunoglobulin genes of the murine hybridoma cell line B6.2, the secreted monoclonal antibody of which reacts with a surface antigen from human breast, lung, and colon carcinomas. The kappa and gamma 1 chain fusion genes were co-introduced into non-antibody producing murine myeloma cells by electroporation. Transfectants that produced murine/human chimeric antibody were obtained at high frequency as indicated by immunoblots probed with an antisera specific for human immunoglobulin. Enzyme-linked immunoabsorbent assay analysis demonstrated that this chimeric antibody was secreted from the myeloma cells and retained the ability to bind selectively to membrane prepared from human tumor cells. The chimeric immunoglobulin was also shown by indirect fluorescence microscopy to bind to intact human carcinoma cells with specificity expected of B6.2. The ability of chimeric antibody to recognize human tumor-associated antigen makes feasible a novel approach to cancer immunotherapy.

Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Isolation of ATP2 coding the F1-ATPase beta subunit.

A yeast nuclear pet mutant of Saccharomyces cerevisiae lacking any detectable mitochondrial F1-ATPase activity was genetically complemented upon transformation with a pool of wild type genomic DNA fragments carried in the yeast Escherchia coli shuttle vector YEp 13. Plasmid-dependent complementation restored both growth of the pet mutant on a nonfermentable carbon source as well as functional mitochondrial ATPase activity. Characterization of the complementing plasmid by plasmid deletion analysis indicated that the complementing gene was contained on adjoining BamH1 fragments with a combined length of 3.05 kilobases. Gel analysis of the product of this DNA by in vitro translation in a rabbit reticulocyte lysate programmed with yeast mRNA hybrid selected by the plasmid revealed a product which could be immunoprecipitated by antisera against the beta subunit of the yeast mitochondrial ATPase complex. A comparison of the protein sequence derived from partial DNA sequence analysis indicated that the beta subunit of the yeast mitochondrial ATPase complex exhibits greater than 70% conservation of protein sequence when compared to the same subunit from the ATPase of E. coli, beef heart, and chloroplast. The gene coding the beta subunit (subunit 2) of yeast mitochondrial adenosine triphosphatase is designated ATP2. The utilization of cloned nuclear structural genes of mitochondrial proteins for the analysis of the post-translational targeting and import events in organelle assembly is discussed.

Heme is necessary for the accumulation and assembly of cytochrome c oxidase subunits in Saccharomyces cerevisiae.

The presence of cytochrome c oxidase subunits and the association of these subunits with each other was studied in a heme-deficient Saccharomyces cerevisiae mutant. This mutant had been isolated by Gollub et al. (1977) J. Biol. Chem. 252, 2846-2854) and had been shown lack delta-aminolevulinic acid synthetase. When grown in the absence of heme or heme precursors, the mutant is respiration-deficient, devoid of cytochrome absorption bands and auxotrophic for all those components whose biosynthesis is dependent on hemoproteins; when grown in the presence of heme or heme precursors, the mutant is phenotypically wild type. Upon growth of the mutant in the absence of heme synthesis, the mitochondria still contained two of the three mitochondrially made cytochrome c oxidase subunits (i.e. II and III) and at least one of the cytoplasmically made cytochrome c subunits (VI). The other subunits were either barely detectable (I, IV) or undetectable (V, VII). The residual subunits were apparently not assembled with each other since an antiserum directed mainly against Subunit VI failed to co-precipitate Subunits II and III which were still present. In contrast, growth of the mutant in the presence of delta-aminolevulinic acid led to the accumulation of active, fully assembled cytochrome c oxidase in the mitochondria. Heme a (or one of its precursors) thus controls the assembly of cytochrome c oxidase from its individual subunits.