Expression of antibody genes in nonlymphoid mammalian cells

A wide variety of mammalian cell lines have the ability to assemble and secrete immunoglobulins. This provides the possibility of adapting highly efficient mammalian cell expression systems available for nonlymphoid host cells to obtain efficient expression of recombinant antibodies. A set of vectors that use a powerful cytomegalovirus promoter enhancer to direct expression of the coding sequence for each immunoglobulin chain is described. The vectors also contain a replication origin from SV40 to generate high copy numbers in COS monkey kidney cells and a marker gene for selection of permanently transfected cell lines. Methods for generating microgram amounts of antibody in 2–4 days from COS cells and for stable introduction of the vectors into CHO cells are described. The use of a glutamine synthetase (GS) selectable marker to obtain vector amplification in CHO cells is also described. This permits the generation of cell lines that can accumulate in excess of 200 mg/liter of antibody in the culture medium.

[1]  L. Hendershot,et al.  Identity of the immunoglobulin heavy-chain-binding protein with the 78,000-dalton glucose-regulated protein and the role of posttranslational modifications in its binding function , 1988, Molecular and cellular biology.

[2]  César Milstein,et al.  Man-made antibodies , 1991, Nature.

[3]  M. Page,et al.  High Level Expression of the Humanized Monoclonal Antibody CAMPATH-1H in Chinese Hamster Ovary Cells , 1991, Bio/Technology.

[4]  S. Colowick,et al.  Methods in Enzymology , Vol , 1966 .

[5]  D. Stacey,et al.  Differences in intracellular DNA ligation after microinjection and transfection. , 1984, Molecular and cellular biology.

[6]  R. Mulligan,et al.  Expression of a bacterial gene in mammalian cells. , 1980, Science.

[7]  G. Wahl,et al.  Recent progress in understanding mechanisms of mammalian DNA amplification , 1989, Cell.

[8]  M. Neuberger,et al.  Polymeric immunoglobulin M is secreted by transfectants of non‐lymphoid cells in the absence of immunoglobulin J chain. , 1987, The EMBO journal.

[9]  J. Kearney,et al.  Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein , 1987, The Journal of cell biology.

[10]  R. Klausner,et al.  Protein degradation in the endoplasmic reticulum , 1990, Cell.

[11]  S. Latt,et al.  Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells , 1985, Molecular and cellular biology.

[12]  E. Brown,et al.  Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. , 1985, Science.

[13]  T. Kadesch,et al.  Effects of the position of the simian virus 40 enhancer on expression of multiple transcription units in a single plasmid , 1986, Molecular and cellular biology.

[14]  M. Cockett,et al.  High Level Expression of Tissue Inhibitor of Metalloproteinases in Chinese Hamster Ovary Cells Using Glutamine Synthetase Gene Amplification , 1990, Bio/Technology.

[15]  J. Schlom,et al.  Expression in COS cells of a mouse-human chimaeric B72.3 antibody. , 1987, Protein engineering.

[16]  R. Dwek,et al.  N-glycosylation and in vitro enzymatic activity of human recombinant tissue plasminogen activator expressed in Chinese hamster ovary cells and a murine cell line. , 1989, Biochemistry.

[17]  S. Reuveny,et al.  Factors affecting cell growth and monoclonal antibody production in stirred reactors. , 1986, Journal of immunological methods.

[18]  B Sollner-Webb,et al.  High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. , 1984, Nucleic acids research.

[19]  A. Dorner,et al.  High level synthesis of immunoglobulins in Chinese hamster ovary cells. , 1990, Journal of immunology.

[20]  N. Proudfoot.  Transcriptional interference and termination between duplicated α-globin gene constructs suggests a novel mechanism for gene regulation , 1986, Nature.

[21]  P. Greenaway,et al.  Human cytomegalovirus DNA: BamHI, EcoRI and PstI restriction endonuclease cleavage maps. , 1982, Gene.

[22]  P. Stephens,et al.  The construction of a highly efficient and versatile set of mammalian expression vectors. , 1989, Nucleic acids research.

[23]  J. Barsoum.  Introduction of stable high-copy-number DNA into Chinese hamster ovary cells by electroporation. , 1990, DNA and cell biology.

[24]  J. Emtage,et al.  The synthesis and in vivo assembly of functional antibodies in yeast , 1985, Nature.

[25]  M. Cockett,et al.  The use of engineered E1A genes to transactivate the hCMV-MIE promoter in permanent CHO cell lines. , 1991, Nucleic acids research.

[26]  H. Lenz,et al.  Reconstitution of functionally active antibody directed against creatine kinase from separately expressed heavy and light chains in non-lymphoid cells. , 1987, Gene.

[27]  Y. Gluzman.  SV40-transformed simian cells support the replication of early SV40 mutants , 1981, Cell.

[28]  A. Dorner,et al.  Reduction of endogenous GRP78 levels improves secretion of a heterologous protein in CHO cells , 1988, Molecular and cellular biology.

[29]  S. Gillies,et al.  Expression of Human Anti-Tetanus Toxoid Antibody in Transfected Murine Myeloma Cells , 1989, Bio/Technology.

[30]  M K Foecking,et al.  Powerful and versatile enhancer-promoter unit for mammalian expression vectors. , 1986, Gene.

[31]  R. Kaufman,et al.  The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins , 1987, The Journal of cell biology.

[32]  M. Kozak,et al.  At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. , 1987, Journal of molecular biology.

[33]  T. Nakaki,et al.  Enhanced transcription of the 78,000-dalton glucose-regulated protein (GRP78) gene and association of GRP78 with immunoglobulin light chains in a nonsecreting B-cell myeloma line (NS-1) , 1989, Molecular and cellular biology.

[34]  S. Fujii,et al.  Structural heterogeneity of sugar chains in immunoglobulin G. Conformation of immunoglobulin G molecule and substrate specificities of glycosyltransferases. , 1990, The Journal of biological chemistry.