title: Cloning and expression of the recombinant FAb fragment of monoclonal antibody K1 that reacts with mesothelin present on mesotheliomas and ovarian cancers.
abstract: The monoclonal antibody (MAb) K1 recognizes an approximate 40 kDa glycoprotein, mesothelin, that is present on the surface of human mesothelial cells, mesotheliomas and ovarian cancers. We have cloned the cDNAs encoding the variable regions of MAb K1 and constructed plasmids for expression of recombinant K1(FAb). Recombinant FAb was produced in Escherichia coli in inclusion bodies that were solubilized and refolded to active protein. Binding of K1 MAb and FAb was compared by radioactive binding and competition assays and by surface plasmon resonance (BIAcore). Recombinant K1(FAb) binds to cells expressing K1-antigen with a similar affinity as papain derived FAb from K1(IgG) and with a 4- to 10-fold reduced affinity compared with bivalent IgG. The cloned FAb can be used to make higher affinity antibodies and immunoconjugates that could be useful for various types of immunotherapies.
authors.affiliation: Laboratory of Molecular Biology, NCI, NIH, Bethesda, MD 20892-4255, USA.
authors.forename: U
authors.initials: U
authors.lastname: Brinkmann
authors.forename: K
authors.initials: K
authors.lastname: Webber
authors.forename: A
authors.initials: A
authors.lastname: Di Carlo
authors.forename: R
authors.initials: R
authors.lastname: Beers
authors.forename: P
authors.initials: P
authors.lastname: Chowdhury
authors.forename: K
authors.initials: K
authors.lastname: Chang
authors.forename: V
authors.initials: V
authors.lastname: Chaudhary
authors.forename: M
authors.initials: M
authors.lastname: Gallo
authors.forename: I
authors.initials: I
authors.lastname: Pastan
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D000951:Antigens, Neoplasm / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D058851:GPI-Linked Proteins; D005803:Genes, Immunoglobulin; D006801:Humans; D018796:Immunoconjugates / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008562:Membrane Glycoproteins / Q000276:immunology*; D000090204:Mesothelin; D008654:Mesothelioma / Q000276:immunology*; D008969:Molecular Sequence Data; D010051:Ovarian Neoplasms / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D018076:DNA, Complementary; D058851:GPI-Linked Proteins; D018796:Immunoconjugates; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D000090204:Mesothelin