title: Baculovirus expression of a functional single-chain immunoglobulin and its IL-2 fusion protein.
abstract: The baculovirus expression system has been used for the production of a variety of proteins, including antibodies. Two single-gene constructs encoding single-chain immunoglobulins have recently been developed. The antibody employed was monoclonal antibody (MAb) CC49 which reacts with the pancarcinoma antigen, tumor associated glycoprotein, TAG-72. One, single-chain construct designated SCA delta CLCH1 (SCIg), consists of the CC49 sFv covalently joined to the human Fc (gamma 1) through the hinge region. The other, SCA delta CLCH1-IL-2 (SCIg-IL-2), has a human IL-2 molecule attached to the carboxyl end of the SCIg. These constructs have been used to test the feasibility of producing biologically active antibodies using the baculovirus expression system. Both constructs have been successfully expressed in insect cells and purified. The baculovirus recombinant single-chain antibodies have been designated, bV-SCA delta CLCH1 (bV-SCIg) and bV-SCA delta CLCH1-IL-2 (bV-SCIg-IL-2) they have been shown to be secreted in the culture supernatant as dimeric molecules of approximately 115 kDa and 140 kDa, respectively. The specificity and antibody dependent cellular cytolytic activity of the baculovirus recombinant single-chain antibodies were shown to be similar to that of the myeloma derived molecules. Glycosylation analysis showed that baculovirus derived proteins were N-glycosylated, but carried few if any high mannose residues. The biological activity of the IL-2 moiety was retained in bV-SCIg-IL-2, as evidenced by its stimulatory effect on the proliferation of the IL-2 dependent cell line HT-2. The observation that a significantly shorter time is required to develop baculovirus recombinant molecules as compared to myeloma derived molecules and that insect cells express single chain MAbs at acceptable levels may have implications for the production of these molecules for clinical use.
authors.affiliation: Laboratory of Tumor Immunology and Biology, National Cancer Institute, National Institutes of Health, Rockville Pike, Bethesda, MD 20892, USA.
authors.forename: R
authors.initials: R
authors.lastname: Bei
authors.forename: J
authors.initials: J
authors.lastname: Schlom
authors.forename: S V
authors.initials: SV
authors.lastname: Kashmiri
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D000920:Antibody-Dependent Cell Cytotoxicity; D000951:Antigens, Neoplasm / Q000276:immunology; D002277:Carcinoma / Q000276:immunology; D002478:Cells, Cultured; D005822:Genetic Vectors; D006023:Glycoproteins / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D010190:Pancreatic Neoplasms / Q000276:immunology; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D006023:Glycoproteins; D007074:Immunoglobulin G; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; C047052:tumor-associated antigen 72