Baculovirus expression of a functional single-chain immunoglobulin and its IL-2 fusion protein

• Published in: Journal of immunological methods 1995-10, Vol.186 (2), p.245-255
• Main Authors: Bei, Roberto, Schlom, Jeffrey, Kashmiri, S.V.S.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal - isolation & purification; Antibodies, Neoplasm - biosynthesis; Antibodies, Neoplasm - genetics; Antibodies, Neoplasm - immunology; Antibodies, Neoplasm - isolation & purification; Antibody Specificity; Antibody-Dependent Cell Cytotoxicity; Antigens, Neoplasm - immunology; baculovirus; Baculovirus recombinant; Biological and medical sciences; Carcinoma - immunology; Cells, Cultured; Fundamental and applied biological sciences. Psychology; Genes. Genome; Genetic Vectors; Glycoproteins - immunology; Glycosylation; Humans; Immunoglobulin G - biosynthesis; Immunoglobulin G - genetics; Immunoglobulin G - immunology; Interleukin-2 - biosynthesis; Interleukin-2 - genetics; Molecular and cellular biology; Molecular genetics; Mucin; Nucleopolyhedrovirus - genetics; Pancreatic Neoplasms - immunology; Protein Processing, Post-Translational; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Recombinant Fusion Proteins - isolation & purification; Single-chain antibody; Spodoptera - cytology; Tumor-associated glycoprotein-72
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/0022-1759(95)00149-5

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 SCAΔC LC H1 (SCIg), consists of the CC49 sFv covalently joined to the human Fc (γ1) through the hinge region. The other, SCAΔC LC H1-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 succesfully expressed in insect cells and purified. The baculovirus recombinant single-chain antibodies have been designated, bV-SCAΔC LC H1 (bV-SCIg) and bV-SCAΔC LC H1-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.