Cassette vectors for conversion of Fab fragments into full-length human IgG1 monoclonal antibodies by expression in stably transformed insect cells.
Phage display technology allows for the production and rapid selection of antigen-specific, Fab antibody fragments. For purposes of immune therapy, though, complete antibodies that retain the Fc domain are often required. In this regard, we designed cassette vectors for converting human Fab fragments selected from combinatorial phage display libraries into full-length IgG(1) monoclonal antibodies (MAbs). Two expression vectors, pIEI-Light and pIEI-Heavy, were engineered to contain respective light- and heavy-chain human signal sequences downstream of the baculovirus immediate early gene promoter, IEI. Vector pIEI-Heavy also contains the coding region for each of the human IgG(1) constant domains. To generate complete antibody genes, the cassette vectors possess convenient restriction enzyme sites for rapid in-frame cloning of coding regions for full-length light chains in pIEI-Light and for the heavy-chain variable domains in pIEI-Heavy of Fab fragments. Using these constructs and a method that allows for stable transformation of insect cells, complete light- and heavy-chain genes can be inserted into the insect cell genome and subsequently expressed under the control of the baculovirus IEI promoter. This cassette vector system was used to generate stably transformed insect cells that continuously secreted functional full-length, IgG(1) MAbs. The expressed antibodies exhibited light and heavy chains of the appropriate molecular sizes and retained the ability to bind antigen. We conclude that our cassette vectors could serve as valuable tools for generating human IgG(1) antibodies.
Guttieri
Mary C
MC
Virology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA. mary.guttieri@det.amedd.army.mil
Sinha
Tanima
T
Bookwalter
Carol
C
Liang
Mifang
M
Schmaljohn
Connie S
CS
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005803:Genes, Immunoglobulin / Q000235:genetics; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology*; D007313:Insecta; D008957:Models, Genetic; D008969:Molecular Sequence Data; D019151:Peptide Library; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D014709:Vero Cells
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D019151:Peptide Library; D004247:DNA