High-level expression of full-length antibodies using trans-complementing expression vectors.
Recombinant antibodies are increasingly used as therapeutics for a wide variety of diseases. Generation of cell lines expressing high levels of recombinant antibody typically requires labor-intensive cloning and screening steps. We describe a mammalian expression system for the high-level production of full-length antibody molecules. It has been shown that the dihydrofolate reductase (DHFR) selectable marker can be divided into two fragments that, with the aid of a leucine zipper, can re-associate to form an active molecule. Using bicistronic vectors, we linked the expression of each antibody chain to the expression of a DHFR fragment. Survival in selective media requires expression of both DHFR fragments that, by virtue of these vectors, also selects for the expression of both antibody chains. Initial pools produced 5 microg of Ab/10(6) cells/d (qP = microg/10(6) cells/d). Expression of each antibody chain in conjunction with a portion of DHFR also leads to concurrent amplification of both antibody chains in the presence of methotrexate, a DHFR inhibitor, and results in a two- to fivefold increase in antibody production with basal qPs ranging from 10-25 ug/10(6) cells/d. Shake-flask cultures of amplified pools produced up to 600 mg/L of antibody in 7 days. This system allows for rapid generation of antibodies without cloning and greatly simplifies selection of cell lines for the production of potential antibody therapeutics.
Bianchi
Allison A
AA
Amgen Incorporated, 51 University Street, Seattle, WA 98101, USA.
McGrew
Jeffrey T
JT
D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D016466:CHO Cells; D003001:Cloning, Molecular / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D007074:Immunoglobulin G / Q000276:immunology; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology; D014162:Transfection / Q000379:methods*
D000906:Antibodies; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D008727:Methotrexate