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.
Amgen Incorporated, 51 University Street, Seattle, WA 98101, USA.
Allison A
AA
Bianchi
Jeffrey T
JT
McGrew
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