Optimized expression and specific activity of IL-12 by directed molecular evolution.
DNA delivery of IL-12 has shown promise in reducing the toxic side effects associated with administration of recombinant human (h)IL-12 protein while maintaining the ability to inhibit tumor growth and abolish tumor metastases in animal models. We have developed a more potent version of IL-12 by using DNA shuffling and screening to improve its expression in human cells and specific activity on human T cells. The most improved evolved IL-12 (EvIL-12) derived from seven mammalian genes encoding both the p35 and p40 subunits of IL-12 showed a 128-fold improvement in human T cell proliferation compared with native hIL-12 during the initial screening of supernatants from transected cells. When purified hIL-12 and EvIL-12 proteins were compared in vitro in human T cell proliferation and Th1 differentiation assays, it was demonstrated that EvIL-12 exhibited a concomitant 10-fold increase in the specific activity of the protein compared with hIL-12. Furthermore, DNA shuffling improved the level of expression and homogeneity of the heterodimer synthesized by 293 human embryonic kidney cells transfected with EvIL-12 by at least 10-fold. Molecular analysis of the variant revealed strategic placement of amino acid substitutions that potentially may facilitate heterodimer formation and product expression. The enhanced expression and biological activity of EvIL-12 may improve the effectiveness of IL-12 gene-based vaccines and therapeutics without the toxic side effects sometimes associated with hIL-12 protein administration.
Leong
Steven R
SR
Department of Vaccines, Maxygen, Incorporated, 515 Galveston Drive, Redwood City, CA 94063, USA.
Chang
Jean C C
JC
Ong
Randal
R
Dawes
Glenn
G
Stemmer
Willem P C
WP
Punnonen
Juha
J
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis*; D019281:Dimerization; D019020:Directed Molecular Evolution; D004305:Dose-Response Relationship, Drug; D015723:Gene Library; D005821:Genetic Techniques; D006031:Glycosylation; D006801:Humans; D018664:Interleukin-12 / Q000096:biosynthesis* / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013601:T-Lymphocytes; D018417:Th1 Cells; D014162:Transfection
D018664:Interleukin-12; D004247:DNA