| Autor: |
Leong, Steven R., Chang, Jean C.C., Ong, Randal, Dawes, Glenn, Stemmer, Willem P.C., Punnonen, Juha |
| Předmět: |
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| Zdroj: |
Proceedings of the National Academy of Sciences of the United States of America; 2/4/2003, Vol. 100 Issue 3, p1163, 6p, 2 Black and White Photographs, 5 Diagrams, 3 Graphs |
| Abstrakt: |
DNA delivery of IL-12 has shown promise in reducing the toxic side effects associated with administration of recombinant human (h)lL-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 (EvIL12) 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 hlL-12 during the initial screening of supernatants from transected cells. When purified hlL-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 hlL-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 hlL-12 protein administration. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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