Optimized expression and specific activity of IL-12 by directed molecular evolution

Abstract

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.

Effects of Recombination on Complex Regulatory Circuits

“…For example, the products of recombination between two distantly related b-lactamases retained function with significantly higher probability than b-lactamase variants that involved the same number of amino acid changes as introduced by recombination, but where these changes were introduced through random point mutation (Drummond et al 2005). More anecdotally, a DNA shuffling experiment involving divergent interleukin-12 genes from seven mammalian species showed that between 40 and 90% of chimeric molecules retained interleukin-12 activity (Leong et al 2003). A similar study creating four random recombinant ainterferons from 20 different human a-interferon genes that differ, on average, at 17 amino acid positions, found that all four recombinant proteins generated were as active as their ''parents.''…”

“…The authors estimate that mutating the same number of positions as are changed in a recombination event among these proteins would abolish protein function (Chang et al 1999). Such weak deleterious effects may contribute to the promise recombination shows in engineering new proteins (Stemmer 1994;Crameri et al 1997Crameri et al , 1998Zhang et al 1997;Chang et al 1999;Kolkman and Stemmer 2001;Leong et al 2003;Raillard et al 2001a,b). Because recombination can reorganize genetic systems on a much larger scale than point mutations, these weak effects on different levels of organization are intriguing.…”

Viral Evolution as a Tool to Improve the Tetracycline-regulated Gene Expression System

“…Directed evolution has been used to change the activity, selectivity, or stability of enzymes (reviewed in Refs. 5 and 6) but also to improve viral vector stability (7), cytokine efficacy (8), and antibody fragment binding (9). Recently, Yokobayashi et al (10) successfully applied directed evolution to genes comprising a simple genetic circuit and demonstrated that a nonfunctional circuit containing improperly matched components could evolve rapidly into a functional one (10,11).…”

Laboratory-Directed Protein Evolution

“…Toxic side effects have been associated with the direct administration of recombinant antitumor interleukin-12 protein. A DNA vaccine based on the interleukin-12 gene has been shown to reduce adverse side effects, while its potency and effectiveness have been further improved by directed evolution (179). In addition, high-affinity T-cell receptor variants can be generated and used for detecting peptidemajor histocompatibility complex complexes on antigen-presenting cells (121).…”