Evolution of a cytokine using DNA family shuffling

Chang, Chia-Chun; Chen, Teddy T.; Cox, Brett; Dawes, Glenn; Stemmer, Willem P.C.; Punnonen, Juha; Patten, Phillip A.

doi:10.1038/11737

Cited by 105 publications

(63 citation statements)

References 30 publications

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“…data not shown), were subjected to secondary and tertiary rounds of quantitative screening in the Th1 differentiation, Daudi antiproliferation, and several antiviral assays (Table 1 and SI Tables 3-5). The most potent variants in the Th1 differentiation assay all came from library BC9, a gene-shuffled library derived from gene shuffling of degenerate PCR products containing the entire human IFN-␣ gene family (9).…”

Section: Resultsmentioning

confidence: 99%

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Directed evolution of gene-shuffled IFN-α molecules with activity profiles tailored for treatment of chronic viral diseases

Brideau-Andersen

Huang

Sun

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Section: Resultsmentioning

confidence: 99%

“…Construction of the gene-shuffled IFN libraries was performed as described in ref. 9. In this study, these libraries were PCR amplified and cloned into pcDNA3.1 to enable CHO expression.…”

Section: Methodsmentioning

confidence: 99%

Directed evolution of gene-shuffled IFN-α molecules with activity profiles tailored for treatment of chronic viral diseases

Brideau-Andersen

Huang

Sun

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Reverse transcription was performed using the cDNA cycle kit (Invitrogen). DNA shuffling was performed as described previously (25)(26)(27). The shuffled sequences were digested with BamHI and Asp-718 and gel-purified, and the resulting chimeric genes were cloned into a pcDNA3.1 Ϫ expression vector (Invitrogen).…”

Section: Isolation Of Mammalian Cdnas and Generation Of Libraries-cd80mentioning

confidence: 99%

“…In addition, the severe phenotype of CD28-and CTLA-4-deficient mice (4,5,8,(12)(13)(14) and the structurally conserved ligand binding sites of CD80 and CD86 (20 -24) suggest that natural evolution has strongly favored the binding of B7 molecules to both of their respective ligands. Directed molecular evolution by DNA shuffling followed by screening has previously been successful in evolving, for example, subtilisin, interferon ␣, viruses, and bacteria (25)(26)(27)(28)(29)(30)(31). We hypothesized that directed molecular evolution of diverse mammalian CD80 genes by DNA shuffling combined with the appropriate screening procedures would enable the generation of chimeric molecules that preferentially bind and signal through either CD28 or CTLA-4.…”

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confidence: 99%

Chimeric Co-stimulatory Molecules That Selectively Act through CD28 or CTLA-4 on Human T Cells

Lazetic

Leong

Chang

et al. 2002

Journal of Biological Chemistry

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CD28 and CTLA-4 (CD152) play a pivotal role in the regulation of T cell activation. Upon ligation by CD80 (B7-1) or CD86 (B7-2), CD28 induces T cell proliferation, cytokine production, and effector functions, whereas CTLA-4 signaling inhibits expansion of activated T cells and induces tolerance. Therefore, we hypothesized that co-stimulatory molecules that preferentially bind CD28 or CTLA-4 would have dramatically altered biological properties. We describe directed molecular evolution of CD80 genes derived from human, orangutan, rhesus monkey, baboon, cat, cow, and rabbit by DNA shuffling and screening. In contrast to wild-type CD80, the evolved co-stimulatory molecules, termed CD28-binding protein (CD28BP) and CTLA-4-binding protein (CTLA-4BP), selectively bind to CD28 or CTLA-4, respectively. Furthermore, CD28BP has improved capacity to induce human T cell proliferation and interferon-␥ production compared with wild-type CD80. In contrast, CTLA-4BP inhibited human mixed leukocyte reaction (MLR) and enhanced interleukin 10 production in MLR, supporting a role for CTLA-4BP in inducing T cell anergy and tolerance. In addition, co-stimulation of purified human T cells was significantly suppressed when CTLA-4BP was cotransfected with either CD80 or CD28BP. The amino acid sequences of CD28BP and CTLA-4BP were 61 and 96% identical with that of human CD80 and provide insight into the residues that are critical in the ligand binding. These molecules provide a new approach to characterization of CD28 and CTLA-4 signals and to manipulation of the T cell response.T cell response is dependent on complex integration of antigen-specific and nonspecific extracellular and intracellular signals. The specificity of the response is determined by recognition of an antigenic peptide in the context of major histocompatibility complex on the plasma membrane of antigen-presenting cells, such as monocytes, dendritic cells, Langerhans cells, or primed B cells. However, a second signal, mediated by co-stimulatory molecules expressed on antigenpresenting cells, is required for induction of a complete T cell response (1-3). The most extensively studied co-stimulatory molecules are CD80 (B7-1) and CD86 (B7-2), which bind the CD28 and CTLA-4 (CD152) ligands expressed on CD4 ϩ and CD8 ϩ T cells. Additional co-stimulatory molecule-ligand pairs such as ICOS-B7-H and PD1-PDL have recently been identified, further illustrating the complexity of the signals that regulate the function of T cells (1-3). Co-stimulatory signal in addition to the T cell receptor signal is a prerequisite for induction of optimal T activation, proliferation, cytokine production, and effector functions.The functional characterization of CD80, CD86, CD28, and CTLA-4 has also illustrated the delicate balance between positive and negative signals mediated through CD28 and CTLA-4, respectively. Upon ligation by CD80 or CD86, CD28 synergizes with T cell receptor signaling to induce activation of both CD4 ϩ and CD8 ϩ T cells (4 -7), whereas CTLA-4 ligation inhibits expansion of...

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“…DNA shuffling followed by screening typically comprises a genetic-combinatorial method that rapidly breeds functional sequence diversity through reiterative mutation and recombination and allows sampling of sequence space not accessible by conventional methods of mutagenesis (30). These technologies can be used for a wide range of applications (31)(32) and have been successful, for example, in evolving subtilisin, IFN-␣, costimulatory molecules, and viruses (33)(34)(35)(36).…”

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

Leong

Chang²,

Ong³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Evolution of a cytokine using DNA family shuffling

Cited by 105 publications

References 30 publications

Directed evolution of gene-shuffled IFN-α molecules with activity profiles tailored for treatment of chronic viral diseases

Directed evolution of gene-shuffled IFN-α molecules with activity profiles tailored for treatment of chronic viral diseases

Chimeric Co-stimulatory Molecules That Selectively Act through CD28 or CTLA-4 on Human T Cells

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

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