EpCAM-specific vaccine response by modified antigen and chimeric costimulatory molecule in cynomolgus monkeys.

Immunization against tumor-associated antigens is a promising approach to cancer therapy and prevention, but it faces several challenges and limitations, such as tolerance mechanisms associated with self-antigens expressed by the tumor cells. Costimulatory molecules B7.1 (CD80) and B7.2 (CD86) have improved the efficacy of gene-based and cell-based vaccines in animal models and are under investigation in clinical trials. However, their efficacy as vaccine adjuvants is likely limited by the fact that they mediate both stimulatory and inhibitory signals to T cells via CD28 and CTLA-4, respectively. To overcome these limitations, we have generated a B7.1-like, chimeric costimulatory molecule with preferential binding to CD28, named CD28-binding protein (CD28BP), which we combined with a modified, nonself tumor antigen variant of epithelial cell adhesion molecule (EpCAM), named TAg25. TAg25 induced a cross-reactive immune response against human wild-type EpCAM upon DNA vaccination in cynomolgus monkeys. However, TAg25 DNA immunization alone or in combination with human (h) B7.1 induced no detectable antigen-specific T cells in the peripheral blood of the animals. In contrast, TAg25 combined with CD28BP induced both CD4 and CD8 T cells specific for EpCAM. Moreover, TAg25 combined with CD28BP induced significantly higher levels of EpCAM-specific antibodies than TAg25 plus hB7.1. These improved adjuvant properties of CD28BP, when compared with hB7.1, illustrate the importance of CD28 costimulation in vaccine responses in nonhuman primates and warrant further studies on the potential of CD28BP in improving the efficacy of cancer vaccines.

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.

Chimeric co-stimulatory molecules that selectively act through CD28 or CTLA-4 on human T cells.

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-gamma 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.