IL2-based fusion proteins as new bi-functional biopharmaceuticals for the therapy of cancer

The growing number of strategies to specifically eliminate tumors by manipulating the host's immune system is tangible evidence of the expectation of a cure for cancer may arise from the field of cancer immunotherapy. The delivery of immune stimulatory genes, such as pro-inflammatory cytokines, co-stimulatory molecules or suicide genes in the tumor site, has been shown to promote specific anti-tumor responses in mouse models of cancer. Total tumor eradication was also found to occur despite subtotal tumor engineering; a phenomenon coined the "bystander effect". The bystander effect in immune competent animals arises mostly from recruitment of a cancer lytic cell-mediated immune response to local and distant tumor cells which escaped gene modification. John Stagg et al. have previously generated and characterized the murine fusion protein between Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Interleukin-2 (IL2, aka GIFT2), which display novel immunological properties compared to both cytokines in combination such as greater melanoma site recruitment of macrophages and functional NK cells. Consequently, GIFT2 prevent tumor formation in mice implanted with genetically modified B16 melanoma cells expressing GIFT2. In the Chapter 2 of my thesis, I evaluated the bystander effect of the murine GIFT2 in vivo to induce an effective antitumor response against non-genetically modified B16 cells present in the tumor site. I observed that GIFT2 secreted by genetically engineered B16 tumor cells induces a bystander effect on non modified B16 cells and such effect was mediated by recruited NK cells in the tumor site. However, the immune bystander effect was completely lost as the total B16 cell number was increased from 104 to 106 which correlated with a sharp reduction in the number of tumor-infiltrating NK cells. I identified that active TGFbeta is the main tumor-derived suppressive factor that downregulated IL-2Rbeta expression and IFNgamma secretion by NK cells, and therefore attenuated GIFT2-dependent bystander effect. We demonstrated that in vivo blockade of B16 originating TGFbeta significantly improved the immune bystander effect arising from GIFT2. Based on the potent immunostimulatory properties of the murine GIFT2 on NK cells, in the chapter 3 I developed, characterized and evaluated the human ortholog of GIFT2, which may serve as a mean to generate oncolytic NK cells for cell-based therapy of cancer. I observed that human GIFT2 induces robust NK cell activation ex vivo with significant secretion of pro-inflammatory cytokines, chemokines and upregulate the expression of activation markers and NK cell activating receptors. This phenotype correlates with significantly greater cytotoxicity against tumor cells. At the molecular level, the human GIFT2 leads to a potent activation of Jak/STAT signaling pathway downstream of IL-2 receptor. In conclusion, hGIFT2 fusokine possesses unique biochemical properties and constitutes a novel and potent tool for ex vivo NK cell activation and maturation. Based on our results, I propose that cancer gene immunotherapy of pre-established tumors will be enhanced by blockade of active TGFbeta, which acts as a potent pro-oncogenic factor and suppresses antitumor immunity. To antagonize TGFbeta dependent effects in tandem with a pro-inflammatory immune stimulus, I described in chapter 4, the generation of a new chimeric protein borne of the fusion of IL-2 and the soluble extracellular domain of TGF-beta receptor II (aka FIST). FIST acts as a decoy receptor trapping active TGF-beta in solution and directly interacts with IL-2-responsive cells, inducing a distinctive hyperactivation of STAT1 downstream of IL-2 receptor, which in turn promotes SMAD7 overexpression. STAT1 hyperactivation further induces significant secretion of CXCL10, upregulates T-Bet and T-Bet target gene expression in NK cells. The synergism of TGFbeta blockade coupled to IL-2(R)-dependent STAT1 hyperagonism leads to potent immune activation contemporaneous to a dominant NK cell-dependent antiangiogenic effect in the B16 murine model of melanoma. Consequently, FIST prevent tumor formation not only in immunocompetent mice but also in several immunodeficient mice including CD4 KO, CD8 KO, B-cell deficient (muMT) and NK-defective beige mice, whereas mice with NK defective functions such as nonobese diabetic--severe combined immunodeficient (NOD-SCID) mice and Rag2/gammac KO mice developed tumors. In the chapter 5, I took advantage of this new technology to generate and characterize FIST-stimulated B cells. FIST-stimulated B cells upregulate co-stimulatory molecules, activation markers and MHC class II molecule expression, which is also supported by robust hyperactivation of Jak/STAT signaling pathway. FIST-stimulated B cells maintain B cell identity based on the expression of PAX5 and CD19 and act as effective APC that induce the activation and cell proliferation of antigen-specific CD4+ and CD8+ T cells. Interestingly, FIST-stimulated B cells confer complete protective immunity to EG.7 tumor challenge in vivo. Therefore, FIST can also be used as stimulator to generate B cells with APC features useful for the cell-based therapy of cancer.;In conclusion, these bi-functional chimeric proteins are potential biopharmaceuticals for the therapy of cancer. Specifically, FIST constitute an advanced technology that not only target several immune system components to generate an effective antitumor but also FIST triggers angiostatic mechanisms that suppresses tumor-derived angiogenesis, an essential process required for tumors to growth and metastasize.