Mutated variant of human vascular endothelial growth factor as a vaccine candidate for cancer immunotherapy

The finding that neo-angiogenesis, -the production of new blood vessels from pre-existing ones-, is an essential mechanism for tumor development and progression [1] has produced a totally new modality for cancer treatment: anti-angiogenic therapy [1]. Novel anti-angiogenic cancer therapies based on synthetic and natural molecules target pro-angiogenic growth factors produced by tumors and/or their cell surface receptors in endothelial cells, or even in some cancer cells. Among these growth factors, vascular endothelial growth factor (VEGF) and its receptors have received special attention, due to: a) their central role in endothelial cell physiology and neo-angiogenesis; b) the detection of VEGF at high concentrations in most of the human tumors and their metastasis; (c) its frequent association with a bad prognosis in cancer; and d) the differential nature of tumor angiogenesis, when compared to normal tissues [2]. Anti-angiogenesis as cancer therapy was validated in 2004, when the FDA first approved Bevacizumab (Avastin, Genentech), a humanized monoclonal antibody that blocks the interaction of circulating VEGFA and VEGF receptors, for the treatment of metastatic colorectal cancer [3].In the following years Bevacizumab has been also approved for the treatment of non-small cell lung carcinoma [4] and metastatic breast cancer [5], and is under phase I, II, and III clinical trials in more than thirty other tumor types. Several other antibodies that block VEGF-VEGFR2 interaction are also under intensive preclinical and clinical investigations [6]. An exciting new approach for antiangiogenic cancer therapy involves active specific immunotherapy. Antiangiogenic cancer vaccines could hopefully not only elicit endogenous antibodies that neutralize VEGF and its receptors, but also induce cytotoxic T lymphocytes-mediated tumor destroying mechanisms. Collateral effects of such vaccines could also be milder than those elicited by externally infused anti-VEGF antibodies. Experimental vaccination using xenogeneic [7] and autologous [8, 9] endothelial cells have initially validated the likelihood of this strategy. But, the heterogeneity of these vaccine preparations have encouraged studies with more defined vaccine antigens, namely, pro-angiogenic factors and their receptors. Xenogeneic [10] and autologous VEGF [11], VEGF receptor 2 [12], in the form of protein or naked DNA vaccines, or infused as antigen-loaded dendritic cells, have resulted in antitumoral and antiangiogenic effects in cancer animal models. Wei et al. [10] using xenogeneic VEGF165 DNA as antigen, and Rad et al. [13], immunizing with autologous VEGF164 protein kinoids, have reported the induction of an antibody-mediated VEGF neutralizing response that led to anti-tumor effects, but with no evidences of breaking T-cell tolerance to this growth factor. On the contrary, Kamstock et al., immunizing an outbreed dog population affected by advanced sarcomas, demonstrated a 30% tumor response rate using human VEGF165 protein mixed with a previously prepared liposome-DNA complex, but found no relevant antibody titers to canine VEGF [11]. Within this rising field of investigation, our group was very early involved in the development of an improved VEGF vaccine strategy, having as centerpiece the use of a functional mutant of the autologous VEGF molecule (or a very high homologous one), impaired for binding to the VEGFR2, as antigen. Our preliminary results demonstrated the feasibility of using a human VEGF variant impaired for VEGFR2 binding as a vaccine antigen in a naked DNA format as an antitumoral approach in mice and provide an insight on the immune mechanism involved [14]. Further work was directed to the development of a new candidate vaccine using a recombinant antigen produced as a fusion protein containing the mutated human VEGF and an amino terminal sequence of Neisseria Meningitidis P64K protein. The antigen was tested with adjuvants of different chemical nature clinically proven or under advanced preclinical testing. With this vaccine candidate, we showed the induction of anti-tumor effects in the C57Bl/6 melanoma B16-F10 tumor system, with the production in mice of neutralizing antibodies against both human and mouse VEGF, and specific T cell responses against murine tumor cells that produce VEGF [15].