Treatment of pre-existing melanoma tumors using allogeneic vaccines expressing alpha(1,3)Gal epitopes. Efficacy and toxicology study

1423 The hyperacute immune response in humans is a potent mechanism of xenograft rejection mediated by complement-fixing natural antibodies that recognize alpha(1,3)Galactosyl epitopes (aGal) not present on human cells. In this study we exploited this immune mechanism to create an allogeneic whole cell cancer vaccine to treat murine melanoma. We used the alpha(1,3)Galactosyltransferase (aGT H-2 bb ) knockout mice, which do not express aGal epitopes on their cell surfaces and develop high titers of anti-aGal antibodies. Melanoma cell lines (B16 and S91M3) naturally lack the expression of aGal epitopes. These cells were retrovirally transduced using lentiviral vectors encoding the murine aGT enzime to produce the aGal (+) variants of these cell lines. Mice with pre-existing subcutaneous (sc) and pulmonary B16 (H-2 bb ) melanoma tumors were treated with allogeneic aGal (+) S91M3 (H-2 dd ) melanoma vaccines or with control aGal (-) S91M3 vaccines. Mice with sc tumors had significant prolonged survival (p (-) S91M3 (p 8 each group). Vaccination with aGal (+) S91M3 induced CD8 + T cells recognizing the “autologous” aGal (-) B16 tumors measured by intracellular TNF-alpha. These cells reduced the pulmonary burden of syngeneic recipients with pre-existing pulmonary melanoma in adoptive transfer experiments. Interestingly, mice vaccinated with allogeneic aGal (+) .S91M3 or “autologous” aGal (+) B16 cells failed to induce DTH responses to B16, despite the induction of a strong T cell dependent mediated anti-tumor immunity. Since effective anti-tumor immunity is associated with autoimmunity in several immunotherapies, we tested whether vaccination with allogeneic aGal (+) vaccines induced autoimmunity or other detectable signs of toxicity in three murine models. Mice (aGT KO H-2 bb ) were vaccinated six times with irradiated aGal (+) EMT-6 (breast H-2 dd ) cells. The aGT KO H-2 dd mice received six vaccine doses of aGal (+) LLC (lung H-2 bb ) or aGal (+) B16 melanoma cells. High titers (>1:200) of anti-aGal Ab titers (IgG and IgM) were demonstrated in all mice. Samples (blood and tissues) were collected at early time points and at 6 months after the last allogeneic vaccine. Analysis of the hematology, chemistry and histopathology of major perfused organs (kidneys, lungs, liver and spleen) data revealed no significant differences among mice receiving allogeneic vaccines and control groups. In addition mice were weighted weekly for a period of 6 months showing no differences in the growth patterns among the groups in three animal models. This study establishes the safety and efficacy data of allogeneic aGal(+) whole cell vaccines and constituted the basis for the initiation of human clinical trials to treat human cancers which are currently open.