The immune factors heat shock protein (HSP)/peptides (HSP/Ps) can induce both adaptive and innate immune responses. Treatment with HSPs in cancer cell-bearing mice and cancer patients revealed antitumor immune activity. We aimed to develop immunotherapy strategies by vaccination with a mixture HSP/Ps (mHSP/Ps, HSP60 HSP70 Gp96 and HSP110) enhanced with cyclophosphamide (CY) and interleukin-12 (IL-12).

We extracted mHSP/Ps from the mouse sarcoma cell line S180 using chromatography. The identity of proteins in this mHSP/Ps was assayed using SDS-PAGE and Western blot analysis with antibodies specific to various HSPs. BALB/C mice bearing S180 cells were vaccinated with mHSP/Ps x3 then were injected intraperitoneally with low-dose CY and subcutaneously with IL-12, 100 ug/day, x5. After vaccination, T lymphocytes in the peripheral blood were analyzed using FACScan and Cytotoxicity (CTL) was analyzed using lactate dehydrogenase assay. ELISPOT assay was used to evaluate interferon gamma (IFN-gamma), and immune cell infiltration in tumors was examined in the sections of tumor specimen.

In mice vaccinated with enhanced vaccine (mHSP/Ps and CY plus IL-12), 80% showed tumor regression and long-term survival, and tumor growth inhibition rate was 82.3% (30 days); all controls died within 40 days. After vaccination, lymphocytes and polymorphonuclear leukocytes infiltrated into the tumors of treated animals, but no leukocytes infiltrated into the tumors of PBS treatment mice. The proportions of natural killer cells, CD8+, and interferon-gamma-secreting cells were all increased in the immune group, and tumor-specific cytotoxic T lymphocyte activity was increased.

In this mice tumor model, vaccination with mHSP/Ps combined with low-dose CY plus IL-12 induced an immunologic response and a marked antitumor response to autologous tumors. The regimen may be a promising therapeutic agent against tumors.

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