Cancer Cell Line–Derived, Pooled Heat Shock Proteins as a Potential Universal Vaccine for Immunotherapy of Cancer
Authors: Qian Jianfei , Hong Sungyoul , Wang Siqing , Zhang Liang , Kwak Larry W. , Yi Qing 
Published: April 2020

Abstract
Tumor cell-derived heat shock proteins (Hsps) such as gp96 have been used as vaccines for immunotherapy of cancer patients. However, current approaches for immunotherapy are individualized and only applicable to certain cancer patients from whom sufficient tumor materials can be obtained to produce the vaccines. To improve the applicability and feasibility of Hsp-based immunotherapy in cancers and to enhance clinical efficacy, we explored using pooled allogeneic cancer cell line-derived gp96 as a universal vaccine for immunotherapy for cancer. We modeled this in a myeloma setting and obtained four murine myeloma cell lines that originated from Balb/c mice. We first examined the ability of the vaccines to protect mice from developing myeloma. Balb/c mice were vaccinated with either normal gp96 (g96N); gp96 from tumor A (gp96A); tumor B (gp96B); tumor C (gp96C); pooled allogeneic gp96 from tumors B and C (gp96BC); or pooled allogeneic gp96 from tumors B, C, and D (gp96BCD), followed by challenge with tumor A. While 100% of mice vaccinated with gp96N (n=10 mice for each group) developed tumors, 20%, 90%, 100%, 30%, and 20% mice vaccinated with gp96A (autologous gp96), gp96B (allogeneic gp96), gp96C (allogeneic gp96), pooled gp96BC or gp96BCD, respectively, developed tumors. These results clearly showed that gp96 vaccination is able to protect mice from tumor challenge, and that pooled allogeneic gp96 was as effective as autologous gp96. These results were reproduced in a repeated experiment by using the same tumor model, and were further verified with myeloma tumor B and C in Balb/c mice, and murine myeloma cells from other strains of mice, including C3H and C57BL mice. These results clearly demonstrated the ability of these vaccines to protect mice with different MHC haplotypes from developing myeloma. Next, we examined the therapeutic effects of gp96 vaccines in our myeloma mouse model. Gp96BCD alone or in combination with adjuvants GM-CSF or IL-12 eradicated established myeloma in 3 out of 5, 4 out of 5, and 3 out of 5 mice, respectively, while gp96BCD plus CpG (ODN 1826) eradicated myeloma in all 5 mice bearing intermediate tumors (≥5 mm in diameter). For mice bearing large tumors (≥10 mm in diameters), pooled gp96BCD plus CpG alone or combined with in vivo treatment with anti-CD25 (to deplete Treg), anti-B7H1 or anti-IL-10 antibodies were able to eradicate myeloma in 60%, 60%, 80%, and 80%, respectively. These results indicate that pooled gp96BCD plus CpG in combination with anti-B7H1 or anti-IL-10 antibodies were much more effective at eradicating established myeloma than that in combination with anti-CD25 antibody in mice with large tumor burdens. Finally, the mechanisms of tumor protection induced by pooled allogeneic gp96 vaccines were investigated. By depleting CD4 or CD8 T cells, or NK cells, or by using IFN-g-deficient mice (IFN-g−/−), we showed that IFN-g and CD8+ T cells were required for gp96-induced antimyeloma immune and clinical responses. In gp96BCD-vaccinated mice, splenocytes contained increased numbers of gp96-specific, IFN-g-secreting and proliferative T cells. The splenic CD8+ T cells exhibited a typical effector phenotype (CD25+CD69+CD62LlowCD44high), and enhanced cytotoxicity against myeloma cells, including the unrelated tumor-A cells. These results demonstrate the presence of myeloma-specific cytotoxic T cells (CTLs) in vaccinated mice and show that pooled allogeneic gp96BCD was as effective as autologous gp96A to induce a potent CTL response capable of killing unrelated myeloma cells. Together, our study lays a basis for future clinical trials in multiple myeloma and possibly other cancers by using pooled allogeneic gp96 from human tumor cell lines as a universal vaccine.