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The development of vaccines against infectious diseases is one of the greatest achievements of modern medicine. However, the results of clinical trials of vaccines against established malignancies have been disappointing, clearly demonstrating a compelling need to discover novel nontoxic vaccine adjuvants as well as to develop innovative vaccination protocols that combine other therapies to create synergistic effects. We have previously shown that a polyphenylpropenoid-polysaccharide complex (PPC) derived from the cones of Pinus sylvestris induces the differentiation and maturation of dendritic cells (DCs) in vitro and enhances DC vaccine-induced antigen-specific CD8+ T-cell responses by oral administration. In the present study, we explored the potential of a combination chemoimmunotherapy, including PPC, a DC-based vaccine and low-dose cyclophosphamide (CTX), to eradicate established large tumors in mice. TC-1 tumor cells were injected subcutaneously into C57BL/6 mice (five mice per group) on day 0. TC-1 is a mouse tumor cell line expressing the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV16). Bone marrow-derived DCs (BMDCs) were prepared by culturing murine BM cells in the presence of GM-CSF. These BMDCs were pulsed with the synthetic tumor antigen peptide HPV16 E743-77. Peptide-pulsed DCs were activated further ex vivo with PPC (PPC-DC(E7)) and then injected into tumor-bearing mice on days 13, 20 and 27. Vaccination with PPC-DC(E7) was followed by oral administration (via gavage) of PPC. Low-dose CTX was administered intraperitoneally 3 days prior to each vaccination with PPC-DC(E7). We observed that 1) all of the untreated control mice died by day 39 post TC-1 injection; 2) treatment with low-dose CTX alone significantly suppressed tumor growth compared to untreated mice (p = 0.0132, day 28) but did not eradicate large established tumors (these mice died by day 56); 3) therapy with low-dose CTX plus oral administration of PPC more strongly suppressed TC-1 tumor growth (p = 0.0008, day 28), and two out of five mice had complete tumor regression by day 42 (these mice were tumor-free on day 231); 4) five out of five mice treated with low-dose CTX and PPC-DC(E7) vaccine had complete tumor regression by day 39, but two of these mice developed tumors again on days 70 and 196 and died on days 98 and 222, respectively (the remaining three mice were tumor-free on day 231); and 5) five out of five mice treated with low-dose CTX and PPC-DC(E7) vaccine plus oral administration of PPC had complete tumor regression by day 39, and all of these mice remained tumor-free on day 231. These results show that therapy with PPC (a DC-activating vaccine adjuvant) and a DC-based vaccine, in combination with low-dose CTX, induced complete eradication of established large tumors in 100% of mice. This may comprise a promising strategy for the design of a safe and effective vaccination protocol to treat established malignancies. Citation Format: Soichi Haraguchi, William Guy Bradley, Akiko Tanaka. A combination chemoimmunotherapy with a dendritic cell-based vaccine activated by a polyphenylpropenoid-carbohydrate complex and low-dose cyclophosphamide completely eradicates established large tumors in mice [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B10.

Immunization with dendritic cell (DC)-based vaccines is a promising vaccination strategy to provide an effective personalized immunotherapy against cancer and other diseases. However, the results of clinical cancer immunotherapy trials carried out using DC-based vaccines to date have shown only modest clinical outcomes. We have previously demonstrated that a polyphenylpropenoid-polysaccharide complex (PPC), a lignin-carbohydrate complex derived from the cones of Pinus sylvestris, induces in vitro the differentiation and maturation of human mononuclear cell-derived DCs (Bradley et al., Int Immunopharmacol 2003;3) and murine bone marrow-derived DCs (BMDCs) (An et al., Anticancer Res 2010;30). Furthermore, oral administration of PPC enhanced a DC vaccine-induced melanoma antigen-specific CD8+ T-cell response (Burrows et al., BMC Complement Altern Med 2009;9) and the proliferative phase of a primary T cell response (Bradley et al., BMC Complement Altern Med 2014;14). In the present study, using a human papillomavirus (HPV)-induced mouse tumor model, we investigated whether ex vivo treatment of BMDCs with the synthetic lignin-like polyphenol polymers poly-caffeic acid (pCA) and poly-coumaric acid (pCoA), as well as PPC, improves the efficacy of DC-based vaccines. Polymerization of the phenylpropenoic acids caffeic acid (CA) and poly-coumaric acid (CoA) was performed by horseradish peroxidase/hydrogen peroxide-mediated enzymatic reactions. BMDCs were prepared by culturing murine bone marrow cells in the presence of GM-CSF for 8-10 days. These BMDCs were pulsed with synthetic tumor antigen peptides HPV16 E743-62 or HPV16 E743-77 that contain both a cytotoxic T lymphocyte (CTL) and a helper T cell epitope. Peptide-pulsed DCs were treated further ex vivo with synthetic polyphenol polymers or PPC and then injected twice into C57BL/6 mice. In addition, splenocytes obtained from immunized mice were restimulated with HPV16 E749-57 peptide (E7 CTL epitope) for 5 days. Tumor antigen-specific CD8+ T cell responses were assessed by HPV16 E749-57/H-2Db pentamer staining. We observed that immunization with PPC-treated HPV16 E743-77 peptide-loaded BMDCs significantly enhanced the induction of pentamer-positive CD8 positive cells in splenocytes (2- to 3-fold increase). Moreover, ex vivo restimulation with E7 CTL epitope peptide dramatically increased the number of pentamer-positive CD8 positive cells in splenocytes obtained from mice immunized with PPC-treated, peptide-loaded DCs (6- to 7-fold increase) when compared to splenocytes from mice immunized with PPC-nontreated, peptide-loaded DCs. Experiments using pCA and pCoA are in progress. We conclude that 1) ex vivo treatment with the natural PPC complex significantly potentiates the in vivo efficacy of DC-based HPV vaccines and 2) tumor antigen-specific CD8+ T cells are significantly expanded ex vivo by restimulating splenocytes from mice primed in vivo with PPC-treated, peptide-loaded BMDCs. In addition, our preliminary studies show that PPC, pCA and pCoA significantly increase the expression levels of CD8α, XCR1 and/or CLEC9A on murine BMDCs that have been demonstrated to be essential for the induction of CTL responses. Our approaches will provide innovative strategies for the design of more effective DC-based vaccines and adaptive T-cell therapies for the treatment of patients with cancer and other immune disorders. Citation Format: Soichi Haraguchi, Tina Pastoor, William Guy Bradley, Akiko Tanaka. Improving the efficacy of dendritic cell-based human papillomavirus vaccines in mice using a natural lignin-carbohydrate complex and/or synthetic lignin-like polyphenol polymers. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B29.