Survival experiments utilized log- rank Mantel Cox test for survival analysis. 3M-052. CD8+ T cells, B cells, Type I IFN, IFN-, and pDC were contributed to efficient tumor suppression whereas perforin, NK cells and CD4 CPI-169 T cells were not required. Finally, 3M-052 therapy potentiated checkpoint blockade therapy with anti-CTLA-4 and anti-PD-L1 antibodies, even when checkpoint blockade alone was ineffective. Our findings suggest that intratumoral treatment with 3M-052 is usually a promising approach for the treatment of cancer and establish a rational strategy and mechanistic understanding for combination therapy with intratumoral, tissue-retained TLR7/8 agonist and checkpoint blockade in metastatic cancer. imiquimod) and TLR7/8 dual agonists (resiquimod), generate tumor-specific T cell immunity and/or kill tumor directly by activation of innate immunity (8, 9). The cream formulation of imiquimod limits its application for deep, non-cutaneous tumors, and systemic administration of TLR agonists is limited by severe toxicity, including cytokine storm (10). Therefore, development of injectable, local-release formulations of TLR7 and TLR7/8 agonists are an area of intense study and drug development. Growing evidence suggests that tumor associated macrophages (TAM) play an important role in tumor growth. TAM can assume M1 or M2 phenotypes, with M1 TAM producing interleukin (IL)-12 to promote tumoricidal responses, whereas M2 TAM produce IL-10 and promote tumor progression (11),(12). One of the factors that may drive M1/M2 TAM ratios in tumors is the chemokine, Chemokine (C-C motif) ligand 2/macrophage chemotactic protein-1 (CCL2/MCP-1). Low CCL2 concentrations can promote accumulation of M2 TAM and tumor growth while high CCL2 secretion results in predominant M1 TAM infiltration and tumor destruction (13). Therefore, shifting TAM phenotype from M2 towards M1 could be an important therapeutic strategy (14C17). Here we report therapeutic activity of a novel TLR7/8 dual agonist, 3M-052, in a preclinical model of melanoma. 3M-052 is an injectable, lipid modified imidazoquinoline that forms a tissue depot with gradual, sustained release, allowing local TLR triggering activity without systemic cytokine release (18). 3M-052 is currently under clinical development by 3M Drug CPI-169 Delivery Systems Division for use in vaccines and cancer therapy. Intratumoral 3M-052 monotherapy induced local innate immune activation as well as systemic, antigen-specific CD8+ T cell responses which suppressed distant, uninjected tumors. Mechanistically, the intratumoral macrophages shifted from a M2-dominant to M1 dominant phenotype, while CCL2 blockade or macrophage depletion abolished therapeutic activity. 3M-052 has promise as monotherapy or in combination with checkpoint blockades, anti-PD-L1 or anti-CTLA-4, for the treatment of metastatic melanoma and other cancers. Materials and Method Mice and cell lines All animal experiments were performed in accordance with NIH guidelines and approved by the MDACC IACUC. C57BL/6 mice were purchased from the NCI. Rag2 KO, B cell KO (IgH), Prf KO, IFN- KO and depletion of pDCs was induced and maintained by DT injection (i.p.; 5 ng DT/g body weight every other day) in = 5, unless otherwise indicated. Statistical analysis was performed with Graph Pad Prism 4 software. Rabbit polyclonal to Synaptotagmin.SYT2 May have a regulatory role in the membrane interactions during trafficking of synaptic vesicles at the active zone of the synapse Data were analyzed using unpaired two-tailed assessments, and differences were considered significant at 0.05. Figures denote statistical significance of p 0.05 as *, p 0.01 as **, and p 0.001 as ***. Survival experiments utilized log- rank Mantel Cox test for survival analysis. All experiments were performed at least twice with comparable results. Results Intratumoral administration of 3M-052 suppresses local injected and distant uninjected melanoma growth Most innate immune cells, including antigen presenting cells (APCs) in mouse and man express TLR7 and/or TLR8 (21, 22). In C57BL/6 mice, TLR8 is usually non-responsive to imidazoquinolines like resiquimod and 3M-052, but both pDCs, mDCs and macrophages in mice express TLR7 and respond to TLR7 agonists (10, 23). Thus, activation of tumor-associated TLR7+ APCs with 3M-052 could generate a range of innate and adaptive anti-tumor immune responses. We tested the anti-tumor effect of 3M-052 against the poorly immunogenic, wild-type B16.F10 melanoma and the more immunogenic version B16.OVA, engineered to express the chicken ovalbumin protein. Palpable 7-day tumors (~20 mm2) were treated with intratumoral 3M-052 or vehicle on day 0 and 4 (treatment CPI-169 schematic; Fig 1A). Growth.
Survival experiments utilized log- rank Mantel Cox test for survival analysis