Background We evaluated whether tumor infiltrating lymphocytes (TIL) could be expanded from surgically resected tumors from pancreatic cancer patients. Methods Tumors were resected from pancreatic cancer patients. Tumors were minced into fragments and cultured in media containing high dose interleukin-2 (IL-2) for up to 6 weeks. T cell phenotype, activation markers, and reactivity were measured. Results TIL expansion was measured in 19 patient samples. The majority of these TIL were CD4⁺ T cells and were highly activated. Purified CD8⁺ T cells produced IFN-γ in response to HLA-matched pancreatic tumor targets. PD-1 blockade and 4-1BB stimulation were demonstrated as effective strategies to improve effective TIL yield, including the production of tumor-reactive pancreatic TIL. Conclusions TIL expanded from pancreatic tumors are functional and able to respond to pancreatic tumor associated antigens. PD-1 blockade, 41BB stimulation, and CD8⁺ T cell enrichment are effective strategies to improve TIL yield and tumor reactivity. These results support the development of adoptive cell therapy strategies using TIL for the treatment of pancreatic cancer.

Inhibition of antitumor T cell responses can be mediated by the productive interaction between the programmed death-1 (PD-1) receptor on T cells and its ligand PD-L1. PD-L1 is highly expressed on both murine bone marrow-derived dendritic cells (DCs) and B16 melanoma. In this study, in vitro blockade of PD-L1 interaction on DCs led to enhanced IFN-γ production and cytotoxicity by Ag-specific T cells. In vivo, the systemic administration of anti–PD-L1 Ab plus melanoma peptide-pulsed DCs resulted in a higher number of melanoma peptide-specific CD8+ T cells, but this combination was insufficient to delay the growth of established B16 melanoma. Although the addition of 600 rad of total body irradiation delayed tumor growth, further adoptive transfer of Ag-specific CD8+ T cells was needed to achieve tumor regression and long-term survival of the treated mice. Lymphopenic mice treated with anti–PD-L1 Ab demonstrated increased activation and persistence of adoptively transferred T cells, including a higher number of CD8+ T cells infiltrating the tumor mass. Together, these studies support the blocking of PD-L1 signaling as a means to enhance combined immunotherapy approaches against melanoma.

Purpose: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) for metastatic melanoma can be highly effective, but attrition due to progression before TIL administration (32% in prior institutional experience) remains a limitation. We hypothesized that combining ACT with CTLA-4 blockade would decrease attrition and allow more patients to receive TIL. Experimental Design: Thirteen patients with metastatic melanoma were enrolled. Patients received four doses of ipilimumab (3 mg/kg) beginning two weeks prior to tumor resection for TIL generation, then one week after resection, and 2 and 5 weeks after pre-conditioning chemotherapy and TIL infusion followed by IL-2. The primary endpoint was safety and feasibility. Secondary endpoints included of clinical response at 12 weeks and at one year after TIL transfer, disease free survival (DFS) and overall survival (OS). Results: All patients received at least 2 doses of ipilimumab, and 12 of the 13 (92%) received TIL. A median of 6.5x10^10 (2.3x10^10 - 1.0x10^11) TIL were infused. At 12 weeks following infusion there were 5 patients who experienced objective response (38.5%), 4 of whom continued in objective response at one year and one of which became a complete response at 52 months. Median progression-free survival was 7.3 months (95%CI 6.1-29.9 months). Grade ≥3 immune-related adverse events included hypothyroidism (3), hepatitis (2), uveitis (1) and colitis (1). Conclusions: Ipilimumab plus ACT for metastatic melanoma is feasible, well tolerated, and associated with a low rate of attrition due to progression during cell expansion. This combination approach serves as a model for future efforts to improve the efficacy of ACT.

Administration of nonmyeloablative chemotherapeutic agents or total body irradiation (TBI) prior to adoptive transfer of tumor-specific T cells may reduce or eliminate immunosuppressive populations such as T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSC). Little is known about these populations during immune reconstitution. This study was designed to understand the reconstitution rate and function of these populations post TBI in melanoma tumor‑bearing mice. Reconstitution rate and suppressive activity of CD4+CD25+Foxp3+ Tregs and CD11b+Gr1+ MDSC following TBI-induced lymphopenia was measured in B16 melanoma tumor‑bearing mice. To ablate the rapid reconstitution of suppressive populations, we treated mice with docetaxel, a known chemotherapeutic agent that targets MDSC, in combination with adoptive T cell transfer and dendritic cell immunotherapy. Both Treg and MDSC populations exhibited rapid reconstitution after TBI-induced lymphopenia. Although reconstituted Tregs were just as suppressive as Tregs from untreated mice, MDSC demonstrated enhanced suppressive activity of CD8+ T cell proliferation compared with endogenous MDSC from tumor-bearing mice. TBI-induced lymphopenia followed by docetaxel treatment improved the efficacy of adoptive T cell transfer and dendritic cell immunotherapy in melanoma-bearing mice, inducing a significant reduction in tumor growth and enhancing survival. Tumor regression correlated with increased CTL activity and persistence of adoptively transferred T cells. Overall, these findings suggest that TBI-induced MDSC are highly immunosuppressive and blocking their rapid reconstitution may improve the efficacy of vaccination strategies and adoptive immunotherapy.

Intralesional (IL) injection of PV-10 has shown to induce regression of both injected and non-injected lesions in patients with melanoma. To determine an underlying immune mechanism, the murine B16 melanoma model and the MT-901 breast cancer model were utilized. In BALB/c mice bearing MT-901 breast cancer, injection of PV-10 led to regression of injected and untreated contralateral subcutaneous lesions. In a murine model of melanoma, B16 cells were injected into C57BL/6 mice to establish one subcutaneous tumor and multiple lung lesions. Treatment of the subcutaneous lesion with a single injection of IL PV-10 led to regression of the injected lesion as well as the distant B16 melanoma lung metastases. Anti-tumor immune responses were measured in splenocytes collected from mice treated with IL PBS or PV-10. Splenocytes isolated from tumor bearing mice treated with IL PV-10 demonstrated enhanced tumor-specific IFN-gamma production compared to splenocytes from PBS-treated mice in both models. In addition, a significant increase in lysis of B16 cells by T cells isolated after PV-10 treatment was observed. Transfer of T cells isolated from tumor-bearing mice treated with IL PV-10 led to tumor regression in mice bearing B16 melanoma. These studies establish that IL PV-10 therapy induces tumor-specific T cell-mediated immunity in multiple histologic subtypes and support the concept of combining IL PV10 with immunotherapy for advanced malignancies.