Robust Antitumor Responses Result from Local Chemotherapy and CTLA-4 Blockade

Abstract

Clinical responses to immunotherapy have been associated with augmentation of preexisting immune responses, manifested by heightened inflammation in the tumor microenvironment. However, many tumors have a noninflamed microenvironment, and response rates to immunotherapy in melanoma have been <50%. We approached this problem by utilizing immunotherapy (CTLA-4 blockade) combined with chemotherapy to induce local inflammation. In murine models of melanoma and prostate cancer, the combination of chemotherapy and CTLA-4 blockade induced a shift in the cellular composition of the tumor microenvironment, with infiltrating CD8⁺ and CD4⁺ T cells increasing the CD8/Foxp3 T-cell ratio. These changes were associated with improved survival of the mice. To translate these findings into a clinical setting, 26 patients with advanced melanoma were treated locally by isolated limb infusion with the nitrogen mustard alkylating agent melphalan followed by systemic administration of CTLA-4 blocking antibody (ipilimumab) in a phase II trial. This combination of local chemotherapy with systemic checkpoint blockade inhibitor resulted in a response rate of 85% at 3 months (62% complete and 23% partial response rate) and a 58% progression-free survival at 1 year. The clinical response was associated with increased T-cell infiltration, similar to that seen in the murine models. Together, our findings suggest that local chemotherapy combined with checkpoint blockade-based immunotherapy results in a durable response to cancer therapy. Cancer Immunol Res; 6(2); 189-200. ©2018 AACR.

Fig. 1.

Melphalan and CTLA-4 blockade in a model of melanoma. (A) B16 cells were treated in vitro with melphalan or vehicle control at 50 μM. After 24 hours, the expression of MHC class I, MHC class II, and PD-L1 was assessed by flow cytometry. This is a representative figure from the experiment performed 3 times. Fold change was calculated based upon mean fluorescence intensity change from untreated to treated. (B) Melphalan synergizes with CTLA-4 blockade. Mice with palpable allografted B16 tumors were treated with single intratumoral dose of melphalan, alone or followed by CTLA-4 blockade, 100 μg IP, every 3 days for 4 doses. Control mice received intratumoral injection of vehicle control and IP injection of isotype antibody. Shown are pooled data of 3 separate experiments (3–5 mice per treatment group per experiment, four experiments). (C,D) Combination therapy enhances the inflammatory environment of the tumor. (C) CD4 effector and CD8⁺ cells per gram of tumor in B16 tumors from the four treatment groups, P < 0.05 for combination versus control treatment. (D) CD4 and FoxP3 expression on cells from tumors from the four treatment groups. Experiments were performed 3 times; shown is representation of one experiment. (E) IFNγR^−/− mice had an improved median survival for melphalan plus CTLA-4 blockade over control or melphalan treated mice (combination (n = 9), median survival 22 days, control (n = 5), median survival 10 days, melphalan (n = 6), 12 days, P < 0.05). However, no combination mice were cured of tumors, suggesting the importance of IFNγ on the host.

Fig. 2.

Clinical benefit from combination limb infusion and CTLA-4 blockade. (A) Schema of the phase II clinical trial. (B) Swimmer plot of all patients, showing responses in the melphalan-treated limb. (C) PFS for patients, including the two patients who died prior to the evaluation point. (D) Example of durable response both in and outside of the limb of the infusion. This patient had an unknown primary melanoma and presented with multiple subcutaneous nodules of the limb, with groin adenopathy, biopsy-proven lung metastasis, and thoracic adenopathy. Three years after limb infusion and systemic ipilimumab, the patient remains free of disease (lower panel).

Fig. 3.

Nanostring analysis of tumors after isolated limb infusion (ILI) and after combination limb infusion and ipilimumab. Gene expression was compared from available pretreatment biopsies (n = 11) to post ILI (n = 4) and post-IPI (n = 14) biopsies. Tumor biopsies were taken just prior to ILI, after ILI (7–15 days) and 3 weeks after the last dose of Ipilimumab. The gene expression pattern after ILI favored up-regulation of costimulatory ligands, and innate immune function, while after combination treatment, there was increased expression of cytotoxic function, particularly granzymes, IFNγ, perforin and ICOS.

Fig. 4.

Analysis of cytokines and immune cell phenotypes in the phase II trial. (A) Increase in serum cytokines after combination therapy. The cytokines indicated were quantitated by Meso Scale Discovery assay in serum taken pretreatment (pre), after isolated limb infusion (ILI), and after the initial ipilimumab (ipi) treatment. *, P < 0.05; ***, P < 0.001 compared with pretreatment or as indicated by brackets. Note the increase in cytotoxic cytokines after combination treatment consistent with nanostring data (B-D) Changes in T cells in blood and tumor after ILI and ipilimumab treatments, assessed by flow cytometry. (B) Increase in percentage of CD4⁺ Ki67⁺ and CD8⁺ Ki67⁺ T cells, of total CD45⁺ cells, in peripheral blood mononuclear cells after ILI and after the first dose of ipilimumab. (C) Increase in percentage of CD4⁺ ICOS⁺ cells after ILI and after the each of the first (of 4) ipilimumab doses. (D) Analysis of tumor samples demonstrating an increase in the ratio of CD8⁺ T cells to CD4⁺FOXP3⁺ T cells (left) and the percentage of CD4⁺ICOS⁺ T cells (right). The bars shown mean values and standard deviation. (E,F) Immune infiltration in tumors after isolated limb infusion and ipilimumab treatment. (E) Representative multiplex immunohistochemistry at 20x showing CD4 (green), CD8 (red), CD68 (yellow), and PD-L1 (white) on samples of a tumor before treatment (top) and after isolated limb infusion and ipilimumab (bottom). (F) Quantitation of the immunohistochemical results from all available tumors. Note the increase in CD4⁺, CD8⁺, and CD68⁺ cells. Left, box-and-whisker plot showing the median, interquartile range, and range (excluding outliers) for cells positive for CD4, CD8, and CD68. Right, mean percentage of positive cells, with dark green indicating the PD-L1-positive subset. G) H&E and immunohistochemistry of a single tumor, demonstrating the pretreatment melanoma tumor cells with a paucity of immune cells but with MHC II expression (top), and an increase in both immune cells and MHC after ILI and IPI (bottom).

Fig. 5.

TCRβ seqencing. (A) TCR fraction in PBMCs and in tumor at baseline (Pre-TX), after limb infusion (post-ILI) and after the fourth dose of Ipilimumab (Post-IPI). Note that there is not a difference in the starting T cells between patients that derived benefit (PFS at one year versus not). Although there was not an overall change in T cells in the periphery, there T cells in the tumors of patients with PFS at one year were somewhat increased, although the change was not significant. (B) Box-and-whisker plots of the clonality of the TCR fraction. The clonality did not change in the PBMC; however, there was a significant increase in clonality, or a less even distribution of T cell clones, in the tumor versus the PBMC (P = 0.015, by Wiloxon signed rank test), after combination treatment.