Combinatorial PD-1 blockade and CD137 activation has therapeutic efficacy in murine cancer models and synergizes with cisplatin

Abstract

There is an urgent need for improved therapy for advanced ovarian carcinoma, which may be met by administering immune-modulatory monoclonal antibodies (mAbs) to generate a tumor-destructive immune response. Using the ID8 mouse ovarian cancer model, we investigated the therapeutic efficacy of various mAb combinations in mice with intraperitoneal (i.p.) tumor established by transplanting 3 × 10(6) ID8 cells 10 days previously. While most of the tested mAbs were ineffective when given individually or together, the data confirm our previous finding that 2 i.p. injections of a combination of anti-CD137 with anti-PD-1 mAbs doubles overall survival. Mice treated with this mAb combination have a significantly increased frequency and total number of CD8(+) T cells both in the peritoneal lavage and spleens, and these cells are functional as demonstrated by antigen-specific cytolytic activity and IFN-γ production. While administration of anti-CD137 mAb as a single agent similarly increases CD8(+) T cells, these have no functional activity, which may be attributed to up-regulation of co-inhibitory PD-1 and TIM-3 molecules induced by CD137. Addition of the anti-cancer drug cisplatin to the 2 mAb combination increased overall survival >90 days (and was probably curative) by a mechanism which included a systemic CD8(+) T cell response with tumor specificity and immunological memory. Strikingly, combined treatment of cisplatin and CD137/PD-1 mAb also gave rise to the long-term survival of mice with established TC1 lung tumors. A similar combination of the 2 mAbs and cisplatin should be considered for clinical 'translation'.

Figure 1. Antitumor effects of anti-CD137/PD-1 mAbs againstne ID8 ovarian cancer.

Mice (5/group) transplanted i.p. with 3 × 10⁶ ID8 cells 10 days previously were injected i.p. twice at 4 days interval with the indicated mAb combinations (0.5 mg of each mAb/mouse); survival was recorded (A, C) and mean survival time was calculated (B, D). The experiment was repeated once with similar results. E, Mice (8-9/group) transplanted i.p. with 3 × 10⁶ ID8 cells 3 days previously were injected i.p. twice at 4 days interval with 0.5 mg of control, anti-PD-1, anti-CD137 and anti-PD-1/CD137 mAb and their survival was recorded. *P < 0.05, **P < 0.01, compared with control mAb treated mice.

Figure 2. Analysis of lymphocyte components in spleens from mice treated with mAb combinations.

Mice (3/group) transplanted i.p. with 3 × 10⁶ ID8 cells 10 days earlier were injected i.p. with 0.5 mg of control, anti-CD137, anti-PD-1 or anti-PD-1/CD137 mAb and the mAb injection was repeated 4 days later. Seven days after the second injection, spleens were harvested and single cell suspensions prepared and stained with fluorescence labeled antibodies against markers of lymphocyte subsets prior to analysis by flow cytometry. The percentages and numbers of CD3⁺, CD4⁺, CD8⁺, CD19⁺, FoxP3⁺/CD4 and GR-1⁺CD11b⁺ cells in spleens are shown in (A) and representative dotplots are shown in (B). Data are presented as M ±SEM from 3 mice/group and are representative of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, The findings with anti- PD-1 or CD137 single mAbs are compared with control mAb, and the findings with anti- PD-1/CD137 mAbs are compared with both control and single mAb.

Figure 3. Analysis of effector CD8⁺ T lymphocyte in spleens from mice treated with mAb combinations.

Mice (3/group) which had been transplanted i.p. with 3 × 10⁶ ID8 cells 10 days earlier were injected i.p. twice at 4 days interval with 0.5 mg of control, anti-CD137, anti-PD-1 or anti-PD-1/CD137 mAb. Spleens from treated mice were analyzed for phenotypes and effector functions of CD8⁺ T lymphocytes by flow cytometry 7 days after the second mAb injection. The percentages and numbers of CD44⁺CD62L^- effector/memory and CD44⁺CD62L⁺ central memory and IFN-γ- and IL-10-producing cells in the CD8⁺ T cell population are shown in (A) and representative dotplots are shown in (B). Data are presented as M±SEM from 3 mice in each group and are representative of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, PD-1 or CD137 mAb compared with control mAb, PD-1/CD137 mAb compared with control and single mAb.

Figure 4. Mice injected with an anti-PD-1/CD137 mAb combination developed a tumor antigen-specific CTL response.

Mice (3/group) transplanted i.p. with 3 × 10⁶ ID8 cells 10 day earlier were injected i.p. twice at 4 days interval with 0.5 mg of anti-PD-1/CD137 mAb. Seven days after the second mAb injection, pooled splenocytes (5 × 10⁶) from 3 mice were incubated with 5 × 10⁵ UV-irradiated ID8 cells for 4 days. The resultant splenocytes were then evaluated for antigen-specific CTL activity by CytoTox 96 Non-radioactive cytotoxicity assay using EL4 cells pulsed with H-2Db-restricted mesothelin or HPV-E7 peptide as target cells (A). The killing activity was also evaluated in the presence of anti-CD4, anti-CD8 or control antibody (B). Data were expressed as M±SEM of triplicate wells.

Figure 5. Analysis of peritoneal lymphocyte subsets from mice injected with the anti-PD-1/CD137 combination.

Mice (3/group) transplanted i.p. with 3 × 10⁶ ID8 cells 10 day earlier were injected i.p. twice at 4 days interval with 0.5 mg of control, anti-CD137, anti-PD-1 or anti-PD-1/CD137 mAb. Two weeks later, peritoneal lavage from treated mice was analyzed by flow cytometry for the percentage and phenotype of peritoneal lymphocytes. The percentages of CD3⁺, CD4⁺, CD8⁺ and CD19⁺ lymphocytes in peritoneal lavage and CD44⁺CD62L^- effector/memory cells in CD4⁺ and CD8⁺ T cells are shown in (A) and (B) respectively. The percentage of PD-1⁺TIM-3⁺ and PD-1^-TIM-3⁺ cells in peritoneal CD4⁺ and CD8⁺ T cells are shown in (C) with representative dotplots in Figure S2. Data are presented as M±SEM from 3 mice of each group and are representative of 2 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, PD-1 or CD137 mAb compared with control mAb, PD-1/CD137 mAb compared with control and single mAb.

Figure 6. Combining anti-PD-1/CD137 mAb with cisplatin induced complete remission of established ID8 cancer with long-lasting systemic tumor-specific immunity.

Mice (10/group) transplanted i.p. with 3 x 10⁶ ID8 cells 10 days earlier were injected i.p. with two doses of control, anti-PD-1, anti-CD137 or anti-PD-1/CD137 mAb (0.5 mg per dose per mouse) at 4 days interval with or without coadministration of cisplatin (10mg/kg) at the first treatment and their survival was evaluated (A). Mice (10/group) treated with combined anti-PD-1/CD137/cisplatin were depleted of lymphocyte subsets by injection of anti-CD4 (0.2 mg/mouse), anti-CD8 (0.2 mg/mouse), anti-NK1.1 (0.1 mg/mouse) or control mAb (0.2 mg/mouse) 48 and 72 hours prior to the first treatment and every 3-4 days thereafter for the duration of the experiments. Untreated tumor-bearing mice were as negative controls (UNT group). The survival of mice was recorded (B). Fifteen long-term surviving mice (120 days after original transplantation of ID8 cells) pooled from 2 experiments were challenged (5 mice/group) with ID8 cells given i.p. or s.c. or with TC1 cells transplanted s.c. (C); naive mice were transplanted with tumor cells as controls (D) and their survival was recorded. Mice (6/group) with established TC1 tumors of 4-5 mm mean diameter were injected i.p. with two doses of control, anti-PD-1, anti-CD137 or anti-PD-1/CD137 mAb (0.5 mg per dose per mouse) at 4 days interval with or without coadministraation of cisplatin (10 mg/kg) at the first treatment; tumor growth was measured (E) and survival was recorded (F). Data are representative of 2 experiments for Figure 6A-D.