Intratumoral CD40 activation and checkpoint blockade induces T cell-mediated eradication of melanoma in the brain

Abstract

CD40 agonists bind the CD40 molecule on antigen-presenting cells and activate them to prime tumor-specific CD8⁺ T cell responses. Here, we study the antitumor activity and mechanism of action of a nonreplicating adenovirus encoding a chimeric, membrane-bound CD40 ligand (ISF35). Intratumoral administration of ISF35 in subcutaneous B16 melanomas generates tumor-specific, CD8⁺ T cells that express PD-1 and suppress tumor growth. Combination therapy of ISF35 with systemic anti-PD-1 generates greater antitumor activity than each respective monotherapy. Triple combination of ISF35, anti-PD-1, and anti-CTLA-4 results in complete eradication of injected and noninjected subcutaneous tumors, as well as melanoma tumors in the brain. Therapeutic efficacy is associated with increases in the systemic level of tumor-specific CD8⁺ T cells, and an increased ratio of intratumoral CD8⁺ T cells to CD4⁺ Tregs. These results provide a proof of concept of systemic antitumor activity after intratumoral CD40 triggering with ISF35 in combination with checkpoint blockade for multifocal cancer, including the brain.

Fig. 1

Antitumor activity and immune response upon intratumoral ISF35 treatment. Mice bearing 8-day s.c. B16.F10 tumors were treated on day 0, 4, 8, and 12 with ISF35 or control rAd/PBS. a Tumor growth (n = 10). b Mouse survival (n = 10). c Tumor growth after i.t. treatment with agonistic CD40 mAb or ISF35 (n = 8). d–f Myeloid cell numbers and activation in tumor 5 days after initiation of ISF35 or PBS treatment. g Intratumoral cytokines and chemokines 7 days after initiation of ISF35 or rAd or PBS treatment, measured by Luminex from a tumor supernatant (n = 5). Data are representative of at least two independent experiments and analyzed by unpaired two-tailed t test or one-way ANOVA. *P < 0.05, **P < 0.005, ***P < 0.0005 and ****P < 0.00005. Error bars are SEM. Survival analysis was performed with the log-rank test

Fig. 2

Induction of tumor-specific CD8⁺ T cell immunity and reduction of regulatory CD4 T cells through intratumoral ISF35 treatment. Mice bearing 8-day s.c. B16.F10 tumors were treated as indicated every 4 days and analyzed 9 days after initiation of treatment. a Tumor antigen (p15E)-specific IFN-γ-producing CD8 T cells in TIL. Percent of CD8⁺IFN-γ⁺ cells (left) and cumulative data (right). b CD62L. c T-bet expression (MFI) in CD8⁺ T cells. d Percentage of CD4 Treg from CD45⁺CD4⁺ T cells in tumor. e Tumor-specific CD8 T cells and f Tregs in spleen. Data are representative of at least 2 independent experiments and analyzed by unpaired two-tailed t test or one-way ANOVA (n = 4 or 5 mice/group). *P < 0.05, **P < 0.005 and ***P < 0.0005. Error bars are SEM

Fig. 3

Mechanism of action of intratumoral ISF35-mediated antitumor activity. Mice bearing 8-day s.c. B16.F10 tumors were treated every 4 days with ISF35 or PBS and depleting antibodies as indicated. The graphs show the survival of a CD8 T cell and b CD11b cell-depleted c CD40 KO mice d CD4 cell and e NK cell depleted and after ISF35 treatment. Survival analysis was performed with the log-rank test (n = 8). **P < 0.005, ***P < 0.0005. Error bars are SEM

Fig. 4

Antitumor effect of intratumoral ISF35 and anti-PD-1 antibody combination therapy. Mice bearing 8-day s.c. B16.F10 tumors were treated as indicated, and tumor-infiltrating leukocytes were stained after 5 days of treatment and analyzed by flow cytometry for the expression of PD-1 and PD-L1. a Mean fluorescence intensity (MFI) and percentage of CD45⁺CD8⁺PD-1⁺cells. b Percentage of CD45⁺CD11b⁺PD-L1⁺ cells. c Mice bearing 8-day s.c. B16.F10 tumors were treated with i.t. ISF35 or rAd and anti-PD-L1. The graph depicts tumor growth at different time points. d Percentage of CD8⁺CTLA-4⁺ cells after indicated treatment. Data are analyzed by unpaired two-tailed t test or one-way ANOVA. *P < 0.05, **P < 0.005 (n = 4–8 mice). Error bars are SEM. Survival analysis was performed with the log-rank test

Fig. 5

Synergistic effect of intratumoral ISF35 with anti-CTLA-4 and anti-PD-1 antibody blockade. Mice bearing 8-day s.c. B16.F10 tumors were treated as indicated. a Mouse survival. b Tumor growth after rechallenge of cured mice. c Vitiligo in a cured mouse (1 cm; represents 3 mice). Mice bearing 8-day s.c. B16.F10 tumors were treated as indicated, and 9 days later, tumors were analyzed for the presence of d Tregs (CD4⁺FoxP3⁺) (e) CD8⁺ T cells and f CD8 to Treg ratio. Data are analyzed by unpaired two-tailed t test. *P < 0.05, **P < 0.005 (n = 4–8 mice). Error bars are SEM

Fig. 6

Abscopal effect after intratumoral ISF35 with anti-CTLA-4 and anti-PD-1 antibody blockade. a Treatment strategy. b Growth of injected and distant, uninjected B16.F10 tumors (n = 5–7). c Mouse survival (n = 5–7). d Tumor antigen (p15E)-specific IFN-γ producing CD8 T cells in circulation (n = 5). e Expression of CD40L on immune cells and tumor cells in treated and untreated tumor after 24 h of ISF35 treatment (n = 3). Data are representative of at least two independent experiments and analyzed by unpaired two-tailed t test or one-way ANOVA. *P < 0.05, **P < 0.005 and ***P < 0.0005. Error bars are SEM. Survival analysis was performed with the log-rank test

Fig. 7

Synergistic effect of intratumoral ISF35 with anti-CTLA-4 and anti-PD-1 antibody therapy against melanoma in the brain. a Treatment strategy. b Mouse survival. c Cause of death (s.c. or brain tumor). d Representative luminescence images from treated animals. Data are from two pooled independent experiments (n = 7 in each experiment). Survival analysis was performed with the log-rank test. ***P < 0.0005