TNFa and IL-2 armed adenoviruses enable complete responses by anti-PD-1 checkpoint blockade

Abstract

Releasing the patient's immune system against their own malignancy by the use of checkpoint inhibitors is delivering promising results. However, only a subset of patients currently benefit from them. One major limitation of these therapies relates to the inability of T cells to detect or penetrate into the tumor resulting in unresponsiveness to checkpoint inhibition. Virotherapy is an attractive tool for enabling checkpoint inhibitors as viruses are naturally recognized by innate defense elements which draws the attention of the immune system. Besides their intrinsic immune stimulating properties, the adenoviruses used here are armed to express tumor necrosis factor alpha (TNFa) and interleukin-2 (IL-2). These cytokines result in immunological danger signaling and multiple appealing T-cell effects, including trafficking, activation and propagation. When these viruses were injected into B16.OVA melanoma tumors in animals concomitantly receiving programmed cell-death protein 1 (PD-1) blocking antibodies both tumor growth control (p < 0.0001) and overall survival (p < 0.01) were improved. In this set-up, the addition of adoptive cell therapy with OT-I lymphocytes did not increase efficacy further. When virus injections were initiated before antibody treatment in a prime-boost approach, 100% of tumors regressed completely and all mice survived. Viral expression of IL2 and TNFa altered the cytokine balance in the tumor microenvironment towards Th1 and increased the intratumoral proportion of CD8+ and conventional CD4+ T cells. These preclinical studies provide the rationale and schedule for a clinical trial where oncolytic adenovirus coding for TNFa and IL-2 (TILT-123) is used in melanoma patients receiving an anti-PD-1 antibody.

Keywords: Adenovirus; Adoptive T cell therapies; Adoptive cell therapy; Checkpoint blockade; Immunotherapy; Immunovirotherapy; Melanoma; Models of immunostimulation; Solid tumors; T cell therapy; Therapeutic antibodies; Virotherapy; anti-PD1.

Figure 1.

Proof of concept antitumor efficacy and overall survival after the combination of virotherapy with checkpoint blockade and adoptive cell therapy. (A) 7–8 animals per group received subcutaneous B16.OVA tumors that were grown for 11 days. Then, 1 × 10⁸ viral particles of non-replicating adenoviruses (coding for mIL2 and mTNFa) or PBS were injected intratumorally. The same day, depending on the group they belonged, 2 × 10⁶ CD8+ OT-1 T cells were adoptively transferred and/or 0.1 mg of anti-PD-1 were injected. Anti-PD-1 treatment was repeated 5 more times every 3 days. (B) Normalized mean tumor volume and SEM at day 15. (C) Overall survival.

Figure 2.

Antitumor efficacy and overall survival after the combination of low dose virotherapy with checkpoint blockade and adoptive cell therapy. (A) Subcutaneous B16.OVA tumors were grown for 10 days. Then mice received i.t. 5 × 10⁶ viral particles of non-replicating adenoviruses coding for mIL2 and mTNFa or PBS. The same day, depending on the group they belonged, 2 × 10⁶ CD8+ OT-1 T cells were adoptively transferred and/or 0.1 mg of anti-PD-1. Checkpoint blockade treatment was repeated 9 more times every 3 days. 6 random animals from each group were euthanized at day 12 and organs were collected for further analysis, the remaining animals (9-12) were maintained for survival studies. (B) Overall survival and statistical significances. (C) Individual tumor growth lines for the different conditions tested and statistical significance of the differences between groups at day 12.

Figure 3.

Phenotypical analysis of tumor infiltrating lymphocytes 13 days after the different treatments started. (A) Percentage CD3+ CD8+ cells of total cells in the tumor. (B) Percentage of PD-1+ lymphocytes of parent population (CD3+ CD8+). (C) Mean fluorescence intensity of the channel used for anti-PD-1. (D) Percentage of CTLA-4+ lymphocytes of parent population (CD3+ CD8+). (E) Percentage of TIM-3+ lymphocytes of parent population (CD3+ CD8+). (F) Percentage of OVA-specific lymphocytes of parent population (CD3+ CD8+). (G) Percentage CD3+ CD4+ cells of total cells in the tumor. (H) Percentage of Regulatory T cells (CD25+ FoxP3+) of parent population (CD3+ CD4+).(I) Percentage of conventional CD4 T cells (CD25+ FoxP3-) of parent population (CD3+ CD4+).

Figure 4.

Antitumor efficacy and overall survival in a clinically relevant set up after the combination of virotherapy with checkpoint blockade. (A) 12–16 animals per group received subcutaneous B16.OVA tumors that were grown for 10 days. Then mice received i.t. 1 × 10⁸ viral particles of non-replicating adenoviruses coding for mIL2 and mTNFa or PBS. The same day, depending on the group they belonged, they received 0.1 mg of anti-PD-1. Checkpoint blockade treatment was repeated every 3 days for a total of 13–15 times. At day 4, 7 untreated animals engrafted with the same tumors were sacrificed to study the status of the tumor at “baseline” time point. 6–7 random animals from each group were euthanized at day 11 (grey line) and organs were collected for further analysis, remaining animals were maintained for survival studies. (B) Overall survival and statistical significances. (C) Individual tumor growth lines for the different conditions tested and statistical significance of the differences between groups at day 39.

Figure 5.

Cytokine profile expression of tumor samples 11 days after the different treatments started. All values are normalized by the cytokine expression of the mock mean value (A) Tumor necrosis factor. (B) Interleukin-2. (C) Interferon gamma. (D) Interleukin-4. (E) Interleukin-6. (F) Interleukin-10. (G) Interleukin-17 A (also studied as Th17 signal representative cytokine). (H) Comparison of pooled Th1 (TNF, IL-2 and IFNg) and Th2 (IL-4, IL-6 and IL-10) cytokines present in the tumor microenvironment.

Figure 6.

Presence of tumor specific CD8 cells in spleens at two different time points. Stacked percentages of OVA/gp100/Trp2 specific CD8+ CD3+ splenocytes. Samples from day 11 (n = 6-7) and from survivors at day 90 (n = 2 from virus alone group, n = 6 from virus + anti-PD-1[simultaneous] and n = 9 from virus + anti-PD-1 [prime and boost]).