Abstract 1795: Combination of cyclophosphamide chemotherapy with immune modulation overcomes resistance to checkpoint blockade in a pre-clinical melanoma model
Authors: Warner Allison Betof , Hirschhorn Daniel , Mangarin Levi M. , Hamadene Linda , Cohen Adam D. , Rizzuto Gabrielle A. , Wolchok Jedd D. , Merghoub Taha 
Published: July 2021

Abstract
Immune checkpoint blockade (ICB) with anti-CTLA-4 and anti-PD-1 is increasingly used to treat a variety of malignancies, but primary and acquired resistance limit the clinical benefit. Mechanism-guided approaches to overcome resistance and re-orchestrate an anti-tumor immune response could expand the clinical impact of ICBs. Cyclophosphamide (CTX) is an alkylating chemotherapeutic which is directly tumoricidal and also modulates immune response. CTX preferentially depletes certain T cell subsets (such as Tregs) and leads to homeostatic proliferation of antigen-specific T cells. We hypothesized that a dose of CTX prior to initiating ICB would counteract several known patterns of resistance and enhance anti-tumor response to ICB.
The B16 murine melanoma model is largely refractory to inhibition of PD-1, CTLA-4, or combination CTLA-4+PD-1, all of which only modestly slow the growth of small tumors. One dose of CTX one day prior to each of these treatments significantly slows tumor growth and prolongs survival compared to CTX or ICB alone. This effect is most pronounced for the triple combination of CTX+ anti-CTLA-4+ anti-PD-1, where tumor regression was observed in 70% of mice and tumors were completely eradicated in 20%.
In addition to standard ICB, we hypothesized that CTX would enhance response to immune modulatory molecules in clinical development. Glucocorticoid-induced TNFR family related gene (GITR) is a costimulatory molecule expressed primarily on the surface of T cell subsets. GITR engagement enhances activation, proliferation, and clonal expansion of effector T cells while hampering the suppressive function of Tregs. However, agonist anti-GITR antibodies have neither effectively controlled tumor growth when given as a monotherapy in preclinical models nor showed efficacy in clinical trials. In the B16 melanoma model, CTX + GITR engagement with the agonist monoclonal antibody DTA-1 slows tumor growth, elicits some tumor regression, and prolongs survival, whereas neither therapy alone controls tumor growth. Similar efficacy is observed in other tumor models (MCP-11 myeloma and CT26 colon carcinoma). Combination therapy with CTX + DTA-1 increases the ratio of CD8+ effector T cells to Tregs, at least in part by inducing Treg-specific activation-induced cell death. Single cell sequencing and flow cytometry demonstrated a marked increase in pro-inflammatory cytokines, cytolytic granules, and activation markers accompanied by reduced exhaustion markers in intratumoral CD8+ T cells. Overall, we demonstrate that a single dose of CTX added to standard and novel immune modulators is a potent combinatorial approach that primes an anti-tumor response, enhances T cell fitness, and overcomes primary resistance to ICB. The data presented here serve as the foundation for mechanistically-driven clinical trials in development.
Citation Format: Allison Betof Warner, Daniel Hirschhorn, Levi M. Mangarin, Linda Hamadene, Adam D. Cohen, Gabrielle A. Rizzuto, Jedd D. Wolchok, Taha Merghoub. Combination of cyclophosphamide chemotherapy with immune modulation overcomes resistance to checkpoint blockade in a pre-clinical melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1795.