Cyclophosphamide enhances the antitumor potency of GITR engagement by increasing oligoclonal cytotoxic T cell fitness
Daniel Hirschhorn, Allison Betof Warner, Rachana Maniyar, Andrew Chow, Levi M.B. Mangarin, Adam D. Cohen, Linda Hamadene, Gabrielle A. Rizzuto, Sadna Budhu, Nathan Suek, Cailian Liu, Alan N. Houghton, Taha Merghoub, Jedd D. Wolchok
Daniel Hirschhorn, Allison Betof Warner, Rachana Maniyar, Andrew Chow, Levi M.B. Mangarin, Adam D. Cohen, Linda Hamadene, Gabrielle A. Rizzuto, Sadna Budhu, Nathan Suek, Cailian Liu, Alan N. Houghton, Taha Merghoub, Jedd D. Wolchok
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Research Article Immunology Oncology

Cyclophosphamide enhances the antitumor potency of GITR engagement by increasing oligoclonal cytotoxic T cell fitness

Abstract

Only a subset of cancer patients responds to checkpoint blockade inhibition in the clinic. Strategies to overcome resistance are promising areas of investigation. Targeting glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) has shown efficacy in preclinical models, but GITR engagement is ineffective in controlling advanced, poorly immunogenic tumors, such as B16 melanoma, and has not yielded benefit in clinical trials. The alkylating agent cyclophosphamide (CTX) depletes regulatory T cells (Tregs), expands tumor-specific effector T cells (Teffs) via homeostatic proliferation, and induces immunogenic cell death. GITR agonism has an inhibitory effect on Tregs and activates Teffs. We therefore hypothesized that CTX and GITR agonism would promote effective antitumor immunity. Here we show that the combination of CTX and GITR agonism controlled tumor growth in clinically relevant mouse models. Mechanistically, we show that the combination therapy caused tumor cell death, clonal expansion of highly active CD8+ T cells, and depletion of Tregs by activation-induced cell death. Control of tumor growth was associated with the presence of an expanded population of highly activated, tumor-infiltrating, oligoclonal CD8+ T cells that led to a diminished TCR repertoire. Our studies show that the combination of CTX and GITR agonism is a rational chemoimmunotherapeutic approach that warrants further clinical investigation.

Authors

Daniel Hirschhorn, Allison Betof Warner, Rachana Maniyar, Andrew Chow, Levi M.B. Mangarin, Adam D. Cohen, Linda Hamadene, Gabrielle A. Rizzuto, Sadna Budhu, Nathan Suek, Cailian Liu, Alan N. Houghton, Taha Merghoub, Jedd D. Wolchok

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Figure 4

High-dose CTX causes tumor cell death and induces in situ vaccination.

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High-dose CTX causes tumor cell death and induces in situ vaccination. (...
(A) Experiment schema: Thy1.2 mice were injected with B16. On day 6, groups mice (n ≥ 5) were treated with CTX or PBS. On day 7, all mice received CFSE-labeled CD8+pmel-1 T cells (Thy1.1) and anti-GITR or rat IgG. On day 12, mice were sacrificed, a single-cell suspension was prepared from inguinal tumor-draining lymph nodes (TDLNs) or contralateral lymph nodes (non-TDLNs), and proliferation was analyzed by flow cytometry. (B) Representative flow plot (left) of proliferating antigen-experienced cells gated on DAPI−, CD8+, and Thy1.1+CFSElo. Bars (right) represent the average of 5–7 mice ± SEM. Symbols represent individual mice and lines represent averages ± SEM. Unpaired 2-tailed Student’s t test was used to compare IgG and anti-GITR groups. *P < 0.05. (C) Experiment schema: Cohorts of mice (n = 10) were injected with CTX at day –2 or +1 relative to B16 tumor implant. Some cohorts were injected with DTA-1 at day 2 after tumor challenge. (D) Kaplan-Meier survival curves demonstrating improved survival in the group treated with a combination therapy after tumor implant. Log-rank (Mantel-Cox) test was used to compare Kaplan-Meier survival curves. (E) Tumor growth curves of individual mice per treatment group demonstrating a regression only when treated with CTX post tumor implant in combination with anti-GITR. *P < 0.05, ***P < 0.001. Experiments were repeated at least twice with similar results.