Immune Checkpoint Blockade Augments Lymphodepleting Chemotherapy–Induced Antitumor Immunity by Expanding Effector CD8+ T-cell Clones

Cancer treatment using immune checkpoint blockade (ICB) with anti-programmed cell death protein 1 (αPD-1) and anti-cytotoxic T lymphocyte-associated protein 4 (αCTLA-4) has been successful. However, primary and acquired resistance limits clinical benefit. To improve the effectiveness of ICB therapies, strategies that reorchestrate antitumor immunity through mechanism-based drug combinations are being actively explored. The alkylating chemotherapeutic agent cyclophosphamide (CTX) has direct tumoricidal and immunomodulatory properties, including the induction of homeostatic proliferation of T cells. As ICB suppresses inhibitory signals in T cells, we hypothesized that ICB could augment CTX-induced homeostatic proliferation of antigen-specific T cells, thereby resetting the T-cell receptor repertoire in favor of tumor-specific T cells. In this study, we showed that a single dose of CTX 1 day prior to starting αPD-1 + αCTLA-4 is sufficient to delay tumor progression in established melanoma and prolong survival in tumor-bearing mouse models. These effects extended to other lymphodepleting treatments, such as gemcitabine and radiotherapy. The antitumor immune response was mainly driven by the clonal expansion of activated/effector CD8+ tumor-infiltrating lymphocytes (TIL). Furthermore, combined CTX and αPD-1 + αCTLA-4 treatment demonstrated efficacy across additional preclinical tumor models, including colorectal cancer and triple-negative breast cancer. Overall, these findings highlight that the combination of CTX and ICB represents a clinically relevant approach in the treatment of immunotherapy-refractory tumors. SIGNIFICANCE: Combining lymphodepleting chemotherapy with dual immune checkpoint blockade enhances clonal expansion of effector CD8+ tumor-infiltrating lymphocytes, delays tumor growth, and improves survival, offering a promising strategy for overcoming resistance to immunotherapy.