BRON: https://www.nature.com/articles/s41523-021-00229-5

We show that treatment with the FDA-approved anti-parasitic drug ivermectin induces immunogenic cancer cell death (ICD) and robust T cell infiltration into breast tumors. As an allosteric modulator of the ATP/P2X4/P2X7 axis which operates in both cancer and immune cells, ivermectin also selectively targets immunosuppressive populations including myeloid cells and Tregs, resulting in enhanced Teff/Tregs ratio. While neither agent alone showed efficacy in vivo, combination therapy with ivermectin and checkpoint inhibitor anti-PD1 antibody achieved synergy in limiting tumor growth (p = 0.03) and promoted complete responses (p < 0.01), also leading to immunity against contralateral re-challenge with demonstrated anti-tumor immune responses. Going beyond primary tumors, this combination achieved significant reduction in relapse after neoadjuvant (p = 0.03) and adjuvant treatment (p < 0.001), and potential cures in metastatic disease (p < 0.001). Statistical modeling confirmed bona fide synergistic activity in both the adjuvant (p = 0.007) and metastatic settings (p < 0.001). Ivermectin has dual immunomodulatory and ICD-inducing effects in breast cancer, converting cold tumors hot, thus represents a rational mechanistic partner with checkpoint blockade.

Introduction

Checkpoint blockade^1,2 has emerged as a revolutionary approach that harnesses a patient’s own immune system to treat cancer. However, checkpoint inhibitors as single agents are only effective in a subset of patients and cancer types². Recent studies suggest that efficacy of checkpoint inhibitors is primarily limited to cancers already infiltrated by T cells—often termed “hot” tumors. In contrast, “cold” tumors have little to no T cell infiltration and generally do not respond to checkpoint blockade. Clinical studies with checkpoint blockade therapy in breast cancer have focused on triple negative breast cancer (TNBC), because this subtype has a higher mutational load and is thought to be more “immunogenic”³. While an early phase Ib study (KEYNOTE-012) of pembrolizumab (anti-PD1 antibody) monotherapy showed preliminary evidence of clinical activity in a small subset (18.5%) of advanced TNBC patients⁴, the phase 3 study (KEYNOTE-119) showed no improvement over chemotherapy⁵. Beyond monotherapy, checkpoint blockade plus chemotherapy combinations are being investigated. Atezolizumab (anti-PDL1 antibody) in combination with nab-paclitaxel demonstrated efficacy for PD-L1-positive unresectable locally advanced or metastatic TNBC in IMpassion130 (ref. ⁶), leading to the first FDA approval of immune checkpoint therapy for breast cancer in March 2019. However, IMpassion131 (atezolizumab + paclitaxel) was recently reported to be negative⁷. These results highlight the continued challenge of breast cancer for immune checkpoint therapies. As such, there is considerable need to identify drugs capable of priming breast tumors (turning “cold” tumors “hot”) to synergize with checkpoint blockade.

A recently described phenomenon, termed immunogenic cell death (ICD)^8,9, is a form of cell death that induces an immune response from the host. ICD is distinguished from classical apoptosis and other non-immunogenic or tolerogenic forms of cell death by several hallmarks, including release of adenosine triphosphate (ATP) and high-mobility group box 1 protein (HMGB1), and surface exposure of calreticulin^8,9,10. In cancer patients, ICD-based anti-tumor immune responses are linked to beneficial outcomes produced by some conventional chemotherapeutic agents^11,12,13,14. For example, efficacy of anthracyclines in breast cancer^15,16,17 and oxaliplatin in colorectal cancer¹⁸ correlates with post-treatment increases in the ratio of cytotoxic CD8⁺ T lymphocytes to FoxP3⁺ regulatory T cells within the tumor. In contrast, poor responses to chemotherapy in solid tumors are associated with lymphopenia¹⁹. Thus, ICD-inducing chemotherapy appears to work in conjunction with the host immune system to achieve efficacy. However, chemotherapy is a double-edged sword: it can suppress as well as stimulate immune cells. An agent that induces ICD of cancer cells without suppressing immune function would be ideal for combination with checkpoint blockade. Seeking such an agent among FDA-approved drugs, our group found that the anti-parasitic agent ivermectin promotes ICD in breast cancer cells²⁰. Among our previous findings was evidence that ivermectin, an anti-parasitic drug used worldwide since 1975, modulates the P2X4/P2X7 purinergic pathway, suggesting that ivermectin may further harness tumors’ intrinsic high extracellular levels of ATP for anti-cancer activity. Of note, P2X purinoceptor 4 and P2X purinoceptor 7 (P2X4/P2X7) are widely expressed on various immune subpopulations, suggesting that ivermectin might also have direct immunomodulatory effects.

Results

Ivermectin can turn “cold” breast tumors “hot”

We studied the effects of ivermectin in vivo using the 4T1 mouse model of TNBC. HMGB1 is a chromatin protein present in all cells and its release is a hallmark of ICD²¹. HMGB1 staining (green) was observed uniformly across the entire tumor from untreated mice (Fig. 1A). In contrast, tumors isolated from mice treated with ivermectin showed large areas of DAPI-positive cells lacking HMGB1 (Fig. 1B), suggesting that HMGB1 had been released into the extracellular space. Ivermectin treatment also altered calreticulin expression, with higher levels (green) observed in tumors from treated animals, indicating a significant increase in this ICD-associated prophagocytic signal and mediator (Fig. 1C, D). Exposure of endosomal calreticulin onto the surface of ER-stressed, damaged or dying cells promotes immunogenic phagocytosis and antigen cross-presentation by antagonizing both the “don’t eat me” signals associated with CD47 and the tolerogenic “eat me” signals associated with phosphatidylserine (PS) exposure, while promoting the interaction with its receptor low-density lipoprotein-receptor related protein (LRP) on phagocytic cells. Robust infiltration of both CD4⁺ and CD8⁺ T cells was seen in ivermectin-treated tumors (Fig. 1F) but not in untreated tumors (Fig. 1E). Significantly higher percentages of cells were positive for CD4 (p < 0.01, Fig. 1G) and CD8 (p < 0.0001, Fig. 1H) in ivermectin-treated than in untreated tumors. Together, these data indicate that treatment with ivermectin induced hallmarks of ICD within 4T1 breast tumors and recruited large numbers of CD4⁺ and CD8⁺ T cells into these tumors. To further confirm that ivermectin induces ICD in vivo, we also utilized a classical vaccination approach considered as the gold standard for detection of ICD: treatment of 4T1 cells with IVM to induce ICD in vitro followed by inoculation into naïve mice, then subsequent challenge with live 4T1 cells to demonstrate prevention of tumor outgrowth²¹. This experiment informed a possible induction of bona fide ICD by demonstrating protection against subsequent challenge with live 4T1 cells (p < 0.01, Fig. 1I).