Neutrophil-activating therapy for the treatment of cancer.

Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils in vitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B4, which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and T cell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer.

Ethanol Ablation Therapy Drives Immune-Mediated Antitumor Effects in Murine Breast Cancer Models.

Ethanol ablation is a minimally invasive, cost-effective method of destroying tumor tissue through an intratumoral injection of high concentrations of cytotoxic alcohol. Ethyl-cellulose ethanol (ECE) ablation, a modified version of ethanol ablation, contains the phase-changing polysaccharide ethyl-cellulose to reduce ethanol leakage away from the tumor. Ablation produces tissue necrosis and initiates a wound healing process; however, the characteristic of the immunologic events after ECE ablation of tumors has yet to be explored. Models of triple-negative breast cancer (TNBC), which are classically immunosuppressive and difficult to treat clinically, were used to characterize the immunophenotypic changes after ECE ablation. In poorly invasive TNBC rodent models, the injury to the tumor induced by ECE increased tumor infiltrating lymphocytes (TILs) and reduced tumor growth. In a metastatic TNBC model (4T1), TILs did not increase after ECE ablation, though lung metastases were reduced. 4T1 tumors secrete high levels of granulocytic colony stimulating factor (G-CSF), which induces a suppressive milieu of granulocytic myeloid-derived suppressor cells (gMDSCs) aiding in the formation of metastases and suppression of antitumor immunity. We found that a single intratumoral injection of ECE normalized tumor-induced myeloid changes: reducing serum G-CSF and gMDSC populations. ECE also dampened the suppressive strength of gMDSC on CD4 and CD8 cell proliferation, which are crucial for anti-tumor immunity. To demonstrate the utility of these findings, ECE ablation was administered before checkpoint inhibitor (CPI) therapy in the 4T1 model and was found to significantly increase survival compared to a control of saline and CPI. Sixty days after tumor implant no primary tumors or metastatic lung lesions were found in 6/10 mice treated with CPI plus ECE, compared to 1/10 with ECE alone and 0/10 with CPI and saline.

Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity.

Innate pattern recognition receptor agonists, including Toll-like receptors (TLRs), alter the tumor microenvironment and prime adaptive antitumor immunity. However, TLR agonists present toxicities associated with widespread immune activation after systemic administration. To design a TLR-based therapeutic suitable for systemic delivery and capable of safely eliciting tumor-targeted responses, we developed immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 dual agonist conjugated to tumor-targeting antibodies. Systemically administered human epidermal growth factor receptor 2 (HER2)-targeted ISACs were well tolerated and triggered a localized immune response in the tumor microenvironment that resulted in tumor clearance and immunological memory. Mechanistically, ISACs required tumor antigen recognition, Fcγ-receptor-dependent phagocytosis and TLR-mediated activation to drive tumor killing by myeloid cells and subsequent T-cell-mediated antitumor immunity. ISAC-mediated immunological memory was not limited to the HER2 ISAC target antigen since ISAC-treated mice were protected from rechallenge with the HER2- parental tumor. These results provide a strong rationale for the clinical development of ISACs.