Purpose: To characterize the ability of PT-112 to induce immunogenic cell death (ICD) in vitro and in vivo. Background: PT-112 is a novel chemical entity consisting of a platinum core conjugated to a pyrophosphate and diaminocyclohexane groups. In vitro studies demonstrated that PT-112 has both cytostatic and cytotoxic effects on human cancer cells, two effects that are seen in the absence of robust binding to nuclear DNA. Accordingly, PT-112 potency is largely unaffected by functional DNA repair pathways, suggesting that PT-112 operates with mechanisms that differ from conventional DNA-damaging chemotherapies. PT-112 is currently under phase I/II clinical development in patients with solid tumors and hematologic malignancies, both as monotherapy and in combination with a PD-L1 inhibitor. Interim reports have established encouraging tolerability and signals of single-agent anticancer activity. Previous work with human colorectal cancer HCT-116 cells demonstrated that PT-112 causes the release of ICD-relevant damage-associated molecular patterns (DAMPs). ICD is a particularly relevant form of cell death for cancer therapy as it can drive tumor-targeting immune responses of clinical relevance. Here, we tested the ability of PT-112 to induce bona fide ICD in murine breast carcinoma TSA cells by in vitro DAMP release studies and in vivo gold-standard vaccination experiments. Methods: TSA cells were exposed to PT-112, cisplatin (negative ICD control) or mitoxantrone (MTX, positive ICD control) at different concentrations for 24 and 48 hours, followed by cytofluorometric assessment of cell number, viability, cell cycle distribution and exposure of the membrane-assocaited DAMP calreticulin (CRT). In addition, culture supernatants were assayed for the release of two other DAMPs, namely ATP and HMGB1, by luminometric tests and ELISA, respectively. BALB/c mice were vaccinated with TSA cells succumbing to optimal PT-112 (or MTX and cisplatin, as positive and negative controls, respectively) concentrations or mock vaccinated (with PBS). After 14 days this was followed by a rechallenge with living TSA cells and scoring of tumor-free survival (TFS) and tumor growth. Results: In vitro, PT-112 caused a robust cytostatic and cytotoxic effect on TSA cells that was accompanied by robust CRT exposure, ATP secretion and HMGB1 release. In line with such an ICD-compatible profile, 10/10 mice vaccinated with PT-112-treated TSA cells were tumor-free after the 1st rechallenge, and also rejected a subsequent challenge performed 1 month later, suggesting the establishment of long-term immunologic anticancer memory. As expected, cisplatin-treated TSA cells failed to mediate a similar effect (TFS at D7 after rechallenge: 10%), as did the mock vaccination arm (TFS at D7: 20%). MTX-treated cells conferred partial protection (TFS at D7: 40%), and the developing tumors had significantly delayed kinetics as compared to tumors evolving in mock-vaccinated mice. Conclusions: In the TSA model system of ICD vaccination, PT-112 is superior to MTX at inducing ICD and the associated establishment of immunologic anticancer memory in vivo. These data provide a robust rationale to combining PT-112 with checkpoint inhibitors in the clinic, to achieve maximal therapeutic effects potentially coupled with long-term protection from metastatic reactivation.
Citation Format: Takahiro Yamazaki, Tyler David Ames, Lorenzo Galluzzi. Potent induction of immunogenic cell death by PT-112 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B199.