Background
Immunogenic cell death (ICD) inducers are an emerging therapeutic modality that kill cancer cells to activate anti-tumor immunity through antigen liberation and presentation. While this dual mechanism of action has strong potential, cytotoxicity to immune cells, as observed with ICD-inducing chemotherapies, has hampered current therapeutic approaches. We previously showed that N17350, a neutrophil elastase-inspired serine protease, leverages a novel cancer-selective killing pathway1 to potently induce ICD in histone H1 rich cancer cells.2ā4 This leads to complete tumor regression, regression of distant metastases, and immune memory following a single intra-tumoral injection.2ā4 N17350 is rapidly inactivated by serine protease inhibitors in blood (eg. alpha-1-antitrypsin; A1AT) of mice and humans. While this inhibition contributes to safety following intratumoral delivery, it stymies therapeutic efficacy following intravenous infusion. To overcome this challenge, we developed NEU-002, an optimized elastase that evades inhibitors and maintains restricted catalytic activity in blood. Here, we investigated the therapeutic properties of NEU-002 in pre-clinical models and in tumors resected from ovarian cancer (OvCa) patients.
Methods
NEU-002 activity was assayed by cleavage of a fluorogenic substrate (AAPV-AMC) or CD95 in the presence and absence of A1AT or plasma. NEU-002 selective cancer cell killing was quantified by calcein-AM using human/murine cell lines and primary cells from OvCa patients. Histone H1 translocation and ICD markers (HMGB1, CALR, ANXA1, HSP70) were assessed by flow cytometry. For in vivo studies, NEU-002 was injected intravenously (I.V.), and its effects on tumor growth, immunology, and survival were assessed in pre-clinical models.
Results
NEU-002 maintained catalytic activity following incubation with A1AT or human plasma in vitro and following I.V. injection into CT26 tumor-bearing mice. Mechanistically, NEU-002 cleaved CD95 and induced histone H1 translocation from the nucleus to the cytosol, hallmarks of the cancer-selective killing pathway. NEU-002 induced ICD in primary cancer cells from OvCa patients, while sparing tumor-adjacent healthy tissue including critical immune cells. Notably, systemically delivered NEU-002 elevated tumor, spleen, and blood CD8+ T cells, and generated complete responses with immune memory (resistant to 90-day rechallenge) in the CT26 colon cancer model. NEU-002 also effectively treated human tumor models including OvCa patient-derived CDX and breast cancer patient-derived PDX models.
Conclusions
Our data demonstrate that NEU-002 overcomes serine protease inhibitors in blood to target a cancer-selective killing pathway and induce ICD in cancer cells following systemic delivery. Its ability to immediately kill cancer cells and induce sustained anti-tumor immunity warrants further clinical testing.
References
Cui C, Chakraborty K, Tang XA, Zhou G, Schoenfelt KQ, Becker KM, Hoffman A, Chang YF, Blank A, Reardon CA, Kenny HA. Vaisar T, Lengyel E, Greene G, Becker L. Neutrophil elastase selectively kills cancer cells and attenuates tumorigenesis. Cell. 2021 Jun 10;184(12):3163ā77.
Gujar R, Cui C, Valdovinos L, Lee C, Arjmand A, Grigaitis N, Khalid A, Reardon CA, Schoenfelt KQ, Feau S, Twitty C and Becker L. N17350 is an emerging therapeutic modality that selectively kills cancer cells and stimulates anti-tumor immunity. Cancer Research 2023;83(7_Supplement):6390ā6390.
Cui C, Gujar R, Lee C, Grigaitis N, Fumagalli M, Martinez N, Arjmand A, Reardon CA, Schoenfelt KQ, Feau S, Becker L. 1344 N17350 combines selective cancer killing with adaptive immune activation to eradicate tumors. Journal for Immunotherapy of Cancer 2023;11(Suppl 1):A1āA1731.
Cui C, Gujar R, Lee C, Fumagalli M, Martinez N, Liu H, Grigaitis N, Feau S, Bahador A, Algazi AP, Becker L. 1344 N17350 combines selective cancer killing with adaptive immune activation to eradicate tumors. Cancer Research 2024;84(6_Supplement):5895ā5895.
Ethics Approval
Human studies were approved by the Institutional Review Board (IRB) with protocol ID # Pro00063741(OP-NTD-01) and Pro00071913 (OP-NTD-02) for Onchilles Pharma Inc., San Diego, CA. All the animal studies were conducted as per institutional guidelines for humane animal treatment and complied with institutional animal care and use committee (IACUC) approved protocols # EB17-010-073 at CRADL vivarium, San Diego, CA.