ABSTRACT The overall objective of this STTR Fast-Track project is to prepare and perform a Phase I clinical trial in metastatic unresectable pancreatic ductal cancer adenocarcinoma (PDAC) patients of a new immunotherapeutic approach based on tetanus recall antigens delivered by bacteria (Listeria-Tetanus Toxoid) with Gemcitabine (GEM). Background: PDAC has a five-year survival of less than 10% in the USA, i.e. standard of care GEM and Abraxane or FOLFIRINOX (FOLinic acid, Fluorouracil, IRINothecan, OXaliplatin) only modestly improve survival. Dr. Gravekamp’s research group (Albert Einstein College of Medicine, Bronx) has developed a unique immunotherapeutic approach that utilizes a safe non-pathogenic attenuated bacterium Listeria monocytogenes to selectively infect tumors and metastases in vivo. We have shown that in mouse models Listeria delivers highly immunogenic antigens, such as tetanus toxoid [TT]), to the immunosuppressed tumor microenvironment (TME), where Listeria-TT spreads from tumor cell to tumor cell expressing TT. By contrast Listeria is quickly eliminated from normal tissue and has few side effects. Paramount to this therapy, people have been vaccinated and boosted against TT during childhood, and memory T cells to TT circulate in blood for life. Acting as a neoantigen surrogate, Listeria-TT reactivates these memory T cells regardless of age, which subsequently kill the infected tumor cells. To further improve T cell responses in the TME, we added a low dose of GEM to the therapy. Preliminary data: This Listeria-TT+GEM combination regimen turned cold into immunologically hot tumors, attracting CD4 T cells to the TME and producing high levels of perforin and granzyme B, two enzymes responsible for killing tumor cells. We have shown in transgenic KPC mice that Listeria-TT+GEM is highly effective against advanced pancreatic cancer (tumors and metastases were reduced by 80% and 87%, respectively, with survival improved by 40% compared to untreated mice (see our recent paper in Sci Transl Med, 2022). The main goal of this proposal is to bring the Listeria-TT+GEM treatment regimen to the clinic. Bringing Listeria-TT+GEM to the clinic: Patents for the Listeria-TT recall antigen concept have been granted in the US, China, Japan, and soon in Europe. All IP has been licensed by the Albert Einstein College of Medicine to Loki Therapeutics to bring the therapy to the clinic. Loki has submitted a pre-IND application to test the Listeria- TT+GEM in PDAC patients in a Phase 1 clinical trial, and a positive response has been received from the FDA. Phase I Aim 1: GMP manufacture of LM-TT+GEM for use in Phase 1 clinical trial (Months 1-12) Aim 2: Establish pathology and toxicology profiles of LM-TT in mice (Months 1-12) Aim 3: Submit an IND application and an IRB protocol (Months 1-12) Phase II Aim 4: Establish safety, toxicity and response profiles of LM-TT+GEM in PDAC patients (Months 13-36)

PROJECT NARRATIVE This project involves preparation and execution of a Fast-Track clinical trial with Listeria-tetanus toxoid (TT) and Gemcitabine (GEM) in patients with unresectable PDAC. This includes production of GMP Listeria-TT, tox and safety profiles in mice, IND and IRB approval, and testing of Listeria-TT+GEM in a Phase 1 clinical trial.