Poster S5 - In Situ Tumor Reprogramming with Poly(beta-amino ester) Nanoparticles to Treat Ovarian Cancer

Introduction: Despite recent advances in cancer immunotherapy, challenges persist in treating many solid tumors, such as ovarian cancer. Ovarian cancer is the second most common gynecological cancer and the fifth leading cause of cancer-related death among women. The prognosis of ovarian cancer is extremely poor, primarily due to a lack of symptoms and screening modalities that can detect the cancer at an earlier stage. Even when treated, ovarian cancer has a high recurrence rate that makes it difficult to cure.

Cytotoxic immune response against tumor cells relies on antigen recognition (signal 1), along with the presence of co-stimulatory (signal 2) and immuno-stimulatory molecules (signal 3). Some strategies have focused on improving signal 1, such as T cell receptor (TCR) engineering and chimeric antigen receptor (CAR) T therapy, to better recognize antigens and improve the specificity of the therapy. These strategies, however, rely on prior knowledge of the tumor antigen, which is not always known and can be difficult to obtain in an ex vivo setting. Furthermore, tumor heterogeneity, between or within patients, makes it difficult to selectively treat tumors.

Here, we propose a solution to co-deliver signals 2 and 3 to stimulate an anti-cancer immune response. We developed a poly(beta-amino ester)s (PBAEs) nanoparticle delivering signal 2 (4-1BBL) and signal 3 (IL-12) to effectively convert tumor cells into tumor-associated antigen-presenting cells (tAPCs) and elicit T-cell driven immune response. Through immunological priming of ovarian cancer cells with signals 2 and 3, our strategy provides a novel off-the-shelf therapy that is safer and efficacious.

Materials and

Methods: PBAEs are a class of synthetic, biodegradable polymers that can transfect and deliver genes of interest to tumor cells with high efficiency and selectivity. PBAEs are synthesized by combining backbone, sidechain, and end-cap monomers in a two-step reaction, making them highly tunable. Through self-assembly, PBAEs can interact with DNA plasmids to form nanoparticles. To find the optimal nanoparticle formulation, we tested multiple PBAEs at various plasmid dosages (0.6 µg/well, 0.9 µg/well, 1.2 µg/well in 96-well plates) and mass ratios (30 w/w, 60 w/w, 90 w/w) with a reporter gene for in vitro transfection efficiencies in ID8 Vegf Defb29, an engineered ovarian cancer cell line. The toxicity of the nanoparticles was assessed using the MTS assay. Next, we delivered signals 2 and 3 with our top nanoparticle formulation to select co-stimulatory molecules that show the highest expression in ID8 Vegf Defb29 using flow cytometry and ELISA. To assess whether reprogramming tumors into tAPCs effectively induce the desired immune response, we transfected ID8 Vegf Defb29 with different combinations of signals 2 and 3, then co-cultured with splenocytes. IFN-γ secretion in the culture medium was measured using ELISA as a surrogate for cytotoxic immune cell activation to assess the anti-tumor activity.

To build our in vivo mouse model, we injected ID8 Vegf Defb29 intraperitoneally (IP) into C57BL/6 mice. Our top nanoparticle formulation was injected into the ID8 Vegf Defb29 mouse model IP to assess the anti-tumor efficacy of tAPCs in vivo.

Results, Conclusions, and Discussions: From the in vitro transfection of ID8 Vegf Defb29, we chose 4-5-6 PBAE nanoparticles at 60 w/w with 900-1200 ng of plasmid DNA to be our top formulations (18-43% transfection and 69-77% viability). (Figure 1A-C) From testing different combinations of signals 2 and 3, 4-1BBL (signal 2) and IL-12 (signal 3) were expressed the highest in ID8 Vegf Defb29. This result was consistent in our tAPC co-culture experiment with splenocytes where the co-delivery of 4-1BBL and IL-12 as signals 2 and 3 respectively elicited the highest IFN-γ secretion that was statistically significant compared to the controls. (Figure 1D)

Injecting C57BL/6 mice with ID8 Vegf Defb29 showed accumulation of ascites and noticeable tumor growth by week 3. We are currently evaluating our top formulation in vivo in combination with immune checkpoint blockades in an ID8 Vegf Defb29 mouse model for tumor reduction.

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