Blocking PD-1 or CTLA-4 inhibitory immune checkpoints has shown remarkable clinical benefit. However, limited response indicates the need for attacking additional checkpoint molecules such as T-cell immune receptor with Ig and ITIM domains (TIGIT). Previously, we have shown the ability of our INTASYL self-delivering RNAi technology to target PD-1 (PH-762) and TIGIT (PH-804) in human immune cells. Here we investigate the effect of silencing TIGIT on tumor growth and the tumor microenvironment (TME) in the CT-26 murine syngeneic tumor model.

TIGIT was targeted with self-delivering RNAi technology, termed INTASYL. INTASYL comprises an asymmetric duplex structure, a small duplex region (≤ 15 base pairs), a single-stranded phosphorothioate tail and chemical modifications to confer stability resulting in efficient cellular uptake allowing in situ delivery. A cell-based screen identified a potent INTASYL compound which was able to knock down both mRNA (≥90%) and protein (≥80%) in the Hepa1-6 TIGIT expressing murine cell line. The lead candidate, mPH-804, was tested in vivo. Briefly, CT-26 tumor cells were implanted into BALB/c mice followed by intratumoral injection of mPH-804 (0.5 mg and 2.0 mg/dose). Controls consisted of PBS (vehicle control), Non-Targeting Control RNAi (NTC; 2 mg/dose), and anti-TIGIT antibody (clone: IG9; 10 mg/kg i.p.) (n=12 per group; 5 doses). Tumor volumes and body weight were recorded throughout the study. Tumor-Infiltrating- Lymphocytes (TILs) were isolated twenty-four hours post the last dose from a satellite group (n=4), and examined for TIGIT expression, TME phenotype analysis and T cell activation by flow cytometry.

In vivo, mPH-804 treatment dose-dependently attenuated tumor growth compared to PBS and NTC control groups (50% less tumor burden on day 11). qPCR analysis on TILs revealed ≥80% TIGIT knockdown. Notably, increased number of CD8+ T cells (≥57%) were detected in mPH-804 dosed group. Furthermore, in mPH-804 treated intratumor T cells, we detected an increase of activation markers, CD25 (≥57%) and CD69 (≥42%).

These findings demonstrate the potential of TIGIT suppression in the TME with mPH-804. Importantly, we demonstrate that mPH-804 is efficiently delivered to intratumor immune cells without the special formulations otherwise required with conventional siRNA. In vivo, mPH-804 intratumoral injection targets TIGIT, overcomes the immunosuppressive microenvironment, enhances effector T cell function and inhibits tumor growth. Together, these novel findings support the hypothesis that local TIGIT silencing is a viable approach to regain T cell effector function and tumor cell kill which warrants further investigation in patients.

Citation Format: Sribalaji Lakshmikanthan, Andrej Jedinak, Melissa Maxwell, Mani Kadiyala, Dingxue Yan, James Cardia, Simon Fricker. Intratumoral delivery of mPH-804 (TIGIT targeting INTASYL compound) inhibits tumor growth and confers an inflammatory tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2239.