Background Despite clinical successes of immune checkpoint blockade (ICB) antibodies blocking the inhibitory receptors CTLA-4, PD-1, or PD-L1, substantial challenges remain. Many patients do not respond, and ICB treatment is associated with serious immune-related adverse effects (irAEs) which are exacerbated by combination therapies. TIGIT blockade has been demonstrated to provide tumor control in pre-clinical studies, sparking ongoing clinical trials, including those targeting TIGIT in combination with anti-PD-1 or anti-PD-L1. The INTASYL™ platform is a self-delivering RNAi technology that (1) provides efficient delivery into target cells bypassing the need for specialized formulations, mechanical perturbation, or drug delivery systems; and (2) specifically and durably silence target gene expression when administered intratumorally (IT), providing in vivo tumor control. IT administration restricts pharmacokinetics to the tumor; an attractive strategy for mitigating ICB-mediated systemic irAEs. Additionally, using INTASYL, multiple targets can be silenced in combination. Here we demonstrate the in vivo efficacy of INTASYL specifically targeting TIGIT (PH-804), PD-1 (PH-762), PD-L1 (PH-790) alone or in combination in a CT26 model of murine colorectal carcinoma.

Methods To assess silencing activity, activated human pan-T cells were incubated in vitro with INTASYL compounds either alone or in combination and mRNA silencing was determined by qRT-PCR and protein silencing by flow cytometry. To assess in vivo tumor efficacy CT-26 cells were implanted subcutaneously into BALB/c mice. INTASYL compounds were administered IT at 1 mg/dose on Days 1, 3, 7, and 10 either as single agents (mPH-804, mPH-762, mPH-790) or in combination (mPH-804 + mPH-762 or mPH-804 + mPH-790). Controls consisted of PBS (vehicle; (IT)), and anti-TIGIT, anti-PD-1, or anti-PD-L1 antibodies (0.2 mg/dose) administered via intraperitoneal injection (IP). Tumor volumes and body weight were recorded throughout the study. Tumors were taken at the end of the study for analysis.

Results Single and combination knockdown of target molecules was validated at the mRNA level (=90%) by qRT-PCR and at the protein level (=80%) in activated human pan-T cells. In vivo, combination treatment with mPH-804 + mPH-762 or mPH-790 improved tumor control compared to individual monotherapies providing evidence of potential synergy. All treatments were well tolerated.

Conclusions n/a

Acknowledgements We demonstrate the potential of INTASYL-mediated combination therapy targeting TIGIT and PD-1/PD-L1. These findings indicate that combination of TIGIT + PD-1/PD-L1 silencing improves tumor control compared to monotherapy. As INTASYL IT is efficacious and may mitigate irAEs caused by antibody ICB, INTASYL combination therapies including PH-804, PH-762 and PH-790 warrant further investigation in patients.