Preclinical evaluation of the efficacy of anti-PVR antibody-drug conjugates combined with anti-PD-1 therapy for bladder cancer.

e16547 Background: Poliovirus receptor (PVR) is a ligand for TIGIT and plays a crucial role in tumor immune evasion. We have identified that PVR is highly expressed in bladder cancer (BC), particularly in cases that are resistant to immunotherapy. This study aims to investigate whether antibody-drug conjugate (ADC) targeting PVR can provide both antitumor activity and inhibition of immune escape in BC. Methods: Forty-one BC patients who received anti-PD1 neoadjuvant therapy were analyzed to identify proteins linked to immunotherapy resistance. Briefly, laser-capture microdissection was employed to isolate 3 urothelial and 3 stromal regions from each FFPE sample, enabling high-sensitivity mass spectrometry to spatially define the proteomic profiles in immunotherapy-responsive and unresponsive samples. Expression levels of PVR and common ADC targets were head-to-head compared by IHC in a cohort includes 347 BC samples. A fully humanized anti-PVR monoclonal antibody was conjugated to the toxin MMAE and the linker MC-VC-PAB to produce the potential therapeutic ADC, PVR-MMAE. The antitumor efficacy of PVR-MMAE was assessed in BC cell lines and organoids. Humanized NGS mice were engrafted with human peripheral blood mononuclear cells following by orthotopic xenografted with 5637 cells. Rats given 0.1% BBN in drinking water for 28 weeks produced bladder tumors. Animals were administered either isotype-MMAE, PVR-MMAE (2 mg/kg), or a combination of PVR-MMAE and anti-PD-1twice weekly for 4 weeks intravenously. Finally, the potential toxicity of PVR-MMAE was evaluated by dose escalation in normal rats. Results: PVR was found to be a significantly upregulated tumor-associated membrane antigen in immunotherapy-unresponsive patients. Objective response rates to immunotherapy were 19% in the PVR high group and 90% in the PVR low group. The positive expression rate of PVR in BC was 76%, comparable to HER-2 (74%), Trop-2 (69%), nectin-4 (82%) and EGFR (80%). High PVR expression correlated with advanced stage and poorer prognosis. Functional assays indicated that PVR is not only a tumor-associated antigen but also conferred strong oncogenic effects. PVR-MMAE showed significant antitumor effect in PVR-positive cells and organoids, with an IC50 of 5-10 µg/mL. Tumor growth was significantly inhibited by PVR-MMAE versus isotype-MMAE in the orthotopic xenograft model and BBN-induced BC model. Combined therapy with PVR-MMAE and anti-PD-1 showed superior efficacy over monotherapy. Additionally, intravenous administration of PVR-MMAE was safe within effective therapeutic dose ranges. Conclusions: A de novo anti-PVR monoclonal antibody conjugated with MMAE was developed. Preclinical data on bladder cancer cell lines, organoids and animal models showed the potent antitumor efficiency of anti-PVR antibody-drug conjugate as a single agent or in combination with ICIs in bladder cancer.