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Background Multiple clinical trials investigating the combination of immunotherapy (IO) with antibody drug conjugates (ADC), with or without concurrent chemotherapy, are underway and showing promising results. Mechanism-based triple combination therapies utilizing ADC, IO and a third agent offer an opportunity for additional patient benefit. We investigated the antitumor efficacy of the B7-H4 targeting topoisomerase ADC puxitatug samrotecan (Puxi-sam), the PD1/TIGIT targeting, Fc-reduced monovalent bispecific antibody rilvegostomig and the PARP1-selective inhibitor (PARP1i) saruparib in combination in experimental cancer models.Methods A 3D tumor spheroid model was generated using RL95-2 endometrial cancer cells and tumor reactive T cells primed on HLA-A24 restricted survivin/BIRC5 tumor antigen peptides or RL95-2 whole cell lysates. Syngeneic MC38 tumor cell lines were engineered to stably express human B7-H4. Tumor cells were implanted in wild-type C57BL6 mice and when tumors reached between 100-200 mm3, mice were randomized to receive either monotherapy doublet or triple combination therapy. Puxi-sam was administered as a single i.v. dose at 7 mg/kg, saruparib was administered orally daily for 21 days at 0.1 mg/kg and the murine surrogate of rilvegostomig was administered twice weekly i.p. at 10 mg/kg. A subset of mice had tumors harvested at 11 days post onset of dosing for pharmacodynamic evaluation by flow cytometry.Results In vitro treatment of endometrial spheroid-T cell co-cultures with ADC-IO, ADC-PARPi and triplet combination induced additive tumor spheroid growth inhibition. In the fully immunocompetent syngeneic mouse model, the combination of Puxi-sam and rilvegostomig induced significant tumor growth inhibition (p<0.0001) and improved survival compared to monotherapies. The addition of saruparib further enhanced the anti-tumor activity and resulted in a majority of animals having complete and durable tumor regression. A follow up pharmacodynamic study assessed a cohort of tumors collected 11 days post treatment initiation. Flow cytometry of the dissociated tumors revealed a greater than 2-fold increase in intratumoral CD3 positive T cells, including a greater than 3-fold increase in cytotoxic effector CD8 positive T cells after triplet therapy compared to untreated mouse tumors. Further, there were decreased intratumoral myeloid derived suppressor cells (MDSCs) and increased macrophages expressing the co-stimulatory molecule CD86 in the triplet therapy group compared to the untreated group.Conclusions Robust anti-tumor activity and tumor immune microenvironment modulation can be achieved with the combination of patuxitug-samrotecan, saruparib and rilvegostomig.Ethics Approval The studies described were approved by the Institutional Animal Care and Use Committee of Astrazeneca under protocol AUP22-25.