Combination of B7-H4-TOP1i ADC, PD-1/TIGIT bispecific antibody, and PARP inhibition orchestrates angiogenesis suppression and interferon-program activation in a syngeneic mouse model

Purpose: To define the pharmacodynamic mechanisms and synergies of a B7-H4 antibody-drug conjugate (ADC) with a topoisomerase I inhibitor (puxitatug samrotecan; AZD8205), a PD-1/TIGIT bispecific antibody (rilvegostomig; PD-1 arm engineered for murine binding), and a PARP1-selective inhibitor (saruparib), using single-cell RNA sequencing (scRNA-seq) in the MC38 hB7-H4 P1A9 murine syngeneic model. Methods: C57BL/6J mice bearing MC38 hB7-H4 P1A9 tumors received vehicle, NIP228 (7 mg/kg ADC control), AZD8205 (7 mg/kg), rilvegostomig (10 mg/kg), saruparib (0.1 mg/kg), or doublet combinations. Tumors were collected on day 11 post-treatment and analyzed through scRNA-seq. scRNA-seq data underwent quality control, log normalization, dimensionality reduction, and clustering. After filtering, 356,930 high‑quality cells across 38 samples were selected for downstream analyses (n = 4-5 per group). Gene set enrichment analysis (GSEA) using MSigDB Hallmark pathways was performed on pseudobulk differential expression analysis (DEA) profiles for each annotated cell type, comparing treatment groups. Enrichment was considered significant at FDR < 0.01 with |NES| > 1.5, and multiple testing correction was applied across all gene sets per cell type. Results: All treatment groups showed increased immune infiltration versus vehicle. Pseudobulk DEA showed the most pronounced transcriptional changes occurred with AZD8205 in tumor cells, rilvegostomig in CD8+ T cells, and saruparib in macrophages. Doublet treatments suppressed epithelial-mesenchymal transition, hypoxia, and angiogenesis hallmarks in tumor cells, with decreased Vegfa expression, particularly in the AZD8205+ rilvegostomig group. CD8+ T cells from combination groups displayed heightened interferon response programs, and macrophages exhibited concordant interferon‑response activation compared to single treatments. Further T cell sub-clustering revealed that rilvegostomig boosts effector T cells and reduces regulatory T cell representation, especially in combination with AZD8205. Conclusions: scRNA-seq results indicate that the studied drug combinations produce a transcriptomic remodeling characterized by coordinated tumor-cell program suppression and system-wide interferon response amplification in immune cells. AZD8205 doublet treatments deliver the most extensive tumor-intrinsic transcriptional changes, while rilvegostomig or saruparib each add orthogonal immune activation, yielding complementary and potentially synergistic pharmacodynamics that are not observed with individual drugs. The ongoing BLUESTAR clinical trial (NCT05123482) is exploring these combinations in patients. Our preclinical findings suggest potential mechanisms of action and support further investigation.