Abstract 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. Citation Format: Octavio Morante-Palacios, Jon Chesebrough, Yoshimi Johnson, Raymond Rothstein, Varsha Shankarappa, Joseph Boland, Elizabeth Galery, Anna Dominika Staniszewska, Mark R. Albertella, Alex Cazes, Paul Chariou, Bilal Omar. 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 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7172.

MET expression in pancreatic cancer

Supplemental data 13: Summary of Gene Set Enrichment analysis results obtained on expression profiling data of CLB-­â€GA on different time points (10' - 30'- 60' - 2h - 4h - 6h) after pharmacological inhibition using TAE-­â€684.

PDF file - 144K, Insert size distributions of fragment lengths after capture and paired-end sequencing for the 10 samples studied

PDF file - 163K, Information on rearrangements detected by capture and paired-end sequencing and validated by PCR

Post-treatment tumor volumes of PDX1342 grafted tumors

PDF file - 110K, FISH analysis on the CLB-Re cell line using the LSI ALK dual color, Break Apart Rearrangement probe (Vysis)

Dose dependent anti-proliferative effect of TR1801-ADC

PDF file - 110K, 2p amplicons in the CLB-Bar cell line

PDF file - 115K, FISH analysis on the N206/Kelly cell line with various BAC probes

Supplemental data 10: Results of gene ontology analysis performed on the 77 ALK signature genes

Post-treatment tumor volumes of PDX1334 grafted tumors

PDF file - 151K, Regions targeted for the capture/paired-end sequencing approach

PDF file - 138K, Supplementary Methods for SNP 6.0 arrays, NimbleGen arrays, analysis of reads obtained using capture and paired-end sequencing, mass spectrometry analysis and inverse PCR

B7-H4 is a cell-surface protein overexpressed in several tumor types and is most prevalent in triple-negative breast (TNBC) cancer (74%), ovarian carcinoma (77%), endometrial carcinoma (94%), and cholangiocarcinoma (89%). In contrast, it has a limited expression in normal tissue, making it an attractive target for an antibody-drug conjugate (ADC). Here we report and characterize the preclinical efficacy of AZD8205, a novel B7-H4 targeted ADC carrying a topoisomerase 1 inhibitor (TOP1i) linker-warhead, AZ’0133 at a drug:antibody ratio (DAR) of 8. The primary mechanism of action of AZD8205 is intracellular delivery of the TOP1i warhead to B7-H4 positive cells, leading to DNA damage and apoptotic cell death. A single IV administration of AZD8205 at 3.5 mg/kg provided robust antitumor activity in patient-derived xenografts from TNBC, ovarian and cholangiocarcinoma tumors, with overall response rates in B7H4-expressing samples of 75%, 64% and 21% respectively. Tumor response was found to be correlated to cell surface target expression and homologous recombination repair deficiency status. To further exploit the DNA damage elicited by the specific delivery of the TOP1i warhead, the combination of AZD8205 with a novel poly-ADP ribose polymerase 1 (PARP1) selective inhibitor, AZD5305, was investigated. Combination of AZD8205 and AZD5305 provided higher antitumor activity than monotherapy, even in PARP inhibition-resistant or low B7-H4 expressing PDX tumors. Finally, cancer cell lines were found to express markers of immunogenic cell death and antigenicity post-AZD8205 treatment in vitro. Therapeutic benefit of combining AZD8205 with checkpoint immunotherapies was then evaluated in syngeneic models, demonstrating enhanced antitumor efficacy of AZD8205 when combined with an anti-PD-L1 antibody. These data demonstrate that AZD8205 is a promising therapeutic candidate for the treatment of B7-H4 positive solid tumors. A first in human phase I/IIa study in patients with advanced solid tumors is currently ongoing (NCT05123482). Citation Format: Alex Cazes, Krista Kinneer, Bilal Omar, Jon Chesebrough, Judith Anderton, Jixin Wang, Anna D. Staniszewska, Steven Arbitman, Kalyani Daita, Himanshi Desai, Yoshimi Johnson, Raymond Rothstein, Marco Gymnopoulos, Giulia Fabbri, Zachary A. Cooper, Mark Albertella, Scott A. Hammond, Sabina Cosulich, Nadia Luheshi, Puja Sapra. Preclinical evaluation of a novel B7-H4 targeted antibody-drug conjugate AZD8205 as a single agent and in combination with novel PARP inhibitor and checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2947.

PDF file - 207K, Primers used to prepare 5-kb PCR probes and BAC used for FISH analysis

Post-treatment tumor volumes of PDX1342 grafted tumors treated with TR1801-ADC and gemcitabine

Supplemental data 12: Results of Connectivity map analysis performed on ALK signature genes.

The MST1R/RON receptor tyrosine kinase is a homologue of the more well-known MET receptor. Like MET, RON orchestrates cell signaling pathways that promote oncogenesis and enable cancer cell survival; however, it has a more unique role in the regulation of inflammation. RON was originally described as a transmembrane receptor expressed on tissue resident macrophages and various epithelial cells. RON is overexpressed in a variety of cancers and its activation modifies multiple signaling pathways with resultant changes in epithelial and immune cells which together modulate oncogenic phenotypes. While several RON isoforms have been identified with differences in structure, activation, and pathway regulation, increased RON expression and/or activation is consistently associated with worse outcomes. Tyrosine kinase inhibitors targeting RON have been developed, making RON an actionable therapeutic target.