Michael Davies

Supplementary Figure S1. Analytical characterization of INT016-ADCs; Supplementary Figure S2. Validation of B7-H4 antibodies for use in IHC; Supplementary Figure S3. Relationship between B7-H4 expression level and heterogeneity in the studied human triple-negative cohort; Supplementary Figure S4. Binding of antibody INT016 to HEK 293 cells stably expressing human, mouse, or cynomolgus monkey B7-H4; Supplementary Figure S5. Binding of antibody INT016 to human breast cancer cell lines and to HT29 cells stably expressing human B7-H4; Supplementary Figure S6. Internalization and lysosomal trafficking of INT016; Supplementary Figure S7. In vitro stability of INT016-ADCs in mouse and cynomolgus monkey serum; Supplementary Figure S8. In vitro cytotoxicity of INT016-ADCs in HT29 and HT29-huB7-H4 isogenic cell lines; Supplementary Figure S9. In vivo efficacy of INT016-ADCs in the MX-1 xenograft model; Supplementary Figure S10. Growth rate analysis of MX-1 in vivo efficacy study comparing INT016-ADCs from days 14–40.

Surface plasmon resonance; In vitro cytotoxicity assays; In vitro proliferation assays; Internalization assays; Subcellular localization microscopy; Immunoblotting; Statistical analysis

Supplementary Table S1. Primary antibodies used for immunoblotting; Supplementary Table S2. Characterization of ADCs evaluated in this study; Supplementary Table S3. B7-H4 expression in human normal tissue; Supplementary Table S4. Binding affinity of anti-B7-H4 antibody INT016 Fab to human, cynomolgus monkey, and mouse B7-H4, measured by surface plasmon resonance.

Supplementary Figure S11. In vitro cytotoxicity of the TOP1i warhead AZ’0132 to HT29 and HT29-huB7-H4 cells; Supplementary Figure S12. Study design for pharmacodynamic and pharmacokinetic analysis of AZD8205 in HT29 and HT29-huB7-H4 mouse xenograft models; Supplementary Figure S13. DNA damage response signaling in HT29-huB7-H4 and MX-1 cells treated with AZD8205 or the TOP1i warhead AZ’0132; Supplementary Figure S14. Accumulation of AZD8205 and changes in epithelial cell density in HT29 and HT29-huB7-H4 tumor xenografts over time; Supplementary Figure S15. Correlation of B7-H4 mean OD and gene expression in TNBC PDX models; Supplementary Figure S16. Impact of homologous recombination deficiency (HRD) on sensitivity to the TOP1i warhead AZ’0132 in vitro with SK-OV-3 ovarian cancer isogenic cells; Supplementary Figure S17. SLFN11 expression in PDX models; Supplementary Figure S18. Analysis of SLFN11 expression and AZD8205 response in PDX.

Purpose:We evaluated the activity of AZD8205, a B7-H4–directed antibody–drug conjugate (ADC) bearing a novel topoisomerase I inhibitor (TOP1i) payload, alone and in combination with the PARP1-selective inhibitor AZD5305, in preclinical models.Experimental Design:IHC and deep-learning–based image analysis algorithms were used to assess prevalence and intratumoral heterogeneity of B7-H4 expression in human tumors. Several TOP1i-ADCs, prepared with Val-Ala or Gly–Gly–Phe–Gly peptide linkers, with or without a PEG8 spacer, were compared in biophysical, in vivo efficacy, and rat toxicology studies. AZD8205 mechanism of action and efficacy studies were conducted in human cancer cell line and patient-derived xenograft (PDX) models.Results:Evaluation of IHC-staining density on a per-cell basis revealed a range of heterogeneous B7-H4 expression across patient tumors. This informed selection of bystander-capable Val-Ala–PEG8–TOP1i payload AZ14170133 and development of AZD8205, which demonstrated improved stability, efficacy, and safety compared with other linker–payload ADCs. In a study of 26 PDX tumors, single administration of 3.5 mg/kg AZD8205 provided a 69% overall response rate, according to modified RECIST criteria, which correlated with homologous recombination repair (HRR) deficiency (HRD) and elevated levels of B7-H4 in HRR-proficient models. Addition of AZD5305 sensitized very low B7-H4–expressing tumors to AZD8205 treatment, independent of HRD status and in models representing clinically relevant mechanisms of PARPi resistance.Conclusions:These data provide evidence for the potential utility of AZD8205 for treatment of B7-H4–expressing tumors and support the rationale for an ongoing phase 1 clinical study (NCT05123482).See related commentary by Pommier and Thomas, p. 991

Supplementary Data from AZD4625 is a Potent and Selective Inhibitor of KRASG12C

Supplementary Data from AZD4625 is a Potent and Selective Inhibitor of KRASG12C

Supplementary Figure from AZD4625 is a Potent and Selective Inhibitor of KRASG12C

Supplementary Figure from AZD4625 is a Potent and Selective Inhibitor of KRASG12C

Supplementary Figure S11. In vitro cytotoxicity of the TOP1i warhead AZ’0132 to HT29 and HT29-huB7-H4 cells; Supplementary Figure S12. Study design for pharmacodynamic and pharmacokinetic analysis of AZD8205 in HT29 and HT29-huB7-H4 mouse xenograft models; Supplementary Figure S13. DNA damage response signaling in HT29-huB7-H4 and MX-1 cells treated with AZD8205 or the TOP1i warhead AZ’0132; Supplementary Figure S14. Accumulation of AZD8205 and changes in epithelial cell density in HT29 and HT29-huB7-H4 tumor xenografts over time; Supplementary Figure S15. Correlation of B7-H4 mean OD and gene expression in TNBC PDX models; Supplementary Figure S16. Impact of homologous recombination deficiency (HRD) on sensitivity to the TOP1i warhead AZ’0132 in vitro with SK-OV-3 ovarian cancer isogenic cells; Supplementary Figure S17. SLFN11 expression in PDX models; Supplementary Figure S18. Analysis of SLFN11 expression and AZD8205 response in PDX.