AZD5335-related intra-tumoral pharmacodynamic analyses.

AZD5335 Pharmacokinetic Parameters

INT-019 HTRF Specificity and Cross-Reactivity Assay

INT-019 SPR Binding Affinity Assay

AZD5335 Statistical Analyses [Figure 3]

Characterization of FRa mAb INT-019 (mAb49).

Assessment of AZD5335-associated bystander activities.

Characterization of 17 ovarian cancer PDX models.

Characterization of FRa ADC AZD5335.

AZD5335 efficacy testing within 17 ovarian cancer PDX models.

Supplementary information to support article findings.

Assessment of AZD5335/TOP1i-mediated DDR.

Abstract Folate receptor alpha (FRα) is a cell surface GPI-anchored protein overexpressed in several solid tumors with highest prevalence in ovarian cancer and lung adenocarcinoma but restricted expression in normal tissues. AZD5335, an FRα-targeting antibody conjugated to AZ’s proprietary topoisomerase 1 inhibitor (TOP1i) payload, AZ14170132, is currently being investigated in the FONTANA Phase 1/2 clinical trial (NCT05797168). Since homologous recombination repair deficiency (HRD) and mutations in genes involved with homologous recombination repair (HRRm+) are associated with sensitivity to DNA damaging agents, we evaluated the efficacy of AZD5335 in ovarian cancer patient-derived xenograft (PDX) models assessed for HRRm, as described1. Sixteen ovarian cancer PDX models were each treated with a single dose of AZD5335 (5 mg/kg IV). Of the HRRm- cohort, 8/11 (73%) cohort responded to treatment with a tumor regression >30% from baseline. In the HRRm+ cohort 4/5 (80%) responded to AZD5335. PARP1 can act as a positive regulator of genomic stability by counteracting TOP1-induced DNA damage (Malanga and Althaus, 2004) that led to the hypothesis that the efficacy of TOP1i drugs can be enhanced by combination with PARP inhibitors. To evaluate for potential synergy of AZD5335 with the PARP1 inhibitor, saruparib (AZD5305): Three FRα-expressing cancer cell lines, KB, IGROV-1 and OVCAR-3, were treated with AZD5335 +/- saruparib, revealing evidence of synergistic cytotoxicity. These results encouraged us to investigate this combination in pre-clinical HRRm+ in vivo xenograft models (OVCAR-3 and OV2022F). In OVCAR-3 xenograft models, single agent activity with saruparib (1 mg/kg PO QD x28) was 37% tumor growth inhibition (TGI) and with AZD5335 (0.625 mg/kg IV x1) was 50% TGI while combined administration at the same doses improved TGI to 86%. Similar combinatorial activity was seen in OV2022F with TGI 61%, 54%, and 97% for saruparib, AZD5335, and saruparib +AZD5335, respectively. In conclusion, AZD5335 exhibits a similar degree of single agent antitumor activity in both HRRm+ and HRRm- preclinical models, and the efficacy can be potentiated by combining with the PARP inhibitor, saruparib. 1. Clarke et al. 2022, NEJM Evid 2022;1(9) DOI: 10.1056/EVIDoa22000432. Malanga and Althaus 2004, JBC 2004; 279(7) DOI: 10.1074/jbc.C3004372003. Citation Format: Marco Gymnopoulos, Ravinder Tammali, Ana De Almeida, Jixin Wang, Claire Myers, Mark Albertella, Paula G. Fraenkel, Puja Sapra. Synergistic antitumor effect of FRα TOP1i antibody drug conjugate, AZD5335, in combination with the PARP1 inhibitor, saruparib (AZD5305), in preclinical models of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB406.

Figure 1: KTN3379 specifically binds to HER3; Figure 2: KTN3379 suppresses cell-growth in HRG-HER3 autocrine cells and HRGinduced VEGF secretion; Figure 3: KTN3379 arrests HER2+ breast cancer cells in G0/G1-phase; Figure 4: KTN3379 is inactive in HER3-positive but HRG-negative Snu16 xenograft model; Figure 5: PTEN knockdown does not impact the growth behavior of HMCB; Figure 6: Full dose response curves of KTN3379, Pertuzumab and Cetuximab in HMCB cells with or without PTEN-knockout in 6-day proliferation assay; Supplementary Table 1 Summary of Kinetic Rate Constants and Dissociation Constants for the Binding of KTN3379 to Human HER3, Cynomolgus Monkey HER3, and Murine Her3 Proteins; Supplementary Table 2 Expression and mutation status of HER3 pathway components of models used in this manuscript

Supplementary Figures 1-14; Supplementary Tables 1-3. Supplementary Figure 1. Lead antibody 80PH3 is a potent inhibitor of recombinant ADAM17 Supplementary Figure 2. 80PH3 is not a potent inhibitor of cellular ADAM17 Supplementary Figure 3. MEDI3622 is highly selective for ADAM17 and does not inhibit ADAM10 or MMP12 Supplementary Figure 4. MEDI3622 does not compete for binding to cellular ADAM17 with the ADAM17 monoclonal antibody 9301 Supplementary Figure 5. MEDI3622 inhibits cellular mouse ADAM17 Supplementary Figure 6. Cell surface levels of HER pathway proteins in OE21 esophageal cancer cells Supplementary Figure 7. The half-life of MEDI3622 in rats is approximately 8 days Supplementary Figure 8. Several phosphorylation sites in EGFR are inhibited by MEDI3622 Supplementary Figure 9. Exposure to MEDI3622 reduces levels of phospho-SRC and phospho-YES Supplementary Figure 10. Percent activated EGFR predicts cetuximab sensitivity in a series of Head and Neck PDX models Supplementary Figure 11. Proliferation of COLO205 colon cancer cells is independent of the HER pathways Supplementary Figure 12. MEDI3622 does not inhibit HER pathways in Colo205 tumors Supplementary Figure 13. Predictive value of ADAM17, EGFR, HER2, and HER3 mRNA levels for sensitivity of cell lines to MEDI3622 Supplementary Figure 14. Sensitivity to MEDI3622 is not predicted my mRNA levels of ADAM1, HER2 OR HER3 in head and neck PDX models Supplementary Table 1. Correlation of MEDI3622 and cetuximab anti-proliferative activity Supplementary Table 2. Identification of ADAM17 substrates in COLO205 cells by SILAC proteomics Supplementary Table 3. Identification of ADAM17 substrates in H358 lung cancer cells by SILAC proteomics

Table S1. P values of comparing time to endpoint survival among all treatment groups in the LNCaP xenograft study Table S2. Summary of PSMA IHC in xenograft tumors Table S3. P values of comparing time to endpoint survival among all treatment groups in the CWR22Rv1 xenograft study Table S4. P values of comparing time to endpoint survival among all treatment groups in PC-3 xenograft study

Supplemental Figure S1: Knockdown of BRCA1 or BRCA2 sensitizes Hela cells to PBD payload and PBD-based ADC in vitro. Supplemental Figure S2: Genetic deletion of BRCA1 sensitizes cells to PBD-based ADC. Supplemental Table S1: BRCA mutation(s) in patient-derived xenograft models. Supplemental Figure S3: Mean tumor volume graphs of 23 BRCA-deficient PDX tumors response to PBD-ADC treatment compared to untreated group. Supplemental Figure S4: Mean tumor volume graphs of BRCA wild-type PDX tumors response to PBD-ADC treatment compared to untreated group. Supplemental Figure S5: Representative IHC images of 5T4 staining in PDX models. Supplemental Figure S6: No in vivo efficacy was observed in tumor model DMS-114 that does not express 5T4.

Methods and figure legends for supplemental data

Supplementary Figure 1 from Aldose Reductase Regulates Growth Factor-Induced Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Human Colon Cancer Cells