Abstract Despite advances in the treatment of B-cell non-Hodgkin lymphomas (B-NHL) and B-cell acute lymphoblastic leukemia (B-ALL), disease resistance, relapse, and treatment-related toxicities remain significant challenges. Antibody-drug conjugates (ADCs) have transformed the treatment of many cancers by enabling the selective delivery of cytotoxic payloads to malignant cells, potentially reducing systemic toxicities and improving patient outcomes. Here we describe AZD4512, a novel, first-in-class ADC targeting CD22, a surface antigen with expression restricted to the B-cell lineage and B-cell malignancies. The consistent expression of CD22 across different B-NHL and B-ALL subtypes and its rapid internalization upon antibody binding make it an ideal target for ADC therapies. AZD4512 consists of an anti-CD22 human monoclonal antibody conjugated to a topoisomerase 1 inhibitor payload via a novel cleavable linker. The primary mechanism of action for AZD4512 involves the intracellular delivery of the topoisomerase 1 inhibitor payload to CD22- expressing tumor cells via target-mediated internalization, leading to drug-release, DNA damage and apoptotic cell death. In vitro, AZD4512 exhibited potent and specific cytotoxicity against CD22-expressing tumor cells, with IC50 values in the picomolar to sub-nanomolar range. Importantly, AZD4512 did not exhibit cross-resistance in B-NHL cell lines resistant to an ADC containing the microtubule inhibitor MMAE, a payload deployed in multiple ADCs used for the treatment of B-NHL. Notably, AZD4512 demonstrated a substantially greater selectivity in primary cell settings by specifically targeting and killing CD22-positive B cells while not killing CD22-negative T cells, in contrast to a benchmark CD22-calicheamicin ADC, which showed a much narrower selective window. In vivo, AZD4512 exhibited robust antitumor activity and provided significant survival benefits across diverse models of B-NHL and B-ALL. In a study of 11 human B-NHL patient-derived xenografts (PDXs) encompassing diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and follicular lymphoma (FL), a single intravenous dose of 1.6 mg/kg achieved a 100% overall response rate, defined as at least a 30% reduction in tumor volume from baseline, with a 36% response rate observed at 0.8 mg/kg. In a study of four B-ALL PDX models, including both Ph-positive, Ph-negative, and KMT2A-rearranged subtypes, treatment with AZD4512 at both 1.0 mg/kg and 2.4 mg/kg resulted in a significant reduction of disease burden in peripheral blood in three of the four models across most assessed time points over a five-week post-treatment period. Subsequently, AZD4512 provided a substantial survival benefit in these responsive models: median survival ranged from 35 to 41 days at 1.0 mg/kg and from 45 to 73 days at 2.4 mg/kg, compared to median survival of 27 to 32 days in untreated groups. Toxicology studies showed that AZD4512 was well-tolerated, with no unexpected side effects. Collectively, these findings highlight AZD4512 as a promising therapeutic candidate for the treatment of patients with B-ALL and B-NHL.

AZD5335-related intra-tumoral pharmacodynamic analyses.

Each year an estimated 1.9 million people are diagnosed with colorectal cancer worldwide and approximately 930, 000 people die due to mCRC. The TOPO1i irinotecan is used frequently in combination chemotherapy regimens to treat mCRC that cannot be managed with surgery. Colorectal tumor cells often overexpress the cell membrane proteins epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition tyrosine kinase receptor (cMET). Overexpression of either EGFR or cMET is associated with cancer progression and poor clinical prognosis in mCRC. AZD9592 is a bispecific antibody-drug conjugate that binds EGFR and cMET and delivers a TOPO1i payload. We evaluated a set of 9 colorectal PDX models with a broad range of EGFR and cMET protein expression. Eight of these models were mCRC obtained from tumors biopsied from the liver (n=7) or lung (n=1). We evaluated these models for a response to AZD9592 (administered in a single dose at 2, 4, or 8 mg/kg), irinotecan, or irinotecan plus oxaliplatin. We also examined whether AZD9592 induced responses in PDX models with tumors that progressed after irinotecan treatment. A response was defined as ≥30% tumor volume reduction from baseline. A single dose of AZD9592 resulted in responses in 100% (9/9) of tumors treated with 8 mg/kg, 67% (6/9) of tumors treated with 4 mg/kg, and 33% (3/9) of tumors treated with 2 mg/kg. A single dose of irinotecan resulted in a response in 33% (3/9) of tumors. Irinotecan plus oxaliplatin resulted in a response in 55% (5/9) of tumors. Subsequent dosing with AZD9592 was feasible in 4 of the PDX models that did not respond to irinotecan; responses to a single dose of AZD9592 were observed in 50% (2/4) and 75% (3/4) of these models treated with 4 or 8 mg/kg, respectively. A similar experiment was conducted in 2 of the 4 models that did not respond to irinotecan plus oxaliplatin; a response was observed in 1 of these models with subsequent AZD9592 dosing at both 4 and 8 mg/kg. We also evaluated the effect of multiple doses of AZD9592 on the magnitude and duration of response. In 22% of mCRC PDX models (2/9), multiple doses of AZD9592 increased the magnitude of response at 4 mg/kg compared to a single dose. In 78% of models (7/9), multiple doses of AZD9592 4 mg/kg increased the duration of response. Dosing AZD9592 every 3 weeks improved the magnitude of response in some models and increased the time until tumor relapse. AZD9592 was effective in mCRC PDX models, even at the lowest doses tested. AZD9592 was effective in mCRC PDX models that did not respond to irinotecan-based treatment. Ongoing research is evaluating whether multiple doses of AZD9592 can induce resistance in PDX models, and potential resistance mechanisms are being assessed by molecular characterization. Mel Ehudin, Lara McGrath, Simon Christ, Italia Grenga, Fernanda Arnaldez, Frank Comer, Edward Rosfjord. Establishing preclinical activity of topoisomerase I inhibitor (TOPO1i) antibody-drug conjugate AZD9592 in patient-derived xenograft (PDX) models of metastatic colorectal cancer (mCRC) in context of irinotecan pretreatment and resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2957.

AZD5335 Pharmacokinetic Parameters

Metastatic castration-resistant prostate cancer (mCRPC) continues to pose a significant clinical challenge and is associated with poor survival rates in patients who failed previous lines of androgen receptor axis-targeted therapies and taxanes. Antibody drug conjugates (ADCs) provide a novel approach to traditional chemotherapy by targeting tumor-specific antigens to deliver cytotoxic payloads while sparing normal tissue and enhancing the therapeutic index. Herein, we describe the preclinical characterization of AZD0516, a first-in-class ADC directed against six-transmembrane epithelial antigen of the prostate-2 (STEAP2), a novel tumor associated antigen that is highly and homogenously expressed across all stages of prostate cancer. The anti-STEAP2 monoclonal antibody (mAb) is conjugated via interchain cysteines to a maleimide-reactive, β-glucuronidase-cleavable linker bearing the topoisomerase 1 inhibitor (TOP1i) payload, exatecan. The STEAP2 antibody binds specifically to the extracellular domains of human, cynomolgus monkey, mouse, and rat STEAP2, but exhibits no detectable binding to other STEAP family members. The fragment crystallizable (Fc) domain of the antibody binder portion carries three amino acid point mutations designed to reduce Fc-mediated immune effector functions. In vitro characterization of the STEAP2 mAb in prostate cancer cells demonstrated specific binding, rapid internalization, and efficient lysosomal trafficking. In vitro cytotoxicity assays with AZD0516 revealed IC50 values in the low nM range and the ADC induced both single- and double-strand DNA breaks, characteristic of the proposed primary mechanism of action (MoA) of the TOP1i payload, namely, DNA damage and apoptotic cell death. Exatecan-driven bystander cell killing from intracellular release of payload was demonstrated in co-culture systems involving STEAP2-positive and negative cell lines. Murine plasma pharmacokinetic data suggest minimal impact of linker-payload conjugation on antibody clearance, while maintaining high plasma stability. The mechanistic and pharmacodynamic effects of AZD0516 were observed in a prostate cancer cell line-derived xenograft (CDX) model via a dose-dependent increase in positive staining for γH2AX foci, indicative of DNA damage. AZD0516 monotherapy administration in prostate cancer CDX and patient-derived xenograft models led to sustained tumor responses. AZD0516 was well-tolerated in both rats and cynomolgus monkeys with no unexpected toxicities observed. Safety findings included hematological and gastrointestinal effects, consistent with the MoA and known effects of TOP1i payloads. Together, these data support exploring AZD0516 clinically for patients with mCRPC. Darlene Monlish, Vanessa Muniz-Medina, Mel Ehudin, Dewald van Dyk, Claire Myers, Rachel Lawrence, Liang Zhang, Linda Irons, Chara Stavraka, Wardha Qureshi, Ruoyan Chen, Asurayya Worrede, Nicolas Giraldo, Miljenka Vuko, Melody Handali, Ali Saleh, Balakumar Vijayakrishnan, Maximillian Lee, Jeong Min Han, Crystal Cheung, Ariel Endlich-Frazier, Amber Lee, Mark Hutchinson, Andrew Dippel, Gilad Kaplan, Keith Rickert, Shraddha Kale, Ryan Fleming, Clare Hoover, Benedicte Recolin, Jan Zaucha, Sreedevi Kesavan, John` Meekin, Aida Mariani, Christian Eisen, John Bullen, Eric Gangl, Jay Harper, Andreas Maderna, Edward Rosfjord, Frank Comer, Elaine Hurt, Neil Gibson, Puja Sapra. Preclinical characterization of AZD0516, a novel STEAP2 antibody-drug conjugate (ADC) for the treatment of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1158.

AZD9592 is a bispecific ADC designed to target both epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition tyrosine kinase receptor (cMET), delivering a topoisomerase 1 inhibitor payload to tumor cells. Despite recent advances in ADC development for CRC, such as the use of trastuzumab deruxtecan to treat HER2-positive CRC, a significant unmet need remains for novel therapeutic approaches, particularly for patients with limited targeted treatment options. We evaluated the antitumor efficacy of AZD9592 and explored biomarker-response relationships in CRC PDX models across a range of very low to high EGFR expression (optical density of membrane [OD] 6.7-162.6) and cMET expression (OD 18.5-142.6) measured by immunohistochemistry-quantitative continuous score (IHC-QCS), as well as other molecular features like oncogene drivers. Responses were defined as ≥30% tumor volume reductions from baseline. AZD9592 demonstrated dose-dependent efficacy, with responses observed in 34.5% of models at 8 mg/kg and 14.8% at 4 mg/kg. Biomarker analyses of tumors revealed a significant association between cMET expression levels by IHC-QCS and responses at 8 mg/kg. cMET levels were significantly higher in AZD9592-responsive models than in antibody-responsive and isotype control-responsive models (p<0.02 for both). An optimized cMET expression cutoff had a predictive performance with an area under the curve of 0.73. Compared to wildtype tumors, response rates were not significantly different in tumors harboring mutations in oncogenes that share signaling pathways with EGFR and cMET and where clinical activity of EGFR antibody therapies is limited, including KRAS and BRAF. Moreover, response rates were not significantly different across tumors derived from patients with or without prior irinotecan treatment, a chemotherapy that also inhibits topoisomerase I. Proteomics identified additional molecular features associated with response. AZD9592 demonstrated dose-dependent antitumor activity in CRC PDX models, including KRAS-mutant and KRAS-wildtype tumors. These responses suggest opportunities for clinical development of AZD9592 in CRC and the potential for biomarker-guided patient selection. Ying Zheng, Edward Rosfjord, Simon Christ, Stephanie Zalesak-Kravec, Italia Grenga, Fernanda Arnaldez, Frank Comer, Lara McGrath. AZD9592, an EGFR/cMET bispecific antibody-drug conjugate (ADC), demonstrates target-dependent efficacy in colorectal cancer (CRC) patient-derived xenograft (PDX) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2961.

Immune checkpoint inhibitors have shown limited success in breast cancer, the most common and deadly cancer in women worldwide. Novel immune therapies, such as CD3-engaging bispecific antibodies, have shown clinical promise in hematologic malignancies. However, developing CD3 bispecifics for solid tumors has been challenging due to the difficulty in identifying tumor-specific antigens. B7-H4 is proposed as an attractive tumor-associated antigen for breast cancer therapeutics with comprehensive coverage regardless of breast cancer molecular subtype. We designed a B7-H4-targeting CD3 bispecific molecule, PF-07260437, and demonstrated B7-H4-dependent pharmacology in vitro by directing cytotoxic T-cell killing to breast cancer cell lines. Treatment of cell line- and patient-derived xenograft in vivo models of human breast cancer with PF-07260437 induced substantial tumoricidal activity, often resulting in complete responses. Mechanistically, PF-07260437 increased T-cell number and activation, leading to efficient tumor killing. Additionally, combining PF-07260437 with standard of care (palbociclib plus fulvestrant) and a checkpoint inhibitor (anti-PD-1) showed combinatorial benefits in an immune-competent in vivo model. Clinically relevant noninvasive PET/CT imaging with a CD8-targeting tracer demonstrated PF-07260437-mediated increases in intratumoral CD8 T cells, highlighting the utility of CD8-PET technology to potentially assess biomarker changes in the clinic. Finally, the manageable toxicity profile of PF-07260437 was highlighted in an exploratory toxicology study in cynomolgus monkeys. These data support the clinical testing of PF-07260437 for treating B7-H4-expressing solid tumors, including breast cancer.

Elevated expression of folate receptor alpha (FRα) in various cancers, including epithelial ovarian cancer (EOC), highlights FRα as a potential target for the development of novel therapeutics to treat FRα-expressing tumor types. AZD5335, an FRα-targeting antibody conjugated to a topoisomerase 1 inhibitor (TOP1i) payload, is currently being investigated in the FONTANA Phase I/IIa clinical trial (NCT#05797168), either as a single agent or in combination with a PARP1-selective inhibitor (saruparib). Using an FRα-expressing EOC xenograft model (OV90), we evaluate additional AZD5335-relevant combined treatment strategies and report pre-clinical assessment of AZD5335 in combination with bevacizumab, paclitaxel and/or carboplatin. Compared to monotherapy treatment, we successfully demonstrate that combination treatments with AZD5335+bevacizumab, AZD5335+paclitaxel or AZD5335+carboplatin result in significant and durable anti-tumor responses. Using the same EOC xenograft model, we extended these studies to include AZD5335+bevacizumab plus either paclitaxel or carboplatin treatments and demonstrated that these triplet combos resulted in superior and durable anti-tumor activities. Combination data in a number of additional CDX/PDX will be presented. A series of in vitro studies will also define the ADC/SOC drug interactions and further delineate underlying mechanisms driving enhanced efficacy. These studies inform our ongoing clinical development of AZD5335 in ovarian cancer. Jason J. Zoeller, Ravinder Tammali, Ana De Almeida, Shane Cronin, Nancy Lee, Alma Andoni, Claire Myers, Elaine Joseph, Michael Lehmann, Rebecca Sargeant, Ina Bisha, Simon Christ, Iris Dino, Xhenifer Guza, Jixin Wang, Lisa Godfrey, Roger Dodd, Neki Patel, Edward Rosfjord, Marco Gymnopoulos, Tim Brier, Sabina Cosulich, Elaine Hurt, Puja Sapra. AZD5335, a folate receptor alpha-targeted ADC, enhances ovarian cancer treatment with bevacizumab, paclitaxel and/or carboplatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 815.

PURPOSE:Folate receptor α (FRα) is expressed in most ovarian cancers. However, only patients with high expression levels are eligible for Elahere, an FRα-targeted microtubule inhibitor antibody-drug conjugate (ADC). Efficacy limitations and safety concerns underscore the need to develop next-generation FRα-targeted ADC to treat tumors expressing variable levels of FRα and incorporate different payloads to reduce safety risks. Herein, we present the characterization of AZD5335, a novel FRα-targeted topoisomerase-1 inhibitor ADC. EXPERIMENTAL DESIGN:The efficacy of AZD5335 was assessed and correlated with FRα expression using cell- and patient-derived models. Focusing on models with low FRα, AZD5335 was directly compared with an Elahere analogue. Additionally, AZD5335 was evaluated in a model of acquired Elahere resistance. Combined treatments, including AZD5335 plus either standard-of-care drugs or a PARP1 inhibitor, were also explored. RESULTS:A single dose of AZD5335 (2.5 mg/kg) achieved an overall response rate of 82% in patient-derived ovarian cancer xenografts (n = 17). Antitumor responses were observed in models expressing both high and low levels of FRα. Specifically within FRα-low models, AZD5335 demonstrated superiority over an Elahere analogue. In the context of acquired Elahere resistance, AZD5335 treatments resulted in complete tumor regressions. Additionally, combining AZD5335 with standard-of-care drugs or a PARP1 inhibitor resulted in enhanced efficacy and sustained durability. Two clinical case studies that demonstrated significant AZD5335 responses in tumors exhibiting high and low FRα expression are also provided. CONCLUSIONS:AZD5335 is a promising next-generation ADC capable of targeting ovarian cancers with both high and low FRα expression. AZD5335 demonstrates efficacy in overcoming Elahere resistance and supports combined treatment strategies.

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.

Supplementary information to support article findings.

Characterization of FRa ADC AZD5335.

AZD5335 efficacy testing within 17 ovarian cancer PDX models.

AZD2284 is a novel, targeted alpha therapeutic directed against six-transmembrane epithelial antigen of prostate-2 (STEAP2), a receptor highly overexpressed in prostate cancer. AZD2284 is comprised of a humanized, STEAP2-targeting rodent cross-reactive IgG1 monoclonal antibody conjugated with a DOTA chelate and radiolabeled with the alpha-particle emitting radionuclide, actinium-225 [225Ac]. Leveraging high STEAP2 expression in prostate cancer and the high linear energy transfer of the alpha emitter, AZD2284 is presumed to deliver the radioisotope to targeted tumor cells, generating clustered DNA double strand breaks and other cellular damage that ultimately leads to cell death. Preclinical pharmacological assessments were conducted with the lutetium-177 [177Lu] labeled analog of AZD2284, FPI-2281. In vitro binding and internalization assays with FPI-2281 were performed in STEAP2-positive prostate cancer cell lines with varying levels of target expression. In vivo biodistribution and uptake were evaluated ex vivo across multiple timepoints following FPI-2281 administration to tumor-bearing cell-line derived xenograft (CDX) models. To assess the radiotherapeutic efficacy of AZD2284, a single dose was administered intravenously to prostate cancer CDX and patient-derived xenograft (PDX) models. Binding and internalization studies demonstrated FPI-2281 effectively binds to the STEAP2-positive prostate cancer cell line models (LNCaP, C4-2, and 22Rv1) with over 30% cellular retention at 24 hours. Corresponding in vivo biodistribution profiles showed sustained tumor uptake in xenograft models, with peak tumor uptakes of 60% ID/g in the 22Rv1 model and 83% ID/g in the C4-2 model at 14 days, respectively, with limited normal organ uptake observed at all timepoints. AZD2284 elicited target- and dose-dependent anti-tumor efficacy and sustained tumor regressions in CDX and PDX models with varying levels of STEAP2 expression. In preclinical models, AZD2284 effectively binds to and internalizes in prostate cancer cells, thereby delivering the radioisotope, [225Ac]. Biodistribution studies confirm sustained tumor uptake of AZD2284 with low normal organ uptake. Single-dose administration of AZD2284 leads to durable anti-tumor efficacy in xenograft models. These data suggest AZD2284 is a promising therapeutic candidate in prostate cancer and support the initiation of clinical trials. Darlene Monlish, Brigitte Thériault, Mel Ehudin, Vanessa Muniz-Medina, Dewald van Dyk, Lisa Godfrey, Dipal Patel, William Turnbull, Liang Zhang, Moditha Nawinne, Teresa Collins, Kenneth Thress, Douglas Ferguson, Rajiv Bhalla, Nicolas Giraldo, Melody Handali, Jeong Min Han, Crystal Cheung, Asurayya Worrede, Clare Hoover, Sujiet Puthenveetil, Edward Rosfjord, Frank Comer, Elaine Hurt, Darshan Dalal, Christopher Leamon, John Valliant, Puja Sapra. AZD2284: A novel, alpha-particle emitting radioconjugate targeting STEAP2 in metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4303.

Assessment of AZD5335/TOP1i-mediated DDR.