Background: NaPi2b is a multi-pass transmembrane sodium-dependent phosphate transport protein encoded by the gene SLC34A2. NaPi2b is involved in normal phosphate homeostasis and its expression is found in the lung, liver, and small intestine. NaPi2b is highly expressed in ovarian carcinomas, lung adenocarcinomas, and colorectal carcinomas. ZW220 is an antibody-drug conjugate (ADC) targeting human NaPi2b, wherein a humanized IgG1 antibody is conjugated to novel camptothecin-based topoisomerase I inhibitor, ZD06519. The drug linker in ZW220 is comprised of a maleimide anchor and a glycyl glycyl phenylalanyl glycine (GGFG)-aminomethyl (AM) cleavable sequence. Materials and Methods: A series of in vitro and in vivo studies were conducted to interrogate the mechanism of action and the therapeutic potential of ZW220. The binding, internalization, potency, and bystander effect of ZW220 were evaluated in vitro using endogenous NaPi2b-expressing ovarian and lung cancer cell lines. In vivo, the anti-tumor activity of ZW220 was evaluated in a panel of cell line derived xenograft (CDX) models and ovarian patient derived xenograft (PDX) models featuring a range of NaPi2b expression. The pharmacokinetic (PK) profile of ZW220 was determined in Tg32 mice, a transgenic mouse expressing human neonatal Fc receptor (hFcRn). Results: ZW220 antibody exhibited cross-reactivity to human and cynomolgus monkey NaPi2b, nanomolar binding affinity and rapid internalization in NaPi2b-expressing cell lines in vitro. ZW220 elicited target-specific, sub-nanomolar cytotoxicity in two-dimensional monolayer and three-dimensional tumor spheroid models and demonstrated bystander-mediated cell killing in cancer cell co-culture assays. The treatment of a panel of ovarian PDXs with a single dose of 6 mg/kg of ZW220 resulted in robust tumor growth inhibition. ZW220 demonstrated a favourable PK profile in Tg32 mice, with comparable half-life to its parental unconjugated antibody. These results support the potential of ZW220 as a novel therapeutic agent which may help address unmet medical need in patients with NaPi2b-expressing tumors. Citation Format: Andrea Hernandez Rojas, Jodi Wong, Dunja Urosev, Sam Lawn, Kaylee Wu, Saki Konomura, Manuel Lasalle, Diego A. Alonzo, Luying Yang, Mark Petersen, Lemlem T. Degefie, Araba P. Sagoe-Wagner, Sohyeong Kang, Chi Wing Cheng, Raffaele Colombo, Daya Siddappa, Stuart D. Barnscher, Jamie R. Rich. ZW220, a novel NaPi2b-targeting antibody drug conjugate bearing a topoisomerase 1 inhibitor payload [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 1533.

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.

Background: Glypican-3 (GPC3) is a cell-surface oncofetal glycoprotein frequently expressed in hepatocellular carcinoma (HCC) with minimal presence in normal adult tissues. ZW251 is an antibody-drug conjugate (ADC) targeting human GPC3, composed of a humanized IgG1 antibody conjugated to a novel camptothecin-based topoisomerase 1 inhibitor, ZD06519, via a maleimide anchor and a glycyl glycyl phenylalanyl glycine (GGFG)-aminomethyl (AM) cleavable linker. Materials and Methods: Extensive functional characterization was performed to assess the mechanism of action and therapeutic potential of the ZW251 ADC. Antibody binding to human and cynomolgus monkey GPC3 was assessed by surface plasmon resonance and flow cytometry. A screen of off-target binding and target specificity was conducted using a membrane proteome array. ZW251 antibody internalization in GPC3-expressing tumor cell lines was assessed by flow cytometry. In vitro ADC cytotoxicity against tumor monolayers and spheroids was assessed in a panel of HCC cell lines. Tumor cell co-culture assays were also performed to assess bystander-mediated cell killing by ZW251. The pharmacokinetic (PK) profile of the ZW251 antibody was assessed in Tg32 mice expressing human FcRn. Anti-tumor activity of ZW251 was investigated in a large panel of cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models representing a range of GPC3 expression. Results: The ZW251 antibody backbone demonstrated nanomolar binding affinity to both human and cynomolgus monkey GPC3, and strong binding to target-expressing cancer cell lines. Rapid internalization of ZW251 antibody was observed in GPC3-expressing HCC cell lines. ZW251 exhibited potent and target-specific cytotoxicity in a panel of HCC cells cultured either in monolayer or as 3D spheroids. ZW251 showed effective bystander-mediated killing of GPC3 negative cancer cells when in co-culture with GPC3 positive cancer cells. The ZW251 antibody demonstrated a favorable PK profile in Tg32 mice. A single administration of ZW251 resulted in robust tumor growth inhibition of a large panel of CDX and PDX models representing a range of GPC3-expression. Overall, these results support the potential of ZW251 as a novel therapeutic agent against GPC3-bearing cancers. Citation Format: Laurence Madera, Andrea Hernández Rojas, Raffaele Colombo, Alex Wu, Chayne L. Piscitelli, Dunja Urosev, Allysha Bissessur, Chi Wing Cheng, Renee Duan, Catrina Kim, Kevin Yin, Vincent Fung, Kaylee Wu, Winnie Cheung, Diego A. Alonzo, Mark E. Petersen, Stuart D. Barnscher, Jamie R. Rich. ZW251, a novel glypican-3-targeting antibody drug conjugate bearing a topoisomerase 1 inhibitor payload [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 2658.

Abstract HER2-directed therapies have improved clinical outcomes for many patients with HER2-positive breast and gastric cancer. Despite these successes, there remains a need to develop improved HER2-targeted therapies for these and other HER2-expressing tumors, particularly in the setting of recurrent or metastatic disease. Zanidatamab (ZW25) is a humanized, bispecific, immunoglobulin (Ig) G1-like antibody directed against the juxtamembrane extracellular domain (ECD4) and the dimerization domain (ECD2) of human epidermal growth factor receptor 2 (HER2), the same domains targeted by trastuzumab (T) and pertuzumab (P), respectively. Data from the ongoing phase 1 study (NCT02892123) demonstrate that zanidatamab is well tolerated and has single agent activity in patients with advanced HER2-expressing cancers that have progressed after standard of care (SOC) therapies, including HER2-targeted agents such as T, P, and trastuzumab emtansine.1,2 We have previously shown that the unique design and bispecific binding of zanidatamab results in multiple mechanisms of action including increased antibody binding density, potent effector function, improved receptor internalization and HER2 downregulation relative to T.3 To better understand the mechanism by which zanidatamab differentiates itself from T, P and T+P, we recently expanded our mechanistic evaluations including cell surface HER2 aggregation, complement-dependant cytotoxicity (CDC) and inhibition of both tumor cell growth and intracellular signaling. Single molecule-sensitive direct stochastic optical reconstruction microscopy (dSTORM) was used to map HER2 receptor distribution and quantitate the size, density, and frequency of receptor clusters induced by antibody binding. In vitro assessments were performed in a panel of HER2-expressing cell lines using standard assays including CDC with human complement serum and inhibition of both tumor cell growth and intracellular signaling. Using dSTORM, we observed that zanidatamab binding resulted in enhanced HER2 aggregation and distinct HER2 capping on the tumor cell surface compared to T, P or T+P. Evaluation of CDC activity in HER2-overexpressing tumor cells demonstrated that zanidatamab, but not T, P or T+P, elicited CDC suggesting that the enhanced HER2 aggregation and capping on the tumor cell surface provides high avidity docking sites to which C1 binds and is activated. Zanidatamab showed further differentiation in the inhibition of both tumor growth and intracellular signaling of HER2-overexpressing cells compared to T, P and T+P. Zanidatamab has novel cell surface binding and additional mechanisms of action compared to T, P and T+P. Zanidatamab is actively being evaluated in clinical trials in multiple HER2-expressing solid tumors, including a registration-enabling clinical trial in HER2 gene amplified biliary tract cancer (NCT04466891). Citation Format: Nina E. Weisser, Grant Wickman, Libin Abraham, Jason O'Toole, Bryant Harbourne, Joy Guedia, Chi Wing Cheng, Peter Chan, Duncan Browman, Michael R. Gold, Neil Josephson, Surjit Dixit, Gerry Rowse. The bispecific antibody zanidatamab's (ZW25's) unique mechanisms of action and durable anti-tumor activity in HER2-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1005.

The androgen receptor (AR) is a member of the nuclear receptor superfamily of transcription factors and is central to prostate cancer (PCa) progression. Ligand-activated AR engages androgen response elements (AREs) at androgen-responsive genes to drive the expression of gene batteries involved in cell proliferation and cell fate. Understanding the transcriptional targets of the AR has become critical in apprehending the mechanisms driving treatment-resistant stages of PCa. Although AR transcription regulation has been extensively studied, the signaling networks downstream of AR are incompletely described. Semaphorin 3C (SEMA3C) is a secreted signaling protein with roles in nervous system and cardiac development but can also drive cellular growth and invasive characteristics in multiple cancers including PCa. Despite numerous findings that implicate SEMA3C in cancer progression, regulatory mechanisms governing its expression remain largely unknown. Here we identify and characterize an androgen response element within the SEMA3C locus. Using the AR-positive LNCaP PCa cell line, we show that SEMA3C expression is driven by AR through this element and that AR-mediated expression of SEMA3C is dependent on the transcription factor GATA2. SEMA3C has been shown to promote cellular growth in certain cell types so implicit to our findings is the discovery of direct regulation of a growth factor by AR. We also show that FOXA1 is a negative regulator of SEMA3C. These findings identify SEMA3C as a novel target of AR, GATA2, and FOXA1 and expand our understanding of semaphorin signaling and cancer biology.

Intratumoral androgen biosynthesis contributes to castration-resistant prostate cancer progression in patients treated with androgen deprivation therapy. The molecular mechanisms by which castration-resistant prostate cancer acquires the capacity for androgen biosynthesis to bypass androgen deprivation therapy are not entirely known. Here, we show that semaphorin 3C, a secreted signaling protein that is highly expressed in castration-resistant prostate cancer, can promote steroidogenesis by altering the expression profile of key steroidogenic enzymes. Semaphorin 3C not only upregulates enzymes required for androgen synthesis from dehydroepiandrosterone or de novo from cholesterol but also simultaneously downregulates enzymes involved in the androgen inactivation pathway. These changes in gene expression correlate with increased production of androgens induced by semaphorin 3C in prostate cancer model cells. Moreover, semaphorin 3C upregulates androgen synthesis in LNCaP cell-derived xenograft tumors, likely contributing to the enhanced in vivo tumor growth rate post castration. Furthermore, semaphorin 3C activates sterol regulatory element-binding protein, a transcription factor that upregulates enzymes involved in the synthesis of cholesterol, a sole precursor for de novo steroidogenesis. The ability of semaphorin 3C to promote intratumoral androgen synthesis may be a key mechanism contributing to the reactivation of the androgen receptor pathway in castration-resistant prostate cancer, conferring continued growth under androgen deprivation therapy. These findings identify semaphorin 3C as a potential therapeutic target for suppressing intratumoral steroidogenesis.

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related deaths in North America. The androgen receptor (AR) is a member of the nuclear receptor superfamily of transcription factors and is heavily implicated in PCa progression. First-line therapies frequently target the AR axis and are initially met with favourable response but restored and aberrant AR signaling fuel disease progression to stages for which treatment is palliative. Transcriptional targets of the AR include genes involved in cell growth and cell fate but are not completely described. The semaphorin family of signaling proteins is a large grouping of cell-surface or secreted signaling proteins that function in neurogenesis and development. The roles of semaphorins in cancer are becoming increasingly evident however mechanistic details surrounding their involvement in cancer are poorly defined. One member of the class 3 semaphorins, SEMA3C, has been shown to confer invasive phenotypes in prostate cancer cells. Using the AR-positive LNCaP cell line we show that SEMA3C is an androgen-responsive gene. Additionally, using RSAT DNA analysis software we identify an androgen response element (ARE) in intron 2 of SEMA3C and show that AR is recruited to this genomic region in an androgen-dependent manner in chromatin immunoprecipitation assays. Furthermore, the isolated ARE binds to the AR-DNA binding domain in gel shift assays and can be transactivated by AR in reporter gene assays. Finally we show that the pioneering factor, GATA2, is necessary for AR-mediated expression of SEMA3C. Collectively, our work identifies SEMA3C as a direct transcriptional target of AR in support of our hypothesis that dysregulated AR signaling drives inadvertent upregulation of SEMA3C and PCa disease progression. Deregulated AR signaling underpins prostate cancer progression and underscores the need to elucidate transcriptional targets of AR. Identification of SEMA3C as a novel target of AR provides the rationale for targeted therapies directed against SEMA3C. Citation Format: Kevin J. Tam, Kush Dalal, Michael Hsing, Chi Wing Cheng, Yan Ting Chiang, Aishwariya Sharma, James W. Peacock, Artem Cherkasov, Yuzhuo Wang, Martin E. Gleave, Paul S. Rennie, Christopher J. Ong. Semaphorin 3C is an androgen receptor-regulated gene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1834.