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Background: Folate Receptor alpha (FRa) is a validated cell surface cancer target that is prevalently expressed in multiple cancers with high unmet need, including ovarian cancer and other gynecological cancers, while exhibiting minimal expression in normal tissues. Due to FRa’s favorable expression profile, multiple antibody-drug conjugates (ADCs) are being explored in this setting. Here we present the preclinical characterization of a new anti-FRa ADC, ZW191. ZW191 is an antibody drug conjugate (ADC) comprised of a humanized IgG1 antibody conjugated to a novel camptothecin-based topoisomerase 1 inhibitor payload, ZD06519, via a maleimidocaproyl (MC) anchor and a glycyl glycyl phenylalanyl glycine (GGFG)-aminomethyl (AM) cleavable linker at a drug-to-antibody ratio (DAR) of 8. Materials and Methods: The novel antibody and drug-linker components of ZW191 were generated, characterized, and optimally integrated. The apparent binding affinity and cellular internalization of the ZW191 antibody, and the intracellular concentration of the released camptothecin payload, ZD06519, were determined in FRa-expressing cells. Additionally, the binding specificity of the ZW191 antibody was determined using a cell microarray technology to test for binding against over 6,000 full length proteins that are individually over-expressed in human cells. Tumor spheroid cancer cell cultures were utilized to determine the cytotoxicity of ZW191 and the ability of ZW191 to penetrate the layers of the three-dimensional (3D) spheroid. The bystander activity of ZW191 was assessed using antigen positive and negative co-culture experiments. The anti-tumor activity of ZW191 was evaluated in a panel of cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) ovarian cancer models spanning a range of FRa expression. ZW191 was evaluated in toxicology and pharmacokinetic (PK) studies performed in rodents and non-human primates (NHP). Results: The antibody component of ZW191 features a favorable binding profile with strong and exclusive binding to FRa, and drives superior tumor spheroid penetration, cellular internalization, and payload delivery compared to FRa targeted antibodies used in other ADCs. ZW191 demonstrates potent activity in FRa expressing 3D tumor spheroid cultures and effective bystander activity. In a panel of CDX and PDX models representing a range of FRa expression, ZW191 demonstrates compelling anti-tumor activity at exposures that are estimated to be readily achievable in the clinic. ZW191 was tolerated up to 200 mg/kg in a two-dose rat study and at 30 mg/kg in a two-dose NHP study, with favorable PK. The promising efficacy, tolerability, and PK supports the potential of ZW191 as a novel therapeutic agent that may help address unmet need in patients with high and low FRa-expressing cancers. Citation Format: Sam Lawn, Andrea Hernandez Rojas, Raffaele Colombo, Dayananda Siddappa, Jodi Wong, Kaylee Wu, Vincent Fung, Dunja Urosev, Luying Yang, Jamie R. Rich, Stuart D. Barnscher. ZW191, a novel FRa-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 2641.

Zanidatamab zovodotin (ZW49) is an antibody drug conjugate (ADC) comprised of a bispecific anti-HER2 IgG1 antibody (ZW25, zanidatamab) conjugated to a microtubule inhibitor auristatin payload (ZD02044) via a protease cleavable linker. Clinical studies of ZW49 in HER2-expressing advanced solid tumors are currently underway (NCT03821233). We have previously presented data illustrating the rapid internalization of ZW49 and the anti-tumor activity of ZW49 in multiple patient-derived xenograft models originating from breast carcinomas. Here we present ZW49’s ability to induce in vitro hallmarks of immunogenic cell death (ICD) including increased calreticulin exposure, increased high mobility group box-1 (HMGB1) exposure, and increased extracellular ATP secretion in a HER2 dependent manner. Additionally, we present the results from a study evaluating the anti-tumor activity of ZW49 in a panel of patient-derived xenografts of gastric cancer. HER2-positive SK-BR-3 and HER2-negative MDA-MB-468 tumor cell lines were treated with ZW49 and cell surface calreticulin was assessed by flow cytometry. ZW49 treatment of SKBR-3 cells induced a significantly higher percentage of cells staining positive for cell surface calreticulin compared to untreated cells. In contrast, ZW49 treatment of MDA-MB-468 cells resulted in no significant difference in the percentage of cells staining positive for calreticulin compared to untreated cells. ZW49 treated HER2-positive cancer cell lines SK-BR-3 and SK-OV-3 induced higher levels of extracellular ATP compared to untreated cells. In the HER2-negative cell line MDA-MB-468, treatment with ZW49 did not alter levels of extracellular ATP compared to untreated cells. ZW49 induced higher levels of HMGB1 in the HER2-positive cancer cell lines SKBR3 and N87 compared to untreated cells, whereas in the HER2-negative cell line MDA-MB-468, ZW49 induced similar levels of HMGB1 to untreated cells. Anti-tumor activity was observed in 5/7 (71%) PDX models of gastric cancer after a single i.v. dose of 6 mg/kg, including in models with moderate and weak HER2 expression. The strong anti-tumor activity of ZW-49 in vivo, together with its ability to induce ICD and potential adaptive immune responses, support ZW49 as a promising ADC for the treatment of HER2-expressing cancers warranting further investigation, including potential combination with checkpoint inhibitors. Citation Format: Stuart D. Barnscher, Andrea Hernández Rojas, Kevin J. Hamblett, Nichole Escalante. Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer [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 2633.

IL-12 is a cytokine produced by antigen-presenting cells that increases T cell proliferation, IFN gamma mediated Th1 effector functions and T and NK cell cytotoxicity. While these effects generate potent anti-tumor immunity in mouse models, the high toxicity of IL-12 in cancer patients has limited its clinical utility. Potency attenuation and intratumoral cytokine localization may improve IL-12 tolerability, but these approaches can reduce efficacy or be limited by intratumoral delivery or tumor antigen expression. To improve both tolerability and efficacy of IL-12, we engineered IL-12Fc fusions with anti-IL-12 antibodies to block IL-12 potency. Using linkers designed to be cleaved by highly active intratumoral proteases, blocking antibodies are released specifically in the tumor microenvironment, thereby increasing intratumoral IL-12 activity. Single chain IL-12 was fused to one C-termini of an Azymetric™ Fc heterodimer. To the other Fc C-termini, an anti-IL-12 scFv was fused via a protease-cleavable linker in order to block IL-12 activity. In addition to antibody blockade, modifications of IL-12Fc fusions were used to further limit IL-12Fc activity. In vitro potency was determined by CD8T cell IFN gamma release. Anti-IL-12 scFv cleavage in the presence of recombinant enzyme or human tumor material was assessed by reducing CE-SDS and LC/MS, respectively. All IL-12Fc fusions were produced with favorable biophysical characteristics. Modified IL-12Fc molecules had up to 500x reduced potency, while antibody blocked IL-12Fc molecules had up to 100,000x reduced potency compared to IL-12Fc control. In vitro cleavage of the anti-IL-12 scFv from IL-12Fc recovered potency to that of the corresponding non-masked IL-12Fc molecules. Reducing CE-SDS confirmed that anti-IL-12 scFvs were cleaved from IL-12Fc in the presence of recombinant enzyme. Cleavage of anti-IL-12 scFvs also occurred when variants were incubated in pancreatic tumor cell supernatant or human tumor tissue lysate. These results indicate that antibody blockade and re-activation by protease cleavage is a promising strategy to localize the activity of IL-12 to the tumor microenvironment while potentially limiting off-tumor toxicities. Further modifying antibody blocked IL-12 has the potential to better fine tune therapeutic index. We are pursuing tumor-specific IL-12 fusions for clinical application in tumors with high intratumoral protease activity. Citation Format: Jennifer Leah Bishop, Ryan Blackler, Gesa Volkers, Maya Poffenberger, Irene Yu, Joel Smith, Akram Khodabandehloo, Sifa Arrafi, Desmond Lau, Liz Stangle, Leisa Stenberg, Patricia Zwierzchowski, Iulia Dude, David Douda, Grant Wickman, Jeff Proctor, Gerry Rowse, Laurence Madera, Genevieve Desjardins, Nicole Afacan, Stuart Barnscher, David Mills, Thomas Spreter, Surjit Dixit. Increasing the therapeutic index of IL12 by engineering for tumor specific protease activation [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 1788.

Antibody drug conjugate (ADC) therapies such as Kadcyla® and Adcetris® have significantly improved outcomes for patients. Despite these early advances, many ADCs have failed due to tolerability and efficacy concerns; therefore, there is a need to develop ADCs with a greater therapeutic window. We have previously reported the increased tolerability of a novel N-acyl sulfonamide auristatin payload conjugated to trastuzumab via a protease cleavable linker. In non-human primates (NHPs), the HNSTD for this ADC was 18 mg/kg compared to 3 mg/kg for the MMAE conjugate control. Separately, we have also reported that a biparatopic antibody targeting a tumor associated antigen (e.g. anti-HER2 bispecific antibody ZW25) can lead to enhanced receptor clustering and improved internalization, thereby increasing the efficiency of payload delivery. Our aim is to develop a series of novel biparatopic ADCs with expanded therapeutic windows against multiple targets. Here we present the proof-of-concept in vitro and in vivo characterization of benchmark ADCs against 3 different targets with improved tolerability and equivalent efficacy. Benchmark antibodies against 3 known clinical targets were conjugated to our N-acyl sulfonamide auristatin (mAb-ADCs) or to MMAE or DM4 controls (mAb-control ADCs) via cleavable linkers and were assessed for in vitro binding affinity and cytotoxicity. The therapeutic windows of mAb-ADCs and mAb-control ADCs were compared by assessing efficacy in mouse xenograft models and tolerability and pharmacokinetics in NHPs. mAb-ADCs had similar binding affinities to recombinant targets and/or to cancer cells expressing low to high levels of target antigen compared to the mAb-control ADCs. mAb-ADCs demonstrated similar in vitro cytotoxicity compared to mAb-control ADCs and this was recapitulated in vivo with similar tumor growth inhibition in mouse xenograft models. In a NHP tolerability/PK study, mAb-ADCs for all 3 targets were tolerated at doses up to 18 mg/kg (single dose IV infusion) compared to the mAb-control ADCs that showed severe to life-threatening neutropenia at lower doses. The increase in maximum tolerated dose for the mAb-ADCs over the mAb-control ADCs, together with comparable efficacy across 3 different targets, demonstrates the broad applicability of the novel N-acyl sulfonamide auristatin payload to expand the therapeutic window. This strategy, together with ongoing efforts to identify synergistic antibody paratopes that more efficiently deliver payload, could lead to next-generation biparatopic ADCs with improved activity. Citation Format: Rupert H. Davies, Stuart D. Barnscher, Peter W. Chan, Laurence Madera, Jamie R. Rich, Marylou Vallejo, Grant R. Wickman, Kevin Yin, Vincent Fung, Kevin J. Hamblett, Patrick G. Kaminker, John S. Babcook. Towards development of next-generation biparatopic ADCs using a novel linker-toxin with expanded therapeutic window [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3912.

Therapies targeting HER2 have transformed the treatment of patients with HER2-expressing breast and gastric cancers. Unfortunately, many patients recur following HER2-targeted treatments and new therapies are needed. Multiple antibody-drug conjugate (ADC) technologies are being explored in this setting, some of which utilize the anti-HER2 antibody trastuzumab. Here we present the preclinical characterization of a new anti-HER2 biparatopic ADC, ZW49, which is generated from the conjugation of a novel N-acyl sulfonamide auristatin payload to the inter-chain disulfide bond cysteines of the bispecific anti-HER2 IgG1 antibody ZW25, via a protease cleavable linker. A series of in vitro and in vivo experiments were performed to characterize ZW49 as a potential therapeutic candidate. In cellular binding assays, it was confirmed that the payload conjugation to ZW25 did not affect the antibody's binding to HER2-expressing cells. ZW49 displayed potent in vitro cytotoxicity in multiple cancer cell lines expressing HER2 and was efficacious in multiple patient-derived xenograft (PDX) models. In mice bearing the HBCx-13b HER2 3+ PDX, two doses of ZW49 administered two weeks apart generated tumor regressions. Furthermore, preliminary results from PDX models with lower levels of HER2 expression treated with ZW49 also generated regressions. In nonhuman primates ZW49 administered intravenously every two weeks for three doses was well tolerated. Based on these findings, we are proceeding with further development of ZW49 as a therapeutic candidate in HER2-expressing cancers. Citation Format: Kevin J. Hamblett, Phil W. Hammond, Stuart D. Barnscher, Vincent K. Fung, Rupert H. Davies, Grant R. Wickman, Andrea Hernandez, Tong Ding, Adam S. Galey, Geoffrey C. Winters, Jamie R. Rich, John S. Babcook. ZW49, a HER2-targeted biparatopic antibody-drug conjugate for the treatment of HER2-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3914.

Antibody drug conjugates (ADCs) combine the specificity of monoclonal antibodies with potent antineoplastic small molecules, and promise efficacy without the systemic toxicity of chemotherapy. Despite this tremendous potential, most clinical ADCs have failed to provide sufficient therapeutic benefit before the onset of off-target dose-limiting platform toxicities. Here we report the development of proprietary protease cleavable N-acyl sulfonamide linked hemiasterlin and auristatin payloads, Zymelink, that allow the generation of an efficacious ADC platform with improved tolerability. Both Zymelink drug-linkers were conjugated via maleimides to endogenous cysteines. As Zymelink drug-linkers are more polar than maleimide valine citrulline monomethyl auristatin E drug-linker, the resulting ADCs can be produced with potential advantages in PK, efficacy and safety. The resulting ADCs exhibit potent in vitro cytotoxicity. A trastuzumab-based ADC prepared with Zymelink drug-linker and demonstrated at least equivalent efficacy compared to a trastuzumab-based ADC prepared with MMAE, promoting durable complete regressions in a patient-derived xenograft model. Zymelink hemiasterlin and auristatin ADCs were tolerated at 5-6 fold higher doses compared to a MMAE ADC in cynomolgus monkeys. The maximum tolerated dose of the MMAE ADC was just 3 mg/kg based on severe neutropenia. Zymelink hemiasterlin ADC was tolerated at 15 mg/kg with no evidence of neutropenia or elevations in transaminases. Zymelink auristatin ADC was tolerated at 18 mg/kg based on increased levels of transaminases at 24 mg/kg. Moreover, Zymelink ADCs exhibited greater serum exposure at equivalent doses. These results suggest Zymelink ADCs have a greatly expanded therapeutic window compared to MMAE conjugates. Citation Format: Stuart Barnscher, John Babcook, Jamie Rich, Geoff Winters, Graham Garnett, Andrea Hernandez, Vincent Fung, Kevin Yin, Kevin Hamblett, Rupert Davies. Zymelink drug conjugate platform: redefining the therapeutic window for ADCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 61. doi:10.1158/1538-7445.AM2017-61