Supplementary Figure 1. Schematic of high-content imaging-based internalization assay. Supplementary Figure 2. OVCAR3 xenografts were grown subcutaneously in NSG mice and treated with a single i.v. dose of 10 mg/kg control IgG1 or CDH6-targeting antibodies conjugated to SMCC-DM1. Supplementary Figure 3. Tumors of the PDX model HOVX2263 were grown subcutaneously in female nude mice randomized into groups of equal mean tumor volume and treated every two weeks with a 5 mg/kg i.v. dose of either IgG1-SPDB-DM4, or CDH6-targeting antibodies conjugated to SPDB-DM4. Supplementary Figure 4. Interaction analysis of anti-CDH6 antibody and CDH6 ECD protein. Supplementary Figure 5. Unenrolled NSG mice bearing OVCAR3 tumors from a separate efficacy study were allowed to grow to ~600 mm3 before being treated with either IgG1-SPDB-DM4 or CDH6-SPDB-DM4 at 8.5 mg/kg i.v. on day 34 post implant and re-dosed as indicated by arrows. Supplementary Figure 6. Representative CDH6 IHC images of the OVCAR3 subcutaneous xenograft grown in NSG mice (A), OVCAR3Luc intraperitoneal xenograft grown in SCID beige mice (B), HOVX2263 ovarian PDX subcutaneous xenograft grown in female nude mice (C), and HOVX4863 ovarian PDX subcutaneous xenograft grown in female nude mice (D). Supplementary Figure 7. (A) Correlation plot of response to HKT288 by best average response vs. CDH6 RNA expression. (B) Waterfall plot of percent best average response to CDH6-sulfoSPDB-DM4 treatment in PCT.

This file contains supplementary tables describing parameters for antitumor activity (T/C analyses), PK and protein crystallography.

This file describes supplementary material and methods used.

This file contains the figure legends for supplementary figures S1-S7.

AbstractDespite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell–cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody–drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models—a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030–45. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920

In an attempt to mine tumor versus normal mRNA expression datasets for novel tumor antigens, we identified the Cadherin-6 (CDH6) gene as frequently overexpressed in ovarian and renal cancers, while featuring a lineage-restricted normal tissue expression pattern. CDH6, also known as K-(kidney)-cadherin, is a member of the cadherin superfamily of calcium-dependent cell-cell adhesion molecules. We hypothesized that based on the combined observation of frequent overexpression of CDH6 in cancer and a restricted normal tissue expression, CDH6 might be an ideal tumor antigen for targeting using an antibody-drug conjugate (ADC) approach. CDH6-ADC is a fully-human anti-CDH6 IgG1, linked via sulfo-SPDB to the maytansinoid payload DM4. The antibody component of CDH6-ADC was selected from a panel of anti-CDH6 antibodies based on a multi-factorial lead selection campaign incorporating readouts of internalization propensity, in vitro cytotoxicity, as well as in vivo PK and efficacy across multiple linker-payload formats. CDH6-ADC features potent, target-dependent in vivo activity consistent with the mechanism of the anti-mitotic, tubulin-targeting sulfo-SPDB-DM4 linker-payload combination used. Specifically, treatment of CDH6-expressing ovarian cancer xenograft models with CDH6-ADC results in the time-dependent generation of intra-tumoral ADC catabolites and concomitant induction of phospho-histone H3 and cleaved caspase-3 - markers of G2/M arrest and apoptosis, respectively. CDH6-ADC induces durable tumor regressions at clinically relevant exposures in multiple human patient-derived tumor xenografts (PDX) across both ovarian and renal cancer lineages. To gain a more thorough understanding of CDH6-ADC activity in pre-clinical models of human ovarian cancer and identify potential molecular correlates for patient stratification, we profiled CDH6-ADC in a PDX clinical trial or PCT comprising 31 individual PDX models. In this unselected population, treatment with CDH6-ADC resulted in robust anti-tumor activity. Integration of PDX response data with CDH6 target expression in both the PDX models and human clinical samples indicate CDH6 expression patterns consistent with in vivo activity are found in a substantial fraction of ovarian, renal and cholangiocarcinoma patients. Together, the encouraging pre-clinical efficacy and tolerability data support the clinical evaluation of CDH6-ADC. Citation Format: Scott D. Collins, Parmita Saxena, Xiao Y. Li, Yeonju Shim, Lance Ostrom, Nicholas C. Yoder, Kalli C. Catcott, Molly A. McShea, Xiuxia Sun, Sanela Bilic, William R. Tschantz, Meghan Flaherty, Keith Mansfield, Tiancen Hu, Vladimir Capka, Markus Kurz, Ivana Liric Rajlic, Anne Serdakowski London, Duc Nguyen, Rebecca Mosher, Matthew J. Meyer, Aaron Bourret, Jamal Saeh, Scott Cameron, Emma Lees, Carl U. Bialucha. Targeting cadherin-6 (CDH6) with an antibody-drug conjugate for the treatment of ovarian and renal cancer. [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 2974.

Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.

ChemMedChemVolume 8, Issue 8 p. 1261-1265 Communication Discovery of NVP-LEQ506, a Second-Generation Inhibitor of Smoothened Dr. Stefan Peukert, Corresponding Author Dr. Stefan Peukert stefan.peukert@novartis.com Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Feng He, Dr. Feng He Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorMiao Dai, Miao Dai Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorRui Zhang, Rui Zhang Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorYingchuan Sun, Yingchuan Sun Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Karen Miller-Moslin, Dr. Karen Miller-Moslin Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorMichael McEwan, Michael McEwan Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Bharat Lagu, Dr. Bharat Lagu Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorKate Wang, Kate Wang Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Naeem Yusuff, Dr. Naeem Yusuff Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorAaron Bourret, Aaron Bourret Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorArun Ramamurthy, Arun Ramamurthy Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Wieslawa Maniara, Dr. Wieslawa Maniara Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorAdam Amaral, Adam Amaral Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorAnthony Vattay, Anthony Vattay Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorAnlai Wang, Anlai Wang Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorRibo Guo, Ribo Guo Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorJing Yuan, Jing Yuan Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorJohn Green, John Green Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Juliet Williams, Dr. Juliet Williams Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Silvia Buonamici, Dr. Silvia Buonamici Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Joseph F. Kelleher III, Dr. Joseph F. Kelleher III Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Marion Dorsch, Dr. Marion Dorsch Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this author Dr. Stefan Peukert, Corresponding Author Dr. Stefan Peukert stefan.peukert@novartis.com Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Feng He, Dr. Feng He Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorMiao Dai, Miao Dai Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorRui Zhang, Rui Zhang Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorYingchuan Sun, Yingchuan Sun Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Karen Miller-Moslin, Dr. Karen Miller-Moslin Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorMichael McEwan, Michael McEwan Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Bharat Lagu, Dr. Bharat Lagu Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorKate Wang, Kate Wang Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Naeem Yusuff, Dr. Naeem Yusuff Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorAaron Bourret, Aaron Bourret Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorArun Ramamurthy, Arun Ramamurthy Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Wieslawa Maniara, Dr. Wieslawa Maniara Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorAdam Amaral, Adam Amaral Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorAnthony Vattay, Anthony Vattay Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorAnlai Wang, Anlai Wang Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorRibo Guo, Ribo Guo Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorJing Yuan, Jing Yuan Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorJohn Green, John Green Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Juliet Williams, Dr. Juliet Williams Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Silvia Buonamici, Dr. Silvia Buonamici Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Joseph F. Kelleher III, Dr. Joseph F. Kelleher III Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this authorDr. Marion Dorsch, Dr. Marion Dorsch Novartis Institutes for Biomedical Research (NIBR), 250 Massachusetts Avenue, Cambridge, MA 02139 (USA)Search for more papers by this author First published: 02 July 2013 https://doi.org/10.1002/cmdc.201300217Citations: 67Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract First disclosure: Continued optimization provided a novel type of Smoothened (Smo) antagonist based on a pyridazine core. The compound, NVP-LEQ506, currently in phase I clinical trials, combines high intrinsic potency and good pharmacokinetic properties resulting in excellent efficacy in rodent tumor models of medulloblastoma. Activity against a Smo mutant conferring resistance observed in a previous clinical trial with a competitor compound suggests additional therapeutic potential. Citing Literature Supporting Information As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Filename Description cmdc_201300217_sm_miscellaneous_information.pdf248.4 KB miscellaneous_information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Volume8, Issue8August 2013Pages 1261-1265 RelatedInformation

Abnormal activation of the Hedgehog (Hh) signaling pathway has been linked to several types of human cancers, and the development of small-molecule inhibitors of this pathway represents a promising route toward novel anticancer therapeutics. A cell-based screen performed in our laboratories identified a new class of Hh pathway inhibitors, 1-amino-4-benzylphthalazines, that act via antagonism of the Smoothened receptor. A variety of analogues were synthesized and their structure-activity relationships determined. This optimization resulted in the discovery of high affinity Smoothened antagonists, one of which was further profiled in vivo. This compound displayed a good pharmacokinetic profile and also afforded tumor regression in a genetic mouse model of medulloblastoma.

Inhibition of receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs) has been validated by recently launched small molecules Sutent(R) and Nexavar(R), both of which display activities against several angiogenesis-related RTKs. EphB4, a receptor tyrosine kinase (RTK) involved in the processes of embryogenesis and angiogenesis, has been shown to be aberrantly up regulated in many cancer types such as breast, lung, bladder and prostate. We propose that inhibition of EphB4 in addition to other validated RTKs would enhance the anti-angiogenic effect and ultimately result in more pronounced anti-cancer efficacy. Herein we report the discovery and SAR of a novel series of imidazo[1,2-a]pyrazine diarylureas that show nano-molar potency for the EphB4 receptor, in addition to potent activity against several other RTKs. (C) 2009 Elsevier Ltd. All rights reserved.

Ortho-biphenyl carboxamides, originally prepared as inhibitors of microsomal triglyceride transfer protein (MTP) have been identified as novel inhibitors of the Hedgehog signaling pathway. Structure-activity relationship studies for this class of compounds reduced MTP inhibitory activity and led to low nanomolar Hedgehog inhibitors. Binding assays revealed that the compounds act as antagonists of Smoothened and show cross-reactivity for both the human and mouse receptor.