Michael Kulke

CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma

Although subtypes of pancreatic ductal adenocarcinoma (PDAC) have been described, this malignancy is clinically still treated as a single disease. Here we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by using immunohistochemistry. Individuals with tumors of these subtypes showed substantial differences in overall survival, and their tumors differed in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and it is highly expressed in several additional malignancies. These findings designate CYP3A5 as a predictor of therapy response and as a tumor cell–autonomous detoxification mechanism that must be overcome to prevent drug resistance.

Antibody–Drug Conjugate Payloads

Toxin payloads, or drugs, are the crucial components of therapeutic antibody-drug conjugates (ADCs). This review will give an introduction on the requirements that make a toxic compound suitable to be used in an antitumoral ADC and will summarize the structural and mechanistic features of four drug families that yielded promising results in preclinical and clinical studies.

Abstract 664: Amanitin-based antibody-drug conjugates targeting the prostate-specific membrane antigen

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Antitumoral activity of monoclonal antibodies can be dramatically enhanced by conjugation to toxic small molecules. Beside the recent approval of Kadcyla (T-DM1) and Adcetris (SGN-35) more than 30 antibody-drug conjugates (ADC) have entered clinical trials, promising to strengthen the therapeutic capabilities for cancer treatment in the next decade. Surprisingly most ADCs are based on one of few toxic compounds only and on an even smaller number of toxicity mechanisms: Most antibodies are coupled to the microtubuli-targeting auristatins and maytansines. Toxins that operate through such a mechanism could suffer from limited activity in different cancer indications and in cells expressing resistance mechanisms. Accordingly the use of new drugs that function via alternative toxicity mechanisms could enhance the therapeutic potential of ADCs. In the present study we evaluated the antitumoral potency of a monoclonal antibody targeting the prostate-specific membrane antigen (PSMA) conjugated to small molecules from the amatoxin family. PSMA is a membrane antigen overexpressed in prostate cancer and an attractive target for an ADC approach, as it shows low expression by most normal tissues and sufficient internalization after antibody binding. Amanitin, the most well-known toxin of the amatoxin family, binds to the eukaryotic RNA pol II and thereby inhibits the cellular transcription at very low concentrations. In our experiments, we tested several random- and site-specific strategies to covalently conjugate amanitin to the antibody and generated conjugates with low aggregation and high affinity for the target antigen. Using a series of PSMA-expressing cells we compared the cytotoxic activity of stable and cleavable linker ADCs and the stability of such constructs in plasma. Overall we observed picomolar activity of ADCs after incubation for three to five days with PSMA-positive prostate cancer cells independent of the hormone-sensitivity status. Moreover we demonstrated high activity of amanitin-based anti-PSMA ADCs in prostate cancer xenograft models. The data encourage the evaluation of these agents in a clinical advanced prostate cancer study. Note: This abstract was not presented at the meeting. Citation Format: Torsten Hechler, Michael Kulke, Christoph Mueller, Andreas Pahl, Jan Anderl. Amanitin-based antibody-drug conjugates targeting the prostate-specific membrane antigen. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 664. doi:10.1158/1538-7445.AM2014-664

Abstract 77: Preclinical evaluation of HDP-101, an anti-BCMA antibody-drug conjugate

Background: ATACs (antibody-targeted Amanitin conjugates) comprise a new class of antibody-drug conjugates using amanitin as toxic payload. Amanitin binds to the eukaryotic RNA pol II and thereby inhibits the cellular transcription process at very low concentrations. In the current study, in vitro and in vivo data of new ATACs targeting BCMA (B Cell Maturation Antigen, also known as CD269) are presented. BCMA is selectively expressed on malignant plasma cells like in multiple myeloma (MM) and hence considered an ideal target for Amanitin based ADCs. Material and methods: MM cell lines: NCI-H929, MM.1S Luc (stable luciferase transfected) and CCRF-CEM (BCMA negative). Antibody: anti-BCMA Thiomab (Max Delbruck Centrum, Berlin; derivatization and production at Heidelberg Pharma). Synthesis of HDP-101: Maleimide amanitin compounds were conjugated to substituted cysteine residues of the anti-BCMA Thiomab. Cell proliferation assay: Quantitative determination of cytotoxicity was performed by CellTiter Glo 2.0 assay (Promega) or WST.1 assay (Roche). Animal models: Subcutaneous and metastatic mouse xenograft tumor models with MM cell lines NCI-H929 and MM.1S Luc were performed in single-dose and multiple-dosing experiments. Tolerability was assessed in mice and nonhuman primates (NHP). Results: HDP-101 showed in vitro cytotoxicity on BCMA+ cell lines in picomolar range, whereas no cytotoxic activity on BCMA- cells was observed. In mouse xenograft models, HDP-101 caused dose-dependent tumor regression and complete remission after a single i.v. dose of 2.0 mg/kg and 4.0 mg/kg in s.c. xenografts and after single i.v. doses from 0.1 mg/kg to 2.0 mg/kg in disseminating xenografts. Safety profiling in Cynomolgus monkeys revealed a good tolerability and therapeutic index after sequentially applied doses of 0.3, 1.0, and multiple dose application of 4 x 3.0 mg/kg. Hematology and clinical chemistry parameters were unaffected except liver enzymes and LDH: A mild to moderate and transient increase was observed. The half-life of the ADC in serum was 7-9 days; the free toxin was detectable at levels close to the lower limit of quantification only (LLOQ = 1.2nM). Conclusions: Targeted cytotoxic drug delivery to BCMA positive MM cell lines was achieved by using HDP-101, an anti-BCMA-ATAC. The mode of action of the payload Amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in NHP studies. Using ADCs in the therapy of multiple myeloma is a promising approach, especially by using a cytotoxic agent whose mode of action differs from other commonly used toxins, like ATACs. First-in-human trial is expected to start in 2018. Citation Format: Torsten Hechler, Aniko Palfi, Christoph Muller, Christian Lutz, Andreas Pahl, Michael Kulke. Preclinical evaluation of HDP-101, an anti-BCMA antibody-drug conjugate [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 77. doi:10.1158/1538-7445.AM2017-77

Abstract 2973: CD269 - A promising target for amanitin based ADCs

Antitumor activity of monoclonal antibodies can be dramatically enhanced by conjugation to toxic small molecules. Beside the recent approval of Kadcyla (T-DM1) and Adcetris (SGN-35) more than 30 antibody-drug conjugates (ADC) have entered clinical trials, promising to strengthen the therapeutic capabilities for cancer treatment in the next decade. Surprisingly most ADCs are based on one of few toxic compounds only and on an even smaller number of toxicity mechanisms: Most antibodies are coupled to the microtubuli-targeting auristatins and maytansines. Toxins that operate through such a mechanism could suffer from limited activity in different cancer indications and in cells expressing resistance mechanisms. Accordingly the use of new drugs that function via alternative toxicity mechanisms could enhance the therapeutic potential of ADCs. Heidelberg Pharma focuses on Amanitin, the most well-known toxin of the amatoxin family. Amanitin binds to the eukaryotic RNA pol II and thereby inhibits the cellular transcription at very low concentrations. In the current study, in vitro and in vivo Data of Amanitin-ADCs targeting CD269 (B cell maturation antigen) are presented. CD269 is expressed on cells of the B cell lineage, predominantly on plasma blasts and plasma cells. It is not expressed on naive B cells, germinal center B cells and memory B-cells (Darce et al. (2007) J Immunol 179:7276-7286). CD269 is highly expressed on malignant plasma cells like multiple myeloma, a B cell non Hodgkin lymphoma of the bone marrow (Novak et al. (2004) Blood 103:689-94). Since multiple myeloma is a usually incurable malignancy of plasma cells, new therapies are urgently needed. Using ADCs in the cure of multiple myeloma could be a promising approach, especially by using a toxin whose mode of action was not applied before, like amanitin based ADCs. In vitro data of anti-CD269-amanitin ADC showed cytotoxicity on CD269 positive cell lines in picomolar range, while up to micromolar concentrations, no cytotoxic activity on CD269 negative cells was observed. In mouse xenograft models, anti-CD269-amanitin showed clear anti-tumorigenic potential. A comprehensive data package will be presented. Citation Format: Aniko Palfi, Torsten Hechler, Christoph Mueller, Andreas Pahl, Michael Kulke. CD269 - A promising target for amanitin based ADCs. [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 2973.

Abstract A69: A novel mechanism mediates drug resistance in the exocrine-like pancreatic ductal adenocarcinoma (PDAC) subtype

PDAC is a highly aggressive disease with dismal prognosis [1, 2]. Despite extensive research and the discovery of several drug candidates, little progress has been reported since the approval of gemcitabine and erlotinib [1]. Moreover, recent trials with targeted therapies have shown only limited or no benefit [1, 2]. For a number of other carcinomas, tumor subclasses have been uncovered that allow the use of targeted therapies. The mutational landscape of PDAC is complex and heterogeneous, raising the question whether subclasses also exist in PDAC [3]. Collisson et al. described three PDAC subtypes that were identified based on their gene-expression profiles: The classical, the quasi-mesenchymal and the exocrine-like subtype [4]. However, not all subtypes could be identified in the previously available model systems. We have established a novel patient-derived model system that allows the analysis of these three human PDAC subtypes in vitro and in vivo. Hence, we provide a systematic workflow to propagate human PDAC in orthotopic xenografts and to derive tumor-initiating primary cell lines of all three PDAC subtypes. HNF-1 and Keratin 81 were identified as markers for subtype stratification by immunohistochemistry. Application of this two-marker set on a 258 large patient cohort confirmed a predominantly non-overlapping staining and revealed a significant difference in overall survival across the three subtypes. Furthermore, a drug screen uncovered subtype-specific drug sensitivities towards a number of drugs, including gemcitabine and dasatinib. Notably, the exocrine-like subtype was resistant towards all compounds tested. Thus, we aimed to identify the underlying cause of the observed drug resistance. Molecular analysis including gene set enrichment analysis (GSEA) allowed us to identify a putative novel mechanism of drug resistance. Analysis by qRT-PCR and Western blot demonstrated the enhanced expression of several genes mediating this mechanism particularly in the exocrine-like subtype in vitro and in vivo. These findings led to the identification of a novel protein target central to this mechanism. Additionally, retrospective immunohistochemical analysis of a large patient cohort confirmed that this target is predominantly found in those patient tumors classified as exocrine-like. Hence, we hypothesized that the observed strong activation of this mechanism in the exocrine-like PDAC subtype could be responsible for the drug resistance observed in this subclass. In line with this, functional inhibition of this mechanism resulted in increased drug sensitivity in the exocrine-like subtype. Hence, our findings may ultimately advance personalized treatment by applying novel marker-based patient selection strategies in combination with tailored drug use, a strategy which will be presented in more detail at the conference. [1] Hidalgo, M. Pancreatic cancer. The New England journal of medicine. 362, 1605-1617, doi:10.1056/NEJMra0901557 (2010). [2] Vincent, A., Herman, J., Schulick, R., Hruban, R. H. & Goggins, M. Pancreatic cancer. Lancet. 378, 607-620, doi:10.1016/S0140-6736(10)62307-0 (2011). [3] Jones, S. et al. Core signalling pathways in human pancreatic cancers revealed by global genomic analyses. Science. 321, 1801-1806, doi:10.1126/science.1164368 (2008). [4] Collisson, E. A. et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nature medicine. 17, 500-503, doi:10.1038/nm.2344 (2011). Citation Format: Elisa M. Noll, Christian Eisen, Elisa Espinet, Vanessa Vogel, Corinna Klein, Albrecht Stenzinger, Franziska Zickgraf, Peter Neuhaus, Marcus Bahra, Bruno V. Sinn, Christian Lutz, Michael Kulke, Andreas Pahl, Nathalia A. Giese, Oliver Strobel, Jens Werner, Wilko Weichert, Andreas Trumpp, Martin R. Sprick. A novel mechanism mediates drug resistance in the exocrine-like pancreatic ductal adenocarcinoma (PDAC) subtype. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A69.