α‐Amanitin and related amatoxins have been studied for more than six decades mostly by isolation from death cap mushrooms. The total synthesis, however, remained challenging due to unique structural features. α‐Amanitin is a potent inhibitor of RNA polymerase II. Interrupting the basic transcription processes of eukaryotes leads to apoptosis of the cell. This unique mechanism makes the toxin an ideal payload for antibody–drug conjugates (ADCs). Only microgram quantities of toxins, when delivered selectively to tumor sites through conjugation to antibodies, are sufficient to eliminate malignant tumor cells of almost every origin. By solving the stereoselective access to dihydroxyisoleucine, a photochemical synthesis of the tryptathion precursor, solid‐phase peptide synthesis, and macrolactamization we obtained a scalable synthetic route towards synthetic α‐amanitin. This makes α‐amanitin and derivatives now accessible for the development of new ADCs.

BackgroundMany studies of the eukaryotic transcription mechanism and its regulation rely on in vitro assays. Conventional RNA polymerase II transcription assays are based on radioactive labelling of the newly synthesized RNA. Due to the inefficient in vitro transcription, the detection of the RNA involving purification and gel electrophoresis is laborious and not always quantitative.ResultsHerein, we describe a new, non-radioactive, robust and reproducible eukaryotic in vitro transcription assay that has been established in our laboratory. Upon transcription, the newly synthesized RNA is directly detected and quantified using the QuantiGene assay. Alternatively, the RNA can be purified and a primer extension followed by PCR detection or qPCR quantification can be performed. When applied to assess the activity of RNA polymerase II inhibitors, this new method allowed an accurate estimation of their relative potency.ConclusionsOur novel assay provides a non-radioactive alternative to a standard in vitro transcription assay that allows for sensitive detection and precise quantification of the newly transcribed, unlabelled RNA and is particularly useful for quantification of strong transcriptional inhibitors like α-amanitin. Moreover, the method can be easily adapted to quantify the reaction yield and the transcription efficiency of other eukaryotic in vitro systems, thus providing a complementary tool for the field of transcriptional research.

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. We accomplished the chemical synthesis of amanitin and were able to synthesize amanitin variants in order to optimize the toxin structure for different tumors and antibodies. We will present in vitro and in vivo data of eight different linker-amanitin constructs attached to three different antibodies targeting solid tumors. Material and methods: Cell lines: JIMT-1, SKBR-3, BT474 and NCI-N87 (used for anti-Her-2-ATACs); LnCap, 22RV1, MDA-PCa2b and C4.2 (used for anti-PSMA-ATACs); Raji, Raji Luc, Nalm-6 and MEC-2 (used for anti-CD19-ATACs) Antibodies: Anti-Her-2 (cysteine engineered monoclonal antibody, Heidelberg Pharma); humanized anti-PSMA (Albert Ludwig University Freiburg, medical center; humanization at Lonza Group AG; cysteine engineered monoclonal antibody, Heidelberg Pharma); chimeric anti-CD19 (DKFZ Heidelberg, Germany; cysteine engineered monoclonal antibody, Heidelberg Pharma). Toxic warhead: Cysteine reactive linker-amanitin constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to the antibodies. Cell proliferation assay: Quantitative determination of cell viability was performed by CellTiter Glo 2.0 assay (Promega). Animal models: Subcutaneous Mouse xenograft tumor models (Her-2-, PSMA- and CD19-positive cell lines) were performed in single-dose experiments. Tolerability was assessed in mice and will be assessed in non-human primates (NHP). Results: Eight different amanitin-linker constructs were synthesized. They differed in the attachment site of the linker at the amanitin as well as in the toxin core structure. All ATACs showed in vitro cytotoxicity on target positive cell lines in the picomolar range. In mouse xenograft models, ATACs with four of the eight linker-amanitin derivatives caused dose-dependent tumor regression and complete remission after a single i.v. dose of 2.0 mg/kg in s.c. xenografts irrespective of the antibody and target used. In contrast the other four linker-amanitin derivatives were only poorly effective in vivo while showing comparable in vitro activities. When comparing subcutaneous and intravenous xenograft models using the same cancer cell line, the different efficacy of the eight linker-amanitin variants was only detected in subcutaneous but not in intravenous xenografts. Mouse tolerability studies of ATACs showed a MTD of at least 10mg/kg for all linker-amanitin variants. Conclusions: Different efficacy of linker-amanitin derivatives with regard to mouse xenograft models was detected. An SAR profile of amanitin could be established which enabled the selection of optimized linker-amanitin variants for the use of ATACs in solid tumors. Citation Format: Michael Kulke, Anikó Pálfi, Christoph Müller, Werner Simon, Susanne Werner-Simon, Christian Lutz, Torsten Hechler, Andreas Pahl. SAR of amanitin and optimization of linker-amanitin derivatives for solid tumors [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 735.

α‐Amanitin und verwandte Amatoxine werden schon über 60 Jahre hauptsächlich durch Isolierung aus dem grünen Knollenblätterpilz untersucht. Die Totalsynthese blieb jedoch wegen der einzigartigen Struktureigenschaften schwierig. α‐Amanitin ist ein potenter Inhibitor der RNA‐Polymerase II. Die Unterbrechung dieses fundamentalen Transkriptionsprozesses von Eukaryonten führt unweigerlich zur Apoptose. Dieser einzigartige Mechanismus macht das Toxin zu einem idealen Kandidaten für Antikörper‐Toxin‐Konjugate (ADCs). Nur Mikrogramm‐Mengen des Toxins, selektiv an den Wirkort des Tumors gebracht, reichen aus, um maligne Tumorzellen nahezu jeder Herkunft in die Apoptose zu zwingen. Indem die stereoselektive Synthese von Dihydroxyisoleucin, der photochemische Zugang zu einer Tryptathionvorstufe, die Festphasensynthese und die Makrozyklisierung realisiert wurden, wurde ein skalierbarer Prozess zu totalsynthetischem α‐Amanitin erhalten. Dies macht α‐Amanitin und seine Derivate zugänglich für die Entwicklung neuer ADCs.