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.