Abstract 2636: Amanitin-based ADCs targeting Guanylyl cyclase C (GCC) as novel therapeutic modality for treatment of colorectal cancer
Authors: Braun Alexandra , Pelz Andreas , Ebeling Franziska , Decker Kristin , Pálfi Anikó , Kulke Michael , Pahl Andreas , Hechler Torsten 
Published: April 2023

Background: Amatoxin-based antibody drug conjugates (so called ATAC®s) constitute a new class of antibody-drug conjugates (ADCs) which utilize amanitin as toxic payload. Amanitin binds to the RNA pol II and thereby efficiently inhibits cellular transcription. In the current study, in vitro and in vivo data of an ATAC® targeting GCC (guanylyl cyclase C) are presented. GCC is a cell surface receptor expressed in &gt;95% of colorectal cancer, and in approximately 65% of esophageal, gastric, and pancreatic tumors. In healthy conditions GCC expression is restricted to the gastrointestinal tract, and more specific to the apical brush border of the intestinal epithelium (luminal site). Thus, GCC in healthy tissue is not exposed to the circulation but upon tumor progression in GI malignancies it becomes accessible for i.v. injected targeted therapeutics. This tumor-specific accessibility for drugs in circulation makes GCC a highly attractive target for ATAC®s.

Material and Methods: ATAC®s: Cysteine-reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to engineered cysteine residues of a proprietary anti-GCC antibody yielding ATAC®s with a DAR of 2.0. Animal models: Subcutaneous (s.c.) mouse xenograft models with the GCC overexpressing cell line HEK293-GUCY2C mono2 or colon cancer PDX models (Charles River) were performed in single-dose and multiple-dosing experiments. Tolerability was assessed in NMRI nude mice and non-human primates (NHP).

Results: All anti-GCC ATAC®s with amanitin-linker derivatives, optimized for use in solid tumors, showed favorable in vitro cytotoxicity with picomolar activity on GCC+ cell lines and no cytotoxic activity on target-negative cells. In mouse xenograft models, the optimized anti-GCC ATAC®s caused dose-dependent tumor regression in HEK293-GUCY2C mono2 s.c. xenografts. Even in colorectal cancer PDX models, anti- GCC ATAC®s led to a substantial anti-tumor effect. Multiple dosing improved this anti-tumor efficacy even further without negative impact on toxicity. The MTD of the ATAC®s tested in mice differed between 7.5 and 50 mg/kg depending on linker chemistry. Safety profiling in cynomolgus monkeys revealed good tolerability for all selected ATAC®s. Hematology and clinical chemistry parameters were unaffected apart from a moderate and transient increase in liver enzymes.

Conclusions: Targeted cytotoxic drug delivery to GCC+ cell lines was achieved by using anti-GCC ATAC®s optimized for the use in solid tumors. The mode of action of the payload amanitin led to a pronounced anti-tumor effect in vitro and in vivo in PDX models with good tolerability in non-human primates. The use of anti-GCC ATAC®s with their novel mode of action in the therapy of GCC positive cancers, such as colorectal cancer represents a promising approach in cancer therapy.

Citation Format: Alexandra Braun, Andreas Pelz, Franziska Ebeling, Kristin Decker, Anikó Pálfi, Michael Kulke, Andreas Pahl, Torsten Hechler. Amanitin-based ADCs targeting Guanylyl cyclase C (GCC) as novel therapeutic modality for treatment of colorectal 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 2636.