Abstract 1754: Amatoxin-based antibody-drug conjugates induce immunogenic cell death and improve the anti-tumor efficacy of immune checkpoint inhibitors in humanized mouse models
Authors: Orlik Christian , Ebeling Franziska , Decker Kristin , Dranova Irina , Pálfi Anikó , Mueller Christoph , Hechler Torsten , Pahl Andreas , Kulke Michael 
Published: June 2022

Abstract
Background: Amatoxin-based antibody-drug conjugates (ATACs) comprise a new class of antibody-drug conjugates (ADCs) using amanitin as a toxic payload. Amanitin binds to the RNA polymerase II and thereby efficiently inhibits the cellular transcription process. In the present study, we show that ATACs belong to the class of immune activating drugs which exhibit synergistic anti-tumor efficacy with immune checkpoint inhibitors (ICIs) in vivo by induction of immunogenic cell death (ICD). ICIs are a new class of cancer therapeutics utilizing patients` immune system to kill cancer cells. ICIs rely on the activity of the immune system to develop their full potential. Therefore, drugs that are heating up cold tumors and make them visible to the patients` immune system and by that enhancing the anti-tumor efficacy of ICIs, e.g., ATACs, are on high demand for the treatment of tumor patients.
Material and methods: Cell line: Her2+ cell lines: NCI-N87; CD19+ cell line: Raji
Antibody: anti-Her2 and anti-CD19 engineered monoclonal antibody produced at Heidelberg Pharma
Toxic warhead: Cysteine reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to the antibody.
Animal models: Raji cells with human peripheral blood mononuclear cells (PBMCs), NCI-N87 cells or HCBx-10/HCBx-11 PDX (Xentech, France) tumor pieces were implanted subcutaneously in mice. Treatment: ATAC: single dose i.v.; immune checkpoint inhibitors: Q3Dx6 or Q3Dx5.
Results: In vivo treatment of heterogenous Her2low PDX tumors with an anti-Her2-ATAC led to a significant tumor growth delay and complete tumor remission. Furthermore, increased expression of HMGB1 and surface exposure of calreticulin (CRT), two hallmarks of ICD, was observed in the same PDX tumors as well as in a Her2+ CDX (NCI-N87) tumor after the treatment with an anti-HER2 ATAC. In addition, the combined treatment of ATACs and different ICIs targeting CTLA-4 (Ipilimumab), PD-L1 (Pembrolizumab) or PD-1 (Avelumab) had a synergistic effect in a humanized lymphoma CDX model in the presence of human PBMCs. Combination treatment led to enhanced tumor growth inhibition and more tumor free animals as compared to single treatments with ATAC or ICI.
Conclusions: The strong anti-tumor efficacy of ATACs even in heterogenous patient-derived xenograft models in combination with the induction of ICD in vitro and in vivo suggest that the anti-tumor effect of ATACs is accompanied by the activation of the immune system. This hypothesis is strengthened by the finding that ATACs and ICIs show a synergistic effect in vivo. The presented data highlights the general concept of the synergistic effect of ATACs and ICIs which applies to several types of ICIs thus strengthening the scientific rationale for combination treatments in clinical trials.
Citation Format: Christian Orlik, Franziska Ebeling, Kristin Decker, Irina Dranova, Anikó Pálfi, Christoph Mueller, Torsten Hechler, Andreas Pahl, Michael Kulke. Amatoxin-based antibody-drug conjugates induce immunogenic cell death and improve the anti-tumor efficacy of immune checkpoint inhibitors in humanized mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1754.