Torsten Hechler

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

Synthesis and biological evaluation of RGD and isoDGR peptidomimetic-α-amanitin conjugates for tumor-targeting

RGD-α-amanitin and isoDGR-α-amanitin conjugates were synthesized by joining integrin ligands to α-amanitin via various linkers and spacers. The conjugates were evaluated for their ability to inhibit biotinylated vitronectin binding to the purified αVβ3 receptor, retaining good binding affinity, in the same nanomolar range as the free ligands. The antiproliferative activity of the conjugates was evaluated in three cell lines possessing different levels of αVβ3 integrin expression: human glioblastoma U87 (αVβ3+), human lung carcinoma A549 (αVβ3−) and breast adenocarcinoma MDA-MB-468 (αVβ3−). In the U87, in the MDA-MB-468, and partly in the A549 cancer cell lines, the cyclo[DKP-isoDGR]-α-amanitin conjugates bearing the lysosomally cleavable Val-Ala linker were found to be slightly more potent than α-amanitin. Apparently, for all these α-amanitin conjugates there is no correlation between the cytotoxicity and the expression of αVβ3 integrin. To determine whether the increased cytotoxicity of the cyclo[DKP-isoDGR]-α-amanitin conjugates is governed by an integrin-mediated binding and internalization process, competition experiments were carried out in which the conjugates were tested with U87 (αVβ3+, αVβ5+, αVβ6−, α5β1+) and MDA-MB-468 (αVβ3−, αVβ5+, αVβ6+, α5β1−) cells in the presence of excess cilengitide, with the aim of blocking integrins on the cell surface. Using the MDA-MB-468 cell line, a fivefold increase of the IC50 was observed for the conjugates in the presence of excess cilengitide, which is known to strongly bind not only αVβ3, but also αVβ5, αVβ6, and α5β1. These data indicate that in this case the cyclo[DKP-isoDGR]-α-amanitin conjugates are possibly internalized by a process mediated by integrins different from αVβ3 (e.g., αVβ5).

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.

Amanitins and their development as a payload for antibody-drug conjugates

Amanitin-based ADCs represent a new class of ADCs using a novel mode of action. This payload introduces a novel mode of action into oncology therapy, the inhibition of RNA Polymerase II. The high potency of the toxin leads to highly efficacious ADCs. The development of the technology around this toxin will be described. These developments support the clinical development of amanitin-based ADCs by using a toxin with a new mode of action and with a favorable therapeutic index. HDP-101 is an Amanitin based ADC directed against BCMA and will be advancing to the clinical phase in 2019.

Abstract 633: Amanitin-based ADCs with an improved therapeutic index

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 a few toxic compounds only and on an even smaller number of toxicity mechanisms: Most antibodies are coupled to the microtubuli-targeting auristatins and maytansines (1). 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 antitumor potency of monoclonal antibodies conjugated to small molecules from the amatoxin family. 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 (2). In our experiments, we tested a site-specific strategy to covalently conjugate amanitin to HER2-targeting trastuzumab and generated conjugates with low aggregation and high affinity for the target antigen. For site-specific conjugation, trastuzumab was engineered at different locations to incorporate cysteines. Trastuzumabs with engineered cysteines at different positions were compared in vitro and in vivo to identify the best localization for drug attachment. Safety profiling of a site-specific amanitin ADC in cynomolgous monkeys revealed good tolerability and a favorable therapeutic index. Cysteine substitutions in trastuzumab heavy chains HER2 targeting trastuzumab antibody was modified by substitution of endogenous amino acids to cysteine at eight different positions (HC1 to HC8) in the constant regions of heavy chains (Fig. 1). The RNA polymerase II binding toxin amanitin was conjugated by stable linker (HDP 30.0880) or cleavable linker (HDP 30.1699) technology and maleimide chemistry, resulting in homogenous ADCs with a DAR of ca. 2 toxins per IgG. Two of the drug positions (HC4 and HC6) were selected for interfering with binding of the antibodies Fc domains to Fcg receptors, thus reducing the cytotoxic activity on Fcg receptor expressing cells, e.g. macrophages.