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Despite major treatment advances in recent years, patients with multiple myeloma inevitably relapse. The RNA polymerase II complex has been identified as a promising therapeutic target in both proliferating and dormant cancer cells. Alpha-amanitin, a toxin so far without clinical application due to high liver toxicity, specifically inhibits this complex. Here, we describe the development of HDP-101, an anti–B-cell maturation antigen (BCMA) antibody conjugated with an amanitin derivative. HDP-101 displayed high efficacy against both proliferating and resting myeloma cells in vitro, sparing BCMA-negative cells. In subcutaneous and disseminated murine xenograft models, HDP-101 induced tumor regression at low doses, including durable complete remissions after a single intravenous dose. In cynomolgus monkeys, HDP-101 was well tolerated with a promising therapeutic index. In conclusion, HDP-101 safely and selectively delivers amanitin to myeloma cells and provides a novel therapeutic approach to overcome drug resistance in this disease.

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 >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.

Background: Amatoxin-based antibody-drug conjugates (so called ATACs) are a new class of ADCs that use the RNA polymerase II inhibitor amanitin as toxic payload. HDP-101 is the first ATAC in the clinic, currently being tested in a phase I/II clinical trial in multiple myeloma patients. HDP-101 is given to patients intravenously (i.v.) as this is the most common administration route for ADCs. I.v. is often selected as route of administration in a clinical setting since it results in a bioavailability of 100%. However, i.v. dosing has disadvantages such as very high Cmax levels which can trigger toxicity, requires hospitalization of the patient for administration, and bears the risk of vessel damage. In this study, we demonstrate that subcutaneous (s.c.) dosing is a promising alternative route of administration for ATACs which is well tolerated and able to improve the therapeutic index (TI) of ATACs. Material and Methods: ATACs: ADCs based on cysteine-reactive and site-specific amatoxin-linker constructs synthesized at Heidelberg Pharma. Monkey Studies: Cynomolgus monkeys, escalating doses of ATACs i.v or s.c. q21d Mouse Studies: CB17 Scid mice, single dose of ATACs i.v. or s.c.; if applicable bleeding at 8 - 12 time points between 5 min and 14 days after dosing; tumor studies in s.c. and i.v. tumor models Results: The impact of the dosing route on organ distribution of ATACs was tested in a PK study after a single i.v. or s.c. ATAC dose. S.c. dosing resulted in increased serum half-life of ATACs and reduced Cmax values as compared to i.v. dosing, while the AUC was comparable. Toxicity of ATACs is most probably driven by its Cmax level in serum, whereas the efficacy is mainly driven by AUC. Thus, we hypothesized that s.c. dosing might improve the TI of ATACs. The maximal tolerated dose (MTD) of ATACs in mice was compared between s.c. and i.v. dosing. S.c. dosing resulted in higher MTDs as compared to i.v. dosing independent of the antibody and amatoxin payload variant used. These findings were also confirmed in cynomolgus monkeys, where s.c. dosing of the ATAC HDP-103 resulted in a reduced Cmax, a comparable AUC, and an improved HNSTD of HDP-103. The impact of s.c. dosing on anti-tumor efficacy of ATACs was investigated in several tumor models in mice. ATACs with different antibodies and amatoxin payloads were applied as single i.v. or s.c. dose. In all models tested, the anti-tumor efficacy of ATACs was similar between groups receiving s.c. or i.v. dosing. Conclusions: The present study demonstrates that s.c. dosing is a promising route of administration for ATACs as it not only refines the pharmacokinetic distribution of ATACs but may improve the TI in patients. By reducing Cmax, s.c. dosing improves the tolerability of ATACs in mice and cynomolgus monkeys. At the same time the anti-tumor efficacy of ATACs is maintained as the AUC is not negatively impacted. Thus, s.c. dosing improves the TI of ATACs and may represent a promising route of administration also in humans. Citation Format: Kristin Decker, Marija Vranic, Marisa Schmitt, Irina Dranova, Anikó Pálfi, Torsten Hechler, Andreas Pahl, Michael Kulke. Subcutaneous dosing of amatoxin-based ADCs increases the therapeutic index [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 1523.

Triple-negative breast cancer (TNBC) is a molecularly diverse and clinically heterogeneous disease. The challenges for developing novel treatment approaches for TNBC are the paucity of actionable targets, lack of targeted therapies, and poor prognosis of patients. Breast cancer genomics revealed that heterozygous deletion of chromosome 17p (Chr17p) is the most prevalent (53%) event in TNBC. Within the 17p deletion region is the tumor suppressor TP53 (encoding p53), whose deletion or mutation has been long known as a primary tumorigenic driver. However, it remains unclear whether the deletion event, which often includes as many as 200 genes, impacts tumorigenesis beyond TP53 loss alone. While p53 aberrancy promotes proliferation, metabolism and metastatic potential of the TNBC cells, we found that heterozygous deletion of Chr17p is tightly correlated with poor cytotoxicity of tumor infiltrating lymphocytes (TILs) and poor clinical outcomes in patients with TNBC. This result suggests that selective advantage due to Chr17p loss reflects the combined impact of TP53 loss and the reduced dosage of co-deleted genes. Our recent study also identified POLR2A in the TP53-neighboring region as a collateral vulnerability target in the TNBC tumors with Chr17p loss, suggesting that inhibition of POLR2A may be a precision therapy approach for TNBC. As a proof-of-concept, we designed pH-activated nanoparticles (termed as nanobomb) for delivery of POLR2A siRNA, which enhances bioavailability and improves endo/lysosomal escape of the siRNA. In this study (Nature Nanotechnology, 2019)1, we demonstrated that suppressing POLR2A expression with the siRNA-laden nanoparticles led to marked growth reduction of TNBC tumors with 17p loss. Despite recent success of the first FDA-approved RNAi-based therapy for polyneuropathy, the issues of RNAi-based drugs, such as non-specific and incomplete knockdown, can make RNAi unpredictable, slow, and risky, in particular in drug discovery, where speed and reliability of results are crucial factors. To accelerate the translational development of our important finding, we will use α-amanitin, a natural small compound isolated from Amanita phalloides, to specifically inhibit POLR2A (Kd ∼10−9 M). However, free form of α-amanitin causes liver toxicity via the interaction with the hepatocyte-specific OATP1B3 transporter, limiting its clinical applications. To overcome the drug toxicity of α-amanitin, we have been developing α-amanitin-based antibody-drug conjugates (ADC). This approach inhibits the specific uptake of α-amanitin into hepatocytes and increases tumor-specific targeting using tumor-specific monoclonal antibodies. This type of ADC showed significant efficacy in inhibiting the growth of colorectal tumors with heterozygous deletion of TP53 and POLR2A (Nature, 2015)2. Here, we propose that heterozygous deletion of Chr17p not only contributes to TNBC tumorigenesis, but also confers therapeutic vulnerabilities, which can be utilized to develop novel targeted cancer therapy. Refernces 1. Xu,J. et al. Precise targeting of POLR2A as a therapeutic strategy for human triple negative breast cancer. Nat. Nanotechnol. 14, 388-397 (2019). 2. Liu,Y. et al. TP53 loss creates therapeutic vulnerability in colorectal cancer. Nature 520, 697-701 (2015). Citation Format: Xiongbin Lu, Yujing Li, Yifan Sun, Kevin Van der Jeught, Michael Kulke, Pahl Andreas, Xinna Zhang, Xiaoming He, Chi Zhang. Precise targeting of POLR2A for treating triple negative breast cancer with Chr17p loss [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-19.

Background: Amatoxin-based antibody-drug conjugates (ATACs) are a new class of ADCs using amanitin, a specific inhibitor of RNA polymerase II, as toxic payload. A first ATAC targeting B-cell maturation antigen (BCMA) is currently being tested in phase I/II clinical trials in multiple myeloma patients. During the clinical trials, the effect of repeated dosing on safety as well as anti-tumor efficacy of the ATAC will be assessed. In preclinical studies a reduction of toxicity was observed after repeated dosing with ATACs regardless of the target or antibody. In the present study, we demonstrate the effect of ATAC pre-treatment on tolerability in pre-clinical models. Material and Methods: Antibody: Engineered monoclonal antibodies 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: Tolerability in CB17 Scid mice and Cynomolgus monkeys; Efficacy in a subcutaneous LNCaP prostate cancer model; Treatment: ADC (ATAC): escalating dosing i.v. q21d (tolerability) or repeated dosing i.v. q7dx4 (efficacy). Results: The impact of repeated dosing on the tolerability of a non-targeting ATAC was tested in CB17 Scid mice. The mice were initially treated with the maximal tolerated dose (MTD) of an ATAC, or PBS followed by a second higher dose 21 days after the first dose. Pre-treatment improved the tolerability of a second, higher dose of the same non-targeting ATAC from 12.5% to 80% survival. As ATAC toxicity is mainly caused by liver toxicity, the liver damage markers AST, ALT and LDH are used as toxicity markers in non-human primate studies. The induction of liver damage markers observed after repeated or escalated dosing was significantly lower than the induction after single or first dose treatment. Taken together, these data indicate that ATAC treatment can induce tolerability in animal models. Furthermore, in a subcutaneous LNCaP prostate cancer model, multiple ATAC dosing did improve the anti-tumor efficacy as compared to single dose treatment, indicating that the induced tolerability is not associated with reduced anti-tumor efficacy. Conclusions: ATAC pre-treatment induces tolerability in preclinical animal models without a loss in efficacy. Since this effect of ATAC pre-treatment was observed across different animal species, it likely translates also into humans. Consequently, if reduced liver toxicity and less adverse effects after repeated dosing with ATACs are also observed in the upcoming clinical trials, it might have a significant impact on treatment regimen and clinical success of ATACs. Citation Format: Kristin Decker, Marisa Schmitt, Can Araman, Franziska Ebeling, Irina Dranova, Torsten Hechler, Anikó Pálfí, Andreas Pahl, Michael Kulke. Treatment with antibody-targeted amanitin conjugates induces tolerability in preclinical models without triggering tolerance [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 1761.

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.

The clinical challenge for treating HER2 (human epidermal growth factor receptor 2)–low breast cancer is the paucity of actionable drug targets. HER2-targeted therapy often has poor clinical efficacy for this disease due to the low level of HER2 protein on the cancer cell surface. We analyzed breast cancer genomics in the search for potential drug targets. Heterozygous loss of chromosome 17p is one of the most frequent genomic events in breast cancer, and 17p loss involves a massive deletion of genes including the tumor suppressor TP53. Our analyses revealed that 17p loss leads to global gene expression changes and reduced tumor infiltration and cytotoxicity of T cells, resulting in immune evasion during breast tumor progression. The 17p deletion region also includes POLR2A, a gene encoding the catalytic subunit of RNA polymerase II that is essential for cell survival. Therefore, breast cancer cells with heterozygous loss of 17p are extremely sensitive to the inhibition of POLR2A via a specific small-molecule inhibitor, α-amanitin. Here, we demonstrate that α-amanitin–conjugated trastuzumab (T-Ama) potentiated the HER2-targeted therapy and exhibited superior efficacy in treating HER2-low breast cancer with 17p loss. Moreover, treatment with T-Ama induced immunogenic cell death in breast cancer cells and, thereby, delivered greater efficacy in combination with immune checkpoint blockade therapy in preclinical HER2-low breast cancer models. Collectively, 17p loss not only drives breast tumorigenesis but also confers therapeutic vulnerabilities that may be used to develop targeted precision immunotherapy.

The leukocyte surface antigen CD37 (TSPAN26), a member of the tetraspanin superfamily, is widely expressed on most malignant B cells, making it an actionable target for treatment of patients with chronic lymphocytic leukemia (CLL) and other B-cell non-Hodgkin lymphoma (NHL) indications. Accordingly, αCD37 antibodies have shown promising results in phase 1/2 clinical trials for CLL and NHL. Richter's syndrome (RS) is the transformation of CLL into an aggressive and rapidly fatal lymphoma, typically a diffuse large B cell lymphoma (DLBCL). RS is a challenging disease since very few effective treatment options exist for these patients and the available regimens, mainly based on R-CHOP scheme, show limited efficacy. We recently established patient-derived xenograft (PDX) models from RS patients and have shown that they can be used to test the efficacy of novel drugs and drug combinations 1,2. All available RS-PDX models were characterized by high-levels of CD37 expression, when assessed by RNA sequencing, reverse-transcriptase-polymerase chain reaction (RT-PCR), flow cytometry (FACS), western blot (WB) and immunohistochemistry (IHC). More precisely, two models (RS1316 and IP867/17) showed slightly higher CD37 levels compared to the others (RS9737 and RS1050). These models were used to test three different αCD37-ATACs®, ADCs which comprise amanitin-derivatives as payload. Amanitin (the main poison in the green deathcap mushroom) belongs to the well-known amatoxin family. Amanitin is taken up by OATP1B3 transporter, solely expressed on hepatocytes. Upon mushroom intoxication, it can lead to severe liver toxicity by inhibiting the RNA polymerase II. Upon conjugation to target-specific antibodies, the maximal tolerated dose is significantly increased by reducing the non-specific liver uptake. By binding to its antigen, ATACs deliver amanitin only into target-positive cancer cells while target negative cells show no off-target toxicity. Consistent with CD37 expression on the cell surface, ex-vivo treatment of freshly purified cells from RS-PDX tumor masses to αCD37-ATACs® resulted in increased apoptosis after 72 hours of treatment, with only minor differences among the 3 ATACs® and the models used. Since alpha-amanitin is a deadly toxin known to target human RNA polymerase II and, at high doses, also RNA polymerase III, we checked messenger RNA levels in basal conditions and after CD37-ATAC® treatment by looking at different housekeeping genes, and confirmed a reduction in global mRNA levels. αCD37-ATAC® efficacy was then assessed in vivo in systemic RS-PDX models where RS cells are intra-venously (i.v.) injected in the tail vein and cells distribute to different tissues (blood, spleen and bone marrow), resembling the human disease. Cells from RS1316, RS1050 and RS9737 models were injected into the tail vein and left to engraft 14 days, before mice were randomly assigned to vehicle or ATAC® groups. A single i.v. treatment for each αCD37-ATAC® was administered, testing two different doses for each compound, and mice were then monitored for survival. Overall, the single administration of all three ATACs® caused highly significant disease regression. In the RS1316 model, independently of the dose or tested ATAC®s, all treated mice, except one, were alive and disease-free until the end of the experiment, 140 days post cells injection, while survival of vehicle-treated mice was 65 days. FACS analysis to trace neoplastic cells in parenchymatous organs and bone marrow confirmed the absence of neoplastic cells. In the other 2 models, RS9737 and RS1050, even though ATAC® treatment did not result in complete disease eradication, a single administration of αCD37-ATAC® resulted in a dramatically increased survival (approximately 35-60 days, depending on the model and ATAC® used). Finally, CD37 expression was confirmed by RNA sequencing on a cohort of 15 primary RS samples, even though with variable levels. Compared to CLL cells, RS samples showed CD37 expression levels comparable to those reported for DLBCL cells. Overall, these data indicate CD37 as a potential target to treat RS patients with selective targeting αCD37-ATACs®. ATACs® is a registered trade mark of Heidelberg Pharma Research GmbH, Germany References Vaisitti T et al. Blood. 2021;137(24):3365-3377. Iannello A, et al. Blood. 2021;137(24):3378-3389. Disclosures Orlik: Heidelberg Pharma: Current Employment. Kulke: Heidelberg Pharma: Current Employment. Pahl: Heidelberg Pharma: Current Employment. Deaglio: Heidelberg Pharma: Research Funding; Astra Zeneca: Research Funding.

Background: ATACs (antibody-targeted amanitin conjugates) comprise a new class of antibody-drug conjugates (ADCs) using amanitin as a toxic payload. Amanitin binds to the RNA pol II and thereby efficiently inhibits the cellular transcription process. In the current study, in vitro and in vivo data of ATACs targeting CD37 (member of the tetraspanin family) are presented. CD37 is a transmembrane protein expressed exclusively on cells of the immune system and mainly on mature B-cells, as well as in many B-cell malignancies, including Richter's Syndrome (RS) or Richter´s transformation. RS is a transformation of B cell chronic lymphocytic leukemia, refractory to treatment and carries a poor prognosis, and hence is considered an ideal target for amanitin-based ADCs. Material and methods: Cell lines: CD37-positive: Mec-2 (B-chronic lymphocytic leukaemia); Raji-Luc, Ramos (Burkitt´s lymphoma); CD37-negative: HL-60 (acute lymphocytic leukemia) Antibody: anti-CD37 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. Cell proliferation assay: Quantitative determination of cell viability was performed by CellTiter Glo 2.0 assay (Promega). Animal models: Disseminating Mouse xenograft tumor models (Mec-2 and Raji-Luc) and CD37-positive patient derived xenograft (PDX; Richter's Syndrome) models were performed in single-dose experiments. Tolerability was assessed in non-human primates (NHP). Results: All tested anti-CD37 ATACs showed in vitro cytotoxicity in the picomolar range on CD37-positive cell lines. No cytotoxic activity was observed on CD37-negative cells. In mouse xenograft models, 80 - 100% overall survival was achieved with two anti-CD37 ATACs at doses of 1/16 MTD (Mec-2 model) and 1/64 MTD (Raji Luc Model). Single-dose treatment caused rapid and complete tumor remission already 7 days after treatment. The tested ATACs revealed good tolerability in mice. In NHP, the anti CD37 ATACs revealed good tolerability. Hematology and clinical chemistry parameters were unaffected except liver transaminases and LDH: A mild to moderate and transient increase was observed. Conclusions: Targeted cytotoxic drug delivery to CD37 positive cell lines was achieved by using anti-CD37 ATACs. The mode of action of the payload amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in non-human primates. Using ADCs in the therapy of B-cell lymphomas, including malignancies that underwent Richter's transformation, is a promising approach, especially by using a payload whose mode of action differs from other commonly used toxins, like ATACs. Citation Format: Stephanie Voss, Aniko Palfi, Christoph Mueller, Andreas Pahl, Torsten Hechler, Michael Kulke. Preclinical evaluation of anti-CD37 antibody-targeted amanitin conjugates (ATAC) in B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 915.

Background: Antibody-targeted amanitin conjugates (ATACs) are 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. The present study highlights the benefit of combining ATACs with immune checkpoint inhibitors. The combination of these two modalities leads to an induction of immunogenic cell death (ICD) in vitro and a synergistic anti-tumor effect in vivo. Hence, the combination of ATACs with immune checkpoint inhibitors (ICI) provides a promising approach for potential further cancer treatment. Material and methods: Cell lines: BT-474 (breast ductal carcinoma); BJAB, Raji, (Burkitt lymphoma) Antibody: 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/without human peripheral blood mononuclear cells (PBMCs) were implanted subcutaneously in mice. Treatment: ADC (ATAC): single dose i.v.; immune checkpoint inhibitor: Q3D x6. Results: The treatment of target-positive cell lines (e.g., BT-474 and BJAB) with corresponding ATACs led to the induction of three ICD hallmarks in vitro. In addition to increased surface expression of calreticulin (CRT), ATAC-treated tumor cells secreted adenosine triphosphate (ATP) and released high-mobility group box 1 protein (HMGB1). In contrast, this was not observed when the same cells were treated with a non-targeting ATAC. In a subcutaneous CDX model, mixed with human PBMCs, the combined administration of target specific ATACs with an immune checkpoint inhibitor led to an increased anti-tumor effect indicated by significant tumor growth inhibition as compared to single treatment with ATAC or ICI. This synergistic effect was not observed in mice bearing tumors without human PBMCs. Conclusions: Antibody-targeted amanitin conjugates (ATACs) induced immunogenic cell death in vitro and resulted in an increased anti-tumor effect in combination with an immune checkpoint inhibitor in a subcutaneous CDX model if human PBMCs were present. Consequently, the data presented provide a rationale to the use of ATACs in combination therapy with immune checkpoint inhibitors. Citation Format: Kristin Decker, Irina Dranova, Christian Orlik, Aniko Palfi, Christoph Mueller, Ram Kumar Singh, Robert Z. Orlowski, Torsten Hechler, Andreas Pahl, Michael Kulke. Combination of antibody-targeted amanitin conjugates (ATAC) with immune checkpoint inhibitors shows synergistic therapeutic effect in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 921.

Background: Antibody-targeted amanitin conjugates (ATACs) comprise a new class of antibody-drug conjugates (ADCs) using amanitin as toxic payload. Amanitin binds to the RNA pol II and thereby efficiently inhibits the cellular transcription process. In the current study, in vitro and in vivo data of an ATAC targeting PSMA (prostate specific membrane antigen) are presented. PSMA is a type II integral membrane glycoprotein and used as tumor marker due to its predominant expression on malignant prostate cells in prostate carcinoma. As PSMA expression increases with tumor aggressiveness, metastatic and disease recurrence, it is considered an ideal target for amanitin-based ADCs. Material and methods: Cell lines: PSMA+ cell lines: LNCap, 22RV1; PSMA- cell line: PC3 Antibody: cysteine engineered monoclonal anti-PSMA antibody (Albert Ludwig University Freiburg, medical center; derivatization and production at Heidelberg Pharma) Toxic warhead: Cysteine reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to engineered cysteine residues of the anti-PSMA antibody yielding ATACs with a DAR of 2. Cell proliferation assay: Quantitative determination of cell viability was performed by BrdU-based chemiluminescent cell proliferation ELISA (Roche). Animal models: Subcutaneous Mouse xenograft tumor models with PSMA-positive cell lines 22RV1 and LNCap were performed in single-dose experiments. Tolerability was assessed in mice and non-human primates (NHP). Results: All anti-PSMA ATACs with optimized amanitin-linker variants for use in solid tumors showed favorable in vitro cytotoxicity with nano- to picomolar activity on PSMA+ cell lines and no cytotoxic activity on PSMA- cells. In mouse xenograft models, the optimized anti-PSMA ATACs caused dose-dependent tumor regression in LNCap s.c. xenografts. Complete remission was achieved after a single i.v. dose of ¼ MTD with 100% overall survival for the full duration of the studies (>160 days). The tolerability of the tested ATACs in mice differed between 15 and > 80 mg/kg Safety profiling in Cynomolgus monkeys revealed a good tolerability and therapeutic index for all selected amanitin-linker variants. Hematology and clinical chemistry parameters were unaffected except liver enzymes and LDH: A moderate and transient increase was observed. Low off target toxicity was confirmed by a non-binding ATAC. Conclusions: Targeted cytotoxic drug delivery to PSMA positive cell lines was achieved by using anti-PSMA ATACs optimized for the use in solid tumors. The mode of action of the payload amanitin led to an efficient anti-tumor potential in vitro and in vivo with good tolerability in non-human primates. Using ATACs in the therapy of PSMA positive prostate cancer is a promising approach, especially by using a cytotoxic agent whose mode of action differs from other commonly used toxins. Citation Format: Alexandra Braun, Aniko Palfi, Christoph Mueller, Torsten Hechler, Andreas Pahl, Michael Kulke. Amanitin-based ADCs targeting PSMA as novel therapeutic modality for prostate cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 910.

Several antibody-drug conjugates (ADCs) are currently being evaluated in clinical trials in a variety of hematologic malignancies. Most of these ADCs are based on only a few toxic compounds, largely limited to microtubule- or DNA-targeting toxins that mainly impact proliferating cells and have limited efficacy in diseases with a low proliferative fraction such as indolent lymphomas or multiple myeloma. Thus, new compounds with alternative toxicity mechanisms and the ability to actively induce cell death in non-proliferating tumor cells could enhance the therapeutic potential of ADCs. We are currently developing amanitin based ADCs. Amanitin specifically inhibits RNA polymerase II thereby inhibiting the cellular transcription process at very low concentrations irrespective of the proliferation status of the target cell. Subsequently tumor cells enter apoptosis and are eliminated. Del(17p) is a frequent deletion in tumor cells, identifying high-risk patients with poor prognosis. TP53, a well-known tumor suppressor gene, is located on the short arm of chromosome 17. The major subunit of the RNA Polymerase II POLR2A is frequently co-deleted with TP53 in del(17p) human cancers, which renders these tumors more vulnerable to amanitin-ADC treatment compared to wild-type tumor and healthy cells. HDP-101 is a new ADC targeting BCMA (B cell maturation antigen) carrying a synthetic version of amanitin as a payload. In vitro cytotoxic potency of HDP-101 was demonstrated on BCMA-positive myeloma cell lines, as well as on non-proliferating primary CD138+ cells isolated from patients with refractory myeloma. Furthermore, the cytotoxic effects of HDP-101 were seen even in non-proliferating myeloma cells with low BCMA density. Toxicity was observed neither in non-BCMA expressing control cells nor in myeloma cells exposed to an amanitin-loaded non-target control antibody. In murine xenograft models of human myeloma, HDP-101 caused dose-dependent tumor regression including complete remissions after a single dose in subcutaneous and as well as in disseminated models. Safety profiling in Cynomolgus monkeys revealed a good therapeutic index after repeated dosing. On safe doses, hematology and clinical chemistry parameters were mainly unaffected except for a mild to moderate and transient increase of liver enzymes and lactate dehydrogenase predominantly after the first dose. Free payload was detectable at levels close to the lower limit of quantification only. GLP toxicity studies of the released payload in rodents revealed a NOAEL, which is well above concentrations observed in non-human primate studies after ADC administration as well as above the toxin doses administered by the ADC. This may be attributed to the hydrophilicity of the toxin, which cannot passively enter any antigen-negative cell and is rapidly cleared without further drug metabolism. Our non-clinical studies concluded that this amanitin-based ADC is a novel promising approach in the therapy of multiple myeloma to overcome drug resistance and improve patient outcome. Patients whose myeloma cells harbor 17p deletion may have a specific therapeutic sensitivity towards HDP-101. HDP-101-01 is a first-in-human, open label, non-randomized, multicenter, phase 1/2a trial in setup phase with HDP-101 in patients with multiple myeloma whose disease has progressed. The aim of the Phase 1 dose escalation part is to determine the Maximum Tolerated Dose and/or establish the Recommended phase 2 Dose. The primary objective of the phase 2 dose expansion phase is to assess the preliminary anti-tumor activity of HDP-101. Patients in phase 2a will be stratified based on del(17p) status. An adaptive Bayesian logistic regression model with overdose control principle will be used to guide the dose escalation steps. The design of the study ensures a safe dose escalation to reach a potential clinical benefit in a patient who have limited or no therapeutic options. The study is expected to enroll patients in early 2021. Disclosures Strassz: Heidelberg Pharma: Current Employment. Raab:Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees. Orlowski:STATinMED Research: Consultancy; Founder of Asylia Therapeutics, Inc., with associated patents and an equity interest, though this technology does not bear on the current submission.: Current equity holder in private company, Patents & Royalties; Laboratory research funding from BioTheryX, and clinical research funding from CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Research Funding; Sanofi-Aventis, Servier, Takeda Pharmaceuticals North America, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen, Inc., AstraZeneca, BMS, Celgene, EcoR1 Capital LLC, Forma Therapeutics, Genzyme, GSK Biologicals, Ionis Pharmaceuticals, Inc., Janssen Biotech, Juno Therapeutics, Kite Pharma, Legend Biotech USA, Molecular Partners, Regeneron Pharmaceuticals, Inc.,: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kulke:Heidelberg Pharma: Current Employment, Current equity holder in publicly-traded company. Schiedner:Heidelberg Pharma: Current Employment. Pahl:Heidelberg Pharma: Current Employment, Current equity holder in publicly-traded company.

We introduce a novel and versatile ADC-linker technology that is based on site-specific enzymatic payload conjugation to ‘off-the-shelf’ antibodies, i.e., without the need to reduce or engineer the antibody. The functionalization takes place site-specifically and stoichiometrically (drug-to-antibody ratio, DAR = 2.0) at the Fc-part of antibodies. The resulting ADCs show favorable biophysical properties such as high solubility and stability using different payloads. Additionally, initial ELISA findings show that our modification does not interfere with Fc-gamma receptor and FcRn binding. Importantly, our ADCs showed superior efficacy in different tumor animal models as compared to control Thiomab™ ADCs. Using native trastuzumab (non-engineered) as the targeting antibody and amanitin as payload, we generated within 36 hours highly homogeneous and pure ADCs with a well-defined DAR of 2.0 as confirmed by LC-MS. In in-vitro assays our ADCs demonstrated potent cytotoxicity in all tested cell-lines (SKBR-3, BT-474, JIMT-1, and NCI-N87) as compared to the control Thiomab™ ADCs, most strikingly for the JIMT-1 cell-line: EC50 of 0.15nM vs 2.5nM. In the mouse JIMT-1 tumor model, our anti-HER2 ADC was highly potent and resulted in complete tumor remission in all mice (10/10 mice) at a single dose of 2mg/kg. In contrast, the control Thiomab™-functionalized ADC showed tumor regrowth in 4 out of 10 animals, starting on day 50. The same high potency was observed for the NCI-N87 xenograft model at a dose of 3mg/kg in which tumor growth inhibition was significantly delayed versus the reference ADC (8/10 vs 4/10 animals alive on day 110). These encouraging results obtained so far indicate that our linker technology a) allows for fast (< 36 hours) and straightforward manufacturing of ADCs using different payloads without protein engineering efforts, b) results in ADCs with favorable biophysical properties and a clear defined drug-to-antibody ratio, and c) enables the generation of highly potent and stable, thus safer, next-generation ADCs. Citation Format: Philipp Rene Spycher, Julia Carina Frei, Jöri Elias Wehrmüller, Isabella Attinger-toller, Dragan Grabulovski, Torsten Hechler, Michael Kulke, Andreas Pahl, Martin Behe, Roger Schibli. Overcoming limitations of current Antibody-Drug Conjugates (ADCs) by a novel linker technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-106.

Background: Triple negative breast cancer (TNBC) is the most difficult to treat subtype of breast cancer with limited therapeutic options. At least 50% of TNBC patients have low epidermal growth factor receptor 2 (HER2; ERBB2) expression with the majority harboring hemizygous loss of POLR2A/chromosome 17p. For these patients the treatment with antibody-targeted amanitin conjugates (ATACs) targeting HER2 is a new promising approach. ATACs comprise a new class of antibody-drug conjugates (ADCs) using amanitin as toxic payload and are able to kill antigen low expressing cells. Amanitin binds to the eukaryotic RNA polymerase II and thereby efficiently inhibits the cellular transcription process. In the current study, in vitro and in vivo data of ATACs targeting human HER2low as well as tolerability studies are presented. HER2low TNBC is considered an interesting target for amanitin-based ADCs. Material and methods: Different HER2 expressing cell lines were treated with anti-HER2 ATACs. Cysteine reactive amanitin-linkers were conjugated site-specifically to engineered cysteine residues of an anti-HER2 antibody yielding ATACs with a DAR of 2.0. Quantitative determination of cell viability was analyzed by BrdU ELISA assay. Subcutaneous mouse xenograft models with HER2-positive cell lines were performed with single-dose treatments. In addition, ATAC efficacy was tested in HER2low heterogeneous TNBC patient derived xenograft (PDX) models with and without POLR2A deletion. Tolerability of ATACs was assessed in mice and non-human primates (NHP). Results: Anti-HER2 ATACs showed in vitro cytotoxicity on HER2+ high and low cell lines in low nanomolar to picomolar range. In mouse xenograft models, the anti-HER2 ATACs caused dose-dependent tumor regression independent of Her2 expression level. In HER2low heterogeneous TNBC PDX models anti-HER2 ATACs caused dose-dependent tumor regression. The efficacy of anti-Her2 ATACs was more pronounced in PDX models with hemizygous loss of TP53 and POLR2A reflecting a 17p deletion. Safety profiling of an optimized anti-Her2 ATAC in cynomolgus monkeys revealed a good tolerability indicating a good therapeutic window for 17p deleted TNBC. Conclusions: Targeted cytotoxic drug delivery to HER2 positive cell lines was achieved by using anti-HER2 ATACs. 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. TNBC PDX models with HER2low expression were sensitive to ATAC treatment. Loss of POLR2A/chromosome 17p increased susceptibility to anti-HER2 ATAC making 17p del TNBC a suitable indication for optimized anti Her2 ATACs. Citation Format: Christian Breunig, Anikó Pálfi, Michael Kulke, Christian Lutz, Christoph Müller, Torsten Hechler, Andreas Pahl. Preclinical evaluation of anti-HER2 Antibody Targeted Amanitin Conjugates (ATACs) on HER2low breast cancer with chromosome 17p deletion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 237.

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 an ATAC targeting PSMA (prostate specific membrane antigen) are presented. PSMA is predominantly expressed on malignant prostate cells in prostate carcinoma and correlates with tumor progression. Hence it is considered an interesting target for Amanitin based ADCs. Material and methods: PSMA cell lines: MDA-PCA-2B, LnCap. PC3 cell line served as PSMA negative control. Antibody: humanized anti-PSMA and cysteine engineered monoclonal antibody (Albert Ludwig University Freiburg, medical center; humanization at Lonza Group AG, derivatization and production at Heidelberg Pharma). Toxic warhead: A cysteine reactive amanitin-linker was conjugated site-specifically to engineered cysteine residues of the anti-PSMA antibody yielding an ATAC with a DAR of 2.0. Cell proliferation assay: Quantitative determination of cell viability was performed by CellTiter Glo 2.0 assay (Promega). Animal models: Subcutaneous Mouse xenograft tumor models with PSMA-positive cell lines MDA-PCA-2B and LnCap were performed in single-dose and multiple-dosing experiments. Tolerability was assessed in mice and non-human primates (NHP). Results: The anti-PSMA ATAC showed in vitro cytotoxicity on PSMA+ cell lines in picomolar range, whereas no cytotoxic activity on PSMA- cells was observed. In mouse xenograft models, the anti-PSMA ATAC caused dose-dependent tumor regression. Complete remission was achieved after a single i.v. dose of 4.0 mg/kg and after repeated i.v. doses of 2.0 mg/kg in s.c. xenografts. Safety profiling in Cynomolgus monkeys revealed a good tolerability and therapeutic index after sequentially applied doses of 0.3, 1.0 and 3.0 mg/kg. Hematology and clinical chemistry parameters were unaffected except liver enzymes and LDH: A moderate and transient increase was observed. The half-life of the ATAC in serum was 7-10 days; the free toxin was detectable at levels close to the lower limit of quantification only (LLOQ = 1.2 nM). Conclusions: Targeted cytotoxic drug delivery to PSMA positive prostate cancer cell lines was achieved by using an anti-PSMA 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. The use of ATACs in the therapy of PSMA positive prostate cancer is a promising approach, especially by using a cytotoxic agent whose mode of action differs from other commonly used toxins. Citation Format: Anikó Pálfi, Christian Breunig, Torsten Hechler, Christoph Müller, Christian Lutz, Andreas Pahl, Michael Kulke. Preclinical evaluation of an anti-PSMA antibody-targeted amanitin conjugate (ATAC) [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 740.

e14527Background: Currently, numerous antibody-drug conjugates (ADCs) are evaluated for hematologic malignancies with toxic payloads mainly affecting only proliferating cells, and thus resulting in...

Although subtypes of pancreatic ductal adenocarcinoma (PDAC) have been described, this malignancy is clinically still treated as a single disease. Here we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers--HNF1A and KRT81--that enable stratification of tumors into different subtypes by using immunohistochemistry. Individuals with tumors of these subtypes showed substantial differences in overall survival, and their tumors differed in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and it is highly expressed in several additional malignancies. These findings designate CYP3A5 as a predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance.

The giant protein titin functions as a molecular spring in muscle and is responsible for most of the passive tension of myocardium. Because the titin spring is extended during diastolic stretch, it will recoil elastically during systole and potentially may influence the overall shortening behavior of cardiac muscle. Here, titin elastic recoil was quantified in single human heart myofibrils by using a high-speed charge-coupled device-line camera and a nanonewtonrange force sensor. Application of a slack-test protocol revealed that the passive shortening velocity ( V p ) of nonactivated cardiomyofibrils depends on: ( i ) initial sarcomere length, ( ii ) release-step amplitude, and ( iii ) temperature. Selective digestion of titin, with low doses of trypsin, decelerated myofibrillar passive recoil and eventually stopped it. Selective extraction of actin filaments with a Ca 2+ -independent gelsolin fragment greatly reduced the dependency of V p on release-step size and temperature. These results are explained by the presence of viscous forces opposing myofibrillar passive recoil that are caused mainly by weak actin–titin interactions. Thus, V p is determined by two distinct factors: titin elastic recoil and internal viscous drag forces. The recoil could be modeled as that of a damped entropic spring consisting of independent worm-like chains. The functional importance of myofibrillar elastic recoil was addressed by comparing instantaneous V p to unloaded shortening velocity, which was measured in demembranated, fully Ca 2+ -activated, human cardiac fibers. Titin-driven passive recoil was much faster than active unloaded shortening velocity in early phases of isotonic contraction. Damped myofibrillar elastic recoil could help accelerate active contraction speed of human myocardium during early systolic shortening.

The PEVK domain of the giant muscle protein titin is a proline-rich sequence with unknown secondary/tertiary structure. Here we compared the force-extension behavior of cloned cardiac PEVK titin measured by single-molecule atomic force spectroscopy with the extensibility of the PEVK domain measured in intact cardiac muscle sarcomeres. The analysis revealed that cardiac PEVK titin acts as an entropic spring with the properties of a random coil exhibiting mechanical conformations of different flexibility. Since in situ, titin is in close proximity to the thin filaments, we also studied whether the PEVK domain of cardiac or skeletal titin may interact with actin filaments. Interaction was indeed found in the in vitro motility assay, in which recombinant PEVK titin constructs slowed down the sliding velocity of actin filaments over myosin. Skeletal PEVK titin affected the actin sliding to a lesser degree than cardiac PEVK titin. The cardiac PEVK effect was partially suppressed by physiological Ca(2+) concentrations, whereas the skeletal PEVK effect was independent of [Ca(2+)]. Cosedimentation assays confirmed the Ca(2+)-modulated actin-binding propensity of cardiac PEVK titin, but did not detect interaction between actin and skeletal PEVK titin. In myofibrils, the relatively weak actin-PEVK interaction gives rise to a viscous force component opposing filament sliding. Thus, the PEVK domain contributes not only to the extensibility of the sarcomere, but also affects contractile properties.

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 CD19 (also known as B4, CVID3) are presented. CD19, a class I transmembrane glycoprotein with no significant homology to any known protein, is expressed in B cells and B-cell malignancies like in B-cell acute lymphocytic leukemia (B-ALL) and B cell chronic lymphocytic leukemia (B-CLL). Therefore it is an ideal target for Amanitin based ADCs. Material and methods Cell lines: Raji (Burkitt Lymphoma), Nalm-6 (B-ALL) and HL-60 (CD19-negative cell line). Antibody: anti-CD19 Thiomab (licensed from the German Cancer Research Center; Heidelberg, Germany). Synthesis of CD19-Thiomab-ATACs: Maleimide amanitin compounds were conjugated site specifically to engineered cysteine residues of the anti-CD19 Thiomab. Cell proliferation assay: Quantitative determination of cytotoxicity was performed by CellTiter Glo 2.0 assay (Promega) or WST-1 assay (Roche). Animal models: Mouse xenograft tumor models were performed. Tolerability was assessed in mice and non-human primates (NHP). Results CD19-Thiomab-ATACs showed in vitro cytotoxicity on CD19+ cell lines in picomolar range, whereas no cytotoxic activity on CD19- cells was observed. In mouse xenograft models, CD19 showed dose-dependent tumor regression and complete remission after single dose i.v. of 2mg/kg and 4mg/kg. In a disseminating Raji xenograft model, median survival was increased from 6 days (vehicle control) to 105 days (CD19-Thiomab-30.2115 treated group at 6mg/kg, single dose). On day 154, 4 animals were still alive and in good overall condition. Safety profiling in Cynomolgus monkey revealed a good tolerability after sequentially applied doses of 0.3, 1.0, 3.0 and 5.0 mg/kg. Hematology and clinical chemistry data show minor alterations: transient increase in liver enzymes (ALT and AST) in combination with a transient increase in LDH. The half-life of the ADC in serum is 7-11 days; the free toxin is detectable at serum levels close to the lower limit of quantification only (LLOQ = 1.2 nM). Conclusions In the current study, in vitro and in vivo data of Amanitin-ADCs targeting CD19 are presented. CD19 is expressed on cells of the B cell lineage, ranging from the pre-B cells until the terminal differentiation to plasma cells. It is expressed in most acute lymphoblastic leukemias (ALL), chronic lymphocytic leukemias (CLL) and B cell lymphomas (Kemeng et al. (2012); Exp Hematol Oncol 1: 36). The preclinical data show high cytotoxicity in picomolar range, very good efficacy in tumor xenograft models and tolerability in an exploratory tolerability study in NHP. The positive findings of these initial experiments encourage Heidelberg Pharma to further proceed with anti-CD19-Amanitin-based ADCs towards clinics. Citation Format: Torsten Hechler, Aniko Palfi, Christoph Müller, Christian Lutz, Andreas Pahl, Michael Kulke. CD19 - a potential target for Amanitin-based ADCs [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 62. doi:10.1158/1538-7445.AM2017-62

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 Delbrück 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 Müller, 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

The elastic section of the giant muscle protein titin contains many immunoglobulin-like domains, which have been shown by single-molecule mechanical studies to unfold and refold upon stretch-release. Here we asked whether the mechanical properties of Ig domains and/or other titin regions could be responsible for the viscoelasticity of nonactivated skeletal-muscle sarcomeres, particularly for stress relaxation and force hysteresis. We show that isolated psoas myofibrils respond to a stretch-hold protocol with a characteristic force decay that becomes more pronounced following stretch to above 2.6-μm sarcomere length. The force decay was readily reproducible by a Monte Carlo simulation taking into account both the kinetics of Ig-domain unfolding and the worm-like-chain model of entropic elasticity used to describe titin’s elastic behavior. The modeling indicated that the force decay is explainable by the unfolding of only a very small number of Ig domains per titin molecule. The simulation also predicted that a unique sequence in titin, the PEVK domain, may undergo minor structural changes during sarcomere extension. Myofibrils subjected to 1-Hz cycles of stretch-release exhibited distinct hysteresis that persisted during repetitive measurements. Quick stretch-release protocols, in which variable pauses were introduced after the release, revealed a two-exponential time course of hysteresis recovery. The rate constants of recovery compared well with the refolding rates of Ig-like or fibronectin-like domains measured by single-protein mechanical analysis. These findings suggest that in the sarcomere, titin’s Ig-domain regions may act as entropic springs capable of adjusting their contour length in response to a stretch.

Kettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and α-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM–I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with μ-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin-null mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.

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.

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.

Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive disease with poor prognosis. Treatment with gemcitabine, the FOLFIRINOX scheme or nab-paclitaxel offer only a modest increase in overall survival. For a number of other carcinomas, tumor subtypes have been uncovered that allow the use of targeted therapies. Although subtypes of PDAC were described, this malignancy is clinically still treated as a single disease. We established patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identified two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by immunohistochemistry. Patients bearing tumors of these subtypes show significant differences in overall survival and their tumors differ in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. The xenobiotic enzyme, cytochrome P450 3A5 (CYP3A5), metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Additionally, retrospective analysis of a large patient cohort confirmed that CYP3A5 is predominantly found in those patient tumors classified as exocrine-like. Whereas the hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. Interfering with these regulatory mechanisms may provide an alternative approach to suppress the CYP3A5 pathway. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and is highly expressed in several additional malignancies. These findings designate CYP3A5 as predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance. Citation Format: Elisa M. Noll, Christian Eisen, Albrecht Stenzinger, Elisa Espinet, Alexander Muckenhuber, Corinna Klein, Vanessa Vogel, Bernd Klaus, Wiebke Nadler, Christoph Rösli, Christian Lutz, Michael Kulke, Jan Engelhardt, Franziska Zickgraf, Octavio Espinosa, Matthias Schlesner, Xiaoqi Jiang, Annette Kopp-Schneider, Peter Neuhaus, Marcus Bahra, Bruno V. Sinn, Roland Eils, Nathalia A. Giese, Thilo Hackert, Oliver Strobel, Jens Werner, Markus W. Büchler, Wilko Weichert, Andreas Trumpp, Martin R. Sprick.{Authors}. CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B77.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with poor prognosis. Treatment with gemcitabine, the FOLFIRINOX scheme or nab-paclitaxel offer only a modest increase in overall survival. For a number of other carcinomas, tumor subtypes have been uncovered that allow the use of targeted therapies. Although subtypes of PDAC were described, this malignancy is clinically still treated as a single disease. We established patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identified two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by immunohistochemistry. Patients bearing tumors of these subtypes show significant differences in overall survival and their tumors differ in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. The xenobiotic enzyme, cytochrome P450 3A5 (CYP3A5), metabolizes these compounds in tumor cells of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Additionally, retrospective analysis of a large patient cohort confirmed that CYP3A5 is predominantly found in those patient tumors classified as exocrine-like (Noll, Eisen et al., Nature Medicine (2016) accepted). Whereas the hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. Interfering with these regulatory mechanisms may provide an alternative approach to suppress the CYP3A5 mediated resistance pathway. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC in vitro and in vivo. Finally, CYP3A5 is highly expressed in several additional malignancies including hepatocellular and cervical carcinomas raising the possibility that the CYP3A5 resistance mechanism is operational in a variety of human cancers. These findings designate CYP3A5 as predictor of therapy response and as a tumor cell-autonomous detoxification mechanism that must be overcome to prevent drug resistance. Citation Format: Elisa M. Noll, Christian Eisen, Albrecht Stenzinger, Elisa Espinet, Alexander Muckenhuber, Corinna Klein, Vanessa Vogel, Bernd Klaus, Wiebke Nadler, Christoph Rösli, Christian Lutz, Michael Kulke, Jan Engelhardt, Franziska Zickgraf, Octavio Espinosa, Matthias Schlesner, Xiaoqi Jiang, Annette Kopp-Schneider, Peter Neuhaus, Marcus Bahra, Bruno Sinn, Roland Eils, Nathalia Giese, Thilo Hackert, Oliver Strobel, Jens Werner, Markus W. Büchler, Wilko Weichert, Andreas Trumpp, Martin R. Sprick. CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma. [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 LB-120.

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 naïve 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.

PDAC is a highly aggressive disease with dismal prognosis [1, 2]. Despite extensive research and the discovery of several drug candidates, little progress has been reported since the approval of gemcitabine and erlotinib [1]. Moreover, recent trials with targeted therapies have shown only limited or no benefit [1, 2]. For a number of other carcinomas, tumor subclasses have been uncovered that allow the use of targeted therapies. The mutational landscape of PDAC is complex and heterogeneous, raising the question whether subclasses also exist in PDAC [3]. Collisson et al. described three PDAC subtypes that were identified based on their gene-expression profiles: The classical, the quasi-mesenchymal and the exocrine-like subtype [4]. However, not all subtypes could be identified in the previously available model systems. We have established a novel patient-derived model system that allows the analysis of these three human PDAC subtypes in vitro and in vivo. Hence, we provide a systematic workflow to propagate human PDAC in orthotopic xenografts and to derive tumor-initiating primary cell lines of all three PDAC subtypes. HNF-1 and Keratin 81 were identified as markers for subtype stratification by immunohistochemistry. Application of this two-marker set on a 258 large patient cohort confirmed a predominantly non-overlapping staining and revealed a significant difference in overall survival across the three subtypes. Furthermore, a drug screen uncovered subtype-specific drug sensitivities towards a number of drugs, including gemcitabine and dasatinib. Notably, the exocrine-like subtype was resistant towards all compounds tested. Thus, we aimed to identify the underlying cause of the observed drug resistance. Molecular analysis including gene set enrichment analysis (GSEA) allowed us to identify a putative novel mechanism of drug resistance. Analysis by qRT-PCR and Western blot demonstrated the enhanced expression of several genes mediating this mechanism particularly in the exocrine-like subtype in vitro and in vivo. These findings led to the identification of a novel protein target central to this mechanism. Additionally, retrospective immunohistochemical analysis of a large patient cohort confirmed that this target is predominantly found in those patient tumors classified as exocrine-like. Hence, we hypothesized that the observed strong activation of this mechanism in the exocrine-like PDAC subtype could be responsible for the drug resistance observed in this subclass. In line with this, functional inhibition of this mechanism resulted in increased drug sensitivity in the exocrine-like subtype. Hence, our findings may ultimately advance personalized treatment by applying novel marker-based patient selection strategies in combination with tailored drug use, a strategy which will be presented in more detail at the conference. [1] Hidalgo, M. Pancreatic cancer. The New England journal of medicine. 362, 1605-1617, doi:10.1056/NEJMra0901557 (2010). [2] Vincent, A., Herman, J., Schulick, R., Hruban, R. H. & Goggins, M. Pancreatic cancer. Lancet. 378, 607-620, doi:10.1016/S0140-6736(10)62307-0 (2011). [3] Jones, S. et al. Core signalling pathways in human pancreatic cancers revealed by global genomic analyses. Science. 321, 1801-1806, doi:10.1126/science.1164368 (2008). [4] Collisson, E. A. et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nature medicine. 17, 500-503, doi:10.1038/nm.2344 (2011). Citation Format: Elisa M. Noll, Christian Eisen, Elisa Espinet, Vanessa Vogel, Corinna Klein, Albrecht Stenzinger, Franziska Zickgraf, Peter Neuhaus, Marcus Bahra, Bruno V. Sinn, Christian Lutz, Michael Kulke, Andreas Pahl, Nathalia A. Giese, Oliver Strobel, Jens Werner, Wilko Weichert, Andreas Trumpp, Martin R. Sprick. A novel mechanism mediates drug resistance in the exocrine-like pancreatic ductal adenocarcinoma (PDAC) subtype. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A69.

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

The giant muscle protein titin contains a unique sequence, the PEVK domain, the elastic properties of which contribute to the mechanical behavior of relaxed cardiomyocytes. Here, human N2-B–cardiac PEVK was expressed in Escherichia coli and tested—along with recombinant cardiac titin constructs containing immunoglobulin-like or fibronectin-like domains—for a possible interaction with actin filaments. In the actomyosin in vitro motility assay, only the PEVK construct inhibited actin filament sliding over myosin. The slowdown occurred in a concentration-dependent manner and was accompanied by an increase in the number of stationary actin filaments. High [Ca 2+ ] reversed the PEVK effect. PEVK concentrations ≥10 μg/mL caused actin bundling. Actin-PEVK association was found also in actin fluorescence binding assays without myosin at physiological ionic strength. In cosedimentation assays, PEVK-titin interacted weakly with actin at 0°C, but more strongly at 30°C, suggesting involvement of hydrophobic interactions. To probe the interaction in a more physiological environment, nonactivated cardiac myofibrils were stretched quickly, and force was measured during the subsequent hold period. The observed force decline could be fit with a three-order exponential-decay function, which revealed an initial rapid-decay component (time constant, 4 to 5 ms) making up 30% to 50% of the whole decay amplitude. The rapid, viscous decay component, but not the slower decay components, decreased greatly and immediately on actin extraction with Ca 2+ -independent gelsolin fragment, both at physiological sarcomere lengths and beyond actin-myosin overlap. Steady-state passive force dropped only after longer exposure to gelsolin. We conclude that interaction between PEVK-titin and actin occurs in the sarcomere and may cause viscous drag during diastolic stretch of cardiac myofibrils. The interaction could also oppose shortening during contraction.

The isolation and identification is described of MHC class II-bound peptides derived from Langerhans cells. A combination of preparative micro-HPLC, MALDI-MS, Edman degradation was used for determining the amino acid sequence of MHC-associated peptides. Sample handling was crucial because fractions containing trace amounts of material require immediate storage at −80 °C to prevent peptide losses.