Merus (Netherlands)

Abstract Inflammation in the context of pancreatic injury drives acinar-to-ductal metaplasia (ADM), a cell state that can be hijacked by mutant Kras to drive the formation of pancreatic cancer. ADM has been described alternatively to bear progenitor features, but the extent to which lineage reversion is a broad feature of the response to injury and early tumorigenesis - particularlyacross the different mutant Kras alleles - remains poorly understood. Herein we implement novel Ptf1a-tdTomato mice to characterize early and late pancreatic progenitors to serve as benchmarks that define cell fate decisions in the context of inflammatory injury and initiation. In so doing, we define ADM as a mixed-lineage transcriptomic and epigenetic state, harboring ductal features but also featuring a lineage reversion to a late progenitor program. By contrast, while inducible mutant Kras alone is able to induce oncogenic gene expression, it does little to alter chromatin in the absence of injury. The most frequent Kras mutant alleles - G12D, G12V, and G12R - display differential cooptation of the injury-induced mixed-lineage state, with mutant KrasG12D able to form ADM and PanIN more robustly than either KrasG12V or KrasG12R. In particular, KrasG12R fails to promote phenotypic transformation on its own or even in concert with inflammatory injury. Whereas KrasG12Dand injury induce enhancer reprogramming at AP-1 chromatin and a lineage reversion to resemble early progenitors, KrasG12Rcan initiate but not sustain the key cell-intrinsic transcriptional and epigenetic programs characteristic of neoplasia. In KrasG12R we identify a decrease in Rac1 signaling, not AKT activation, as the key molecular defect. Together, these data demonstrate that the PDAC-associated Kras variants are not functionally equivalent and distinctly drive lineage reversion and epigenetic reprogramming, such that allele-specific approaches will be essential to therapeutic targeting in pancreatic cancer. Citation Format: David Falvo, Adrien Grimont, Rohit Chandwani. Molecular dynamics driving phenotypic divergence among KRAS mutants in pancreatic tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6416.

Therapeutic antibodies have broad indications across diverse disease states, such as oncology, autoimmune diseases, and infectious diseases. New research continues to identify antibodies with therapeutic potential as well as methods to improve upon endogenous antibodies and to design antibodies de novo. On April 27-30, 2022, experts in antibody research across academia and industry met for the Keystone symposium "Antibodies as Drugs" to present the state-of-the-art in antibody therapeutics, repertoires and deep learning, bispecific antibodies, and engineering.

Abstract Neuregulin 1 (NRG1) fusion proteins are oncogenic drivers in multiple cancer types, including non-small cell lung cancer, pancreas adenocarcinoma, and other solid tumors. NRG1 fusion proteins bind to HER3 and signal through HER2/HER3 heterodimers, leading to oncogenic transformation. Zenocutuzumab is an HER2/HER3 humanized IgG1 bispecific antibody that specifically and potently blocks NRG1 fusion-driven signaling of the heterodimeric complex. Zenocutuzumab binds via its anti-HER2 monovalent Fab arm to domain I of HER2. Docking of zenocutuzumab in this position increases the effective affinity of the anti-HER3 Fab arm, which binds to domain 3, a critical epitope that blocks binding of the HER3 ligand NRG1. Consequently, zenocutuzumab displaces NRG1 binding to HER3, even at high ligand concentrations, keeping HER3 in an inactive state and blocking HER2/HER3 heterodimerization and NRG1-mediated signaling. The clinical efficacy and safety of zenocutuzumab were recently demonstrated in patients with NRG1 fusion-driven cancers who were enrolled in the ongoing global multicenter eNRGy study and a global early access program NCT02912949 (J Clin Oncol 39;15_suppl; abstract 3003). Treatment with zenocutuzumab led to rapid and major radiologic tumor regression and biomarker responses in heavily pretreated patients with multiple NRG1 fusion-positive cancers with several different N-terminal fusion partners. Zenocutuzumab has been enhanced for antibody-dependent cell-mediated cytotoxicity (ADCC) to recruit and bolster immune effector cells to aid in tumor elimination. Here we explored the different Fc-mediated immune effector functions of zenocutuzumab. In several cancer cell lines, zenocutuzumab induced Fc-mediated dose-dependent ADCC and antibody-dependent cellular phagocytosis. Insertion of ATP1B1-NRG1 and SLC3A2-NRG1 into immortalized pancreatic ductal epithelial cells (H6c7) led to activation of the HER3 oncogenic signaling pathways including AKT. The H6c7-SLC3A2-NRG1 cells formed tumors when implanted in the subcutaneous flank of immune compromised mice (after about 10 weeks). Furthermore, a dependency of the transformed cell on this oncogene for growth and survival was also observed. Zenocutuzumab effectively inhibited proliferation of these NRG1 fusion-expressing, transformed cells. In addition, proteomic analysis showed that zenocutuzumab inhibited the HER3 and AKT-related downstream signaling pathways. In conclusion, these data provide strong mechanistic evidence supporting the clinical efficacy of zenocutuzumab in patients with NRG1 fusion-driven cancers. Citation Format: Jan Gerlach, Igor Odintsov, Ron Schackmann, Marc Ladanyi, Jeroen Lammerts van Bueren, Romel Somwar, Cecile Geuijen. Zenocutuzumab is an effective HER2/HER3 Biclonics® antibody in cancers with NRG1 fusions [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P201.

Heparan sulfate proteoglycan (HSPG) expression is found in many animal tissues and regulates growth factor signaling such as of Fibroblast growth factors (Fgf), Wingless/Int (Wnt) and Hedgehog (HH). Glypicans, which are GPI (glycosylphosphatidylinositol)-anchored proteins, and transmembrane-anchored syndecans represent two major HSPG protein families whose involvement in development and disease has been demonstrated. Their participation in regenerative processes both of the central nervous system and of regenerating limbs is well documented. However, whether HSPG are expressed in regenerating zebrafish fins, is currently unknown. Here, we carried out a systematic screen of glypican and syndecan mRNA expression in regenerating zebrafish fins during the outgrowth phase. We find that 8 of the 10 zebrafish glypicans and the three known zebrafish syndecans show specific expression at 3 days post amputation. Expression is found in different domains of the regenerate, including the distal and lateral basal layers of the wound epidermis, the distal most blastema and more proximal blastema regions. HSPG expression is prevalent in regenerating zebrafish fins. Further research is needed to delineate the function of glypican and syndecan action during zebrafish fin regeneration.

CD137 (4-1BB) is a transmembrane costimulatory receptor on T and NK cells that enhances adaptive immune responses and is a critical mediator of antitumor immunity. The development of CD137 targeted agents for cancer therapy has been hampered by on-target off-tumor toxicity in the case of agonist monospecific, bivalent mAbs or limited antitumor activity in the case of crosslinking mAbs. Here we have developed an Fc-silenced bispecific IgG1 antibody to CD137 and PD-L1 with monovalent binding specificity to each target. MCLA-145 drives transactivation of CD137 in the vicinity of cells expressing PD-L1, such as in the immunosuppressive tumor microenvironment. The degree of CD137 agonistic activity in T cells correlated with the expression level of PD-L1 on neighboring cells, as demonstrated in transactivation assays whereby reporter T cells were co-cultured with cells expressing different levels of PD-L1. PD-L1 expression as low as 6000 receptors per cell was sufficient to activate CD137 in neighboring T cells. In contrast, MCLA-145 blocked PD-1 signaling without requirement for CD137 binding in a PD-1/PD-L1 reporter assay. CD137 signaling was induced by MCLA-145 in multiple primary human immune cell assays including the mixed lymphocyte reaction, human PBMC, and whole blood SEB stimulation assays. MCLA-145 reversed T cell suppression mediated by M2 macrophages or Tregs, in vitro. In addition, MCLA-145 enhanced Ag-specific expansion and differentiation of human naïve CD8+ T cells in vitro.In vivo, MCLA-145 treatment resulted in significant tumor immune activation and antitumor responses in two separate humanized mouse tumor models. In one model, human T cells expressing NY-ESO specific TCR were adoptively transferred to mice bearing A549 tumors which expressed NY-ESO antigen and human PD-L1. MCLA-145 treatment at 5 mg/kg resulted in 54% tumor growth inhibition (TGI) as compared to T cell only treated mice. In the tumors of MCLA-145 treated mice, the percentage of NY-ESO specific CD8+ T cells were significantly increased compared to controls. In a second model, mice engrafted with human CD34+ cells were implanted with the breast tumor cell line MDA-MB-231. MCLA-145 at 0.5 mg/kg and 5 mg/kg induced significant tumor growth inhibition (55 and 57% respectively) as compared to vehicle control or Fc-silenced huIgG1 controls. Additionally, two out of nine animals in the 5 mg/kg MCLA-145-treated group had complete tumor regression. MCLA-145 increased the number of infiltrating CD8+ T cells, as well as the percentage of central memory CD8+ T cells. The cured animals were then re-challenged with MDA-MB-231 tumor cells, and tumors of previously cured mice were rejected as compared to no growth inhibition in treatment-naïve CD34+ NSG mice. In conclusion, these data support the clinical evaluation of MCLA-145 as a novel, PD-L1 dependent CD137 agonist immune therapy.Citation Format: Patrick Mayes, Paul Tacken, Steve Wang, Pieter-Fokko van Loo, Thomas Condamine, Hans van der Maaden, Eric Rovers, Steef Engels, Floris Fransen, Ashwini Kulkarni, Yao-bin Liu, Arpita Mondal, Leslie Hall, Soyeon Kim, Marina Martinez, Shaun O'Brien, Edmund Moon, Steven Albelda, Peggy Scherle, Gregory Hollis, Reid Huber, Mark Throsby, Cecile A. Geuijen. A bispecific Fc-silenced IgG1 antibody (MCLA-145) requires PD-L1 binding to activate CD137 [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 539.

SummaryIntroduction MCLA-128 is a bispecific monoclonal antibody targeting the HER2 and HER3 receptors. Pharmacokinetics (PK) and pharmacodynamics (PD) of MCLA-128 have been evaluated in preclinical studies in cynomolgus monkeys and mice. The aim of this study was to characterize the PK and PD of MCLA-128 and to predict a safe starting dose and efficacious clinical dose for the First-In-Human study. Methods A PK-PD model was developed based on PK data from cynomolgus monkeys and tumor growth data from a mouse JIMT-1 xenograft model. Allometric scaling was used to scale PK parameters between species. Simulations were performed to predict the safe and efficacious clinical dose, based on AUCs, receptor occupancies and PK-PD model simulations. Results MCLA-128 PK in cynomolgus monkeys was described by a two-compartment model with parallel linear and nonlinear clearance. The xenograft tumor growth model consisted of a tumor compartment with a zero-order growth rate and a first-order dying rate, both affected by MCLA-128. Human doses of 10 to 480 mg q3wk were predicted to show a safety margin of >10-fold compared to the cynomolgus monkey AUC at the no-observed-adverse-effect-level (NOAEL). Doses of ≥360 mg resulted in predicted receptor occupancies above 99% (Cmax and Cave). These doses showed anti-tumor efficacy in the PK-PD model. Conclusions This analysis predicts that a flat dose of 10 to 480 mg q3wk is suitable as starting dose for a First-in-Human study with MCLA-128. Flat doses ≥360 mg q3wk are expected to be efficacious in human, based on receptor occupancies and PK-PD model simulations.

Abstract Background: The high failure rate in cancer drug research has been linked to the poor predictive capacity of in vitro models using 2D cultures of cancer cell lines. Compared to 2D monolayer cultures, 3D cultured tissues show gene expression patterns, differentiation- and functional characteristics which more closely reflect the situation in vivo. Furthermore, patient-derived organoid cultures retain the gene expression profiles and histological characteristics of the original tumor tissues which are often lost during long term selection on tissue culture plastics. Organoid cultures therefore increase the scope for predicting drug responses in patients, discriminating different drug responses and flagging toxicity. We used a high content screening platform and a panel of 40 colorectal cancer organoids to characterize the responses to signaling pathway inhibitors and a panel of 545 bispecific antibodies. These antibodies comprised a HER3 or EGFR targeting arm combined with a LGR4, LGR5, ZNRF3 or RNF43 targeting arm to target stem cells. Active antibodies were rescreened and candidate leads were selected. Results: The broad mutation spectrum of the organoids was reflected in a broad heterogeneity of organoid phenotypes. Some CRC organoids formed well-differentiated spheroids with a single lumen that resembled the phenotype of normal wild type organoids, whereas others had multiple lumens or were poorly differentiated without a luminal cavity. A rich set of morphological features was extracted from 3D image data, including organoid size and shape, planar cell polarity, lumen formation as well as cell number and nucleus shape. Some features, such as those that described lumen formation, were more sensitive in detecting drug treatment than features associated with cell proliferation, improving the sensitivity of the assay to detect active molecules. A set of 10 features was selected to create a drug response profile. We observed that the absence of activating mutations did not always correlate with sensitivity to corresponding pathway inhibitors, underscoring the need for empirical testing of drugs to predict patient sensitivity. The bispecific antibody screen was performed in two stages: a primary screen in three different tumoroid models (18,908 wells) and a validation screen in 25 different tumoroid models (23,040 wells). These screens simultaneously measured morphological alterations associated with growth, differentiation and survival (e.g. apoptosis) and identified a panel of bispecific antibodies that potently inhibited a significant majority of colorectal cancer tumoroid models tested. Conclusions: These results demonstrate that high content screening of CRC organoids is an effective strategy to identify novel inhibitors of CRC tumor outgrowth and enable identification of bispecific antibodies that target colorectal cancer stem cells with different mutational backgrounds. This abstract is also being presented as Poster A35. Citation Format: Bram Herpers, Rob Roovers, Berina Eppink, Marc Van de Wetering, Kuan Yan, Lucia Salinaro, Wim De Lau, Hans Clevers, Robert Vries, Mark Throsby, Leo Price. A 3D image-based phenotypic screen of bi-specific antibodies targeting stem cells in a panel of patient derived colon carcinoma organoids. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr PR05.