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Background. Colorectal cancer (CRC) is the third most common cancer and remains a large unmet need. Dysregulation of Wnt and receptor tyrosine kinase (RTK) signalling pathways are important oncogenic driving events in CRC. Due to this dysregulation, Wnt target genes are expressed at higher levels in CRC particularly in tumor initiating cells. We previously performed an unbiased screen of bispecific antibodies (bAbs) targeting Wnt and RTK targets that resulted in the selection of MCLA-158. Methods. A cohort of 32 genetically and transcriptionally annotated patient-derived colorectal cancer and normal colon organoids were used to functionally characterize responses to antibodies based on morphological changes with high content 3D imaging. Binding affinity was measured by surface plasma resonance and cell based assays. The antibody binding epitopes were mapped by shotgun mutagenesis and FACS based screening. Ligand (R-spondin or EGF) blocking activity was measured in vitro by competition for ligand binding or functional inhibition of ligand dependent growth. In vivo activity was evaluated in xenograft models generated from organoids subcutaneously implanted into immunocompromised mice. Safety was evaluated via once weekly intravenous administration of MCLA-158 to cynomolgus monkeys for 4 weeks and monitoring for pathological changes. Results. MCLA-158, an ADCC enhanced common light chain IgG1 bispecific antibody, binds in domain III of EGFR and in the N-Cap/1st LRR of LGR5, both ligand binding regions, however, only EGF binding was blocked by MCLA-158. MCLA-158 demonstrated inhibitory activity in 74% of tumor organoids independent of KRAS mutational status but was not active on organoids of the cohort harboring both KRAS and PIK3CA mutations. MCLA-158 was significantly more active on organoids derived from tumors than from normal tissue in contrast to cetuximab, which demonstrated equivalent activity on both (range 20-100 fold, n=4). In vivo activity was evaluated against tumor organoids with different KRAS mutation status shown to be sensitive to MCLA-158 in vitro. In all cases, MCLA-158 significantly inhibited the growth of the tumor compared to both control and cetuximab treatment. Inhibitors of both the Wnt and EGFR pathways have shown significant toxicity in humans. An initial evaluation of MCLA-158 toxicity in cynomolgus monkeys did not demonstrate any pathological finding after repeated dosing at 25mg/kg. Conclusions. MCLA-158 demonstrates superior activity compared to reference antibodies in both in vitro and in vivo tumor organoid based assays regardless of KRAS status and was well tolerated in non-human primates. These preclinical data suggest MCLA-158 could benefit patients with metastatic CRC and warrant clinical evaluation. Citation Format: Rob Roovers, Bram Herpers, Mark James, Berina Eppink, Carme Cortina, David Maussang-Detaille, Ingrid Kolfschoten, Sylvia Boy, Marc van de Wetering, Wim De Lau, Robert Doornbos, Kuan Yan, Lucia Salinaro, Lex Bakker, john de Kruif, Hans Clevers, Robert Vries, Eduard Batlle, Leo Price, Mark Throsby. Preclinical evaluation of MCLA-158: A bispecific antibody targeting LGR5 and EGFR using patient-derived colon carcinoma organoids [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 32. doi:10.1158/1538-7445.AM2017-32

Background: MCLA-128 is an ADCC-enhanced humanized common light chain bispecific IgG1 antibody that targets the HER2:HER3 dimer with nanomolar affinity, potently inhibiting tumor growth in vitro and in vivo. MCLA-128 shows superior activity to the combination trastuzumab/ pertuzumab and HER3 targeting monoclonal antibodies and is currently being evaluated in a Phase I clinical trial. This study investigated the mechanism of action of MCLA-128. Methods: Phosphorylation of HER receptors and downstream signaling molecules was studied in vitro and in vivo on HER2-amplified cancer cell lines by Pathscan arrays, luminex beads and Western blot analysis. Inhibition of MCLA-128 cell growth in combination with tyrosine kinase inhibitors and small molecules targeting the MAPK and PI3 kinase/Akt pathway was determined by proliferation inhibition and high content imaging assays. The potential effect of MCLA-128 on primary cardiomyocytes in the presence of Doxorubicin was analyzed by measuring ATP. Binding of MCLA-128 to a panel of cell lines in comparison to HER2 and HER3 antibodies was determined by FACS. Results: In contrast to other HER2 and HER3 targeted agents, only MCLA-128 inhibited phosphorylation of HER3 and downstream Akt and ERK in HER2 amplified cell lines cultured with high concentrations of heregulin in vitro. In xenograft studies, growth inhibition of the trastuzumab-resistant cell line JIMT-1 by MCLA-128 was correlated with reduced HER2:HER3 dimerization and a profound inhibition of the PI3K pathway. Synergistic growth inhibition in vitro was observed when tyrosine kinase inhibitors or inhibitors of the PI3K pathway were added to HER2 amplified cancer cells in the presence of MCLA-128. MCLA-128 did not show any evidence of cardiotoxicity in vitro in contrast to trastuzumab. MCLA-128 binds and coats breast cancer cell lines with differing levels of HER2 expression more efficiently in comparison to monospecific HER2 or HER3 monoclonal antibodies. Conclusions: The unique simultaneous targeting of MCLA-128 to HER2 and HER3 on HER2-overexpressing breast cancer cells leads to severe impairment of PI3K signaling and reduced cell growth whereas proliferation of primary cardiomyocytes is unaffected. The enhanced coating effect of MCLA-128 also supports its ADCC activity. Citation Format: Cecile Geuijen, Eric Rovers, Tristan Gallenne, David Maussang-Detaille, Arjen Kramer, Nellie Nieuwenhuizen, Carina Clements, Katinka van Zoest, Roy Nijhuis, Therese Visser, Renate Den Blanken-Smit, Willem Bartelink, Vanessa Zondag-van der Zande, Linda Kaldenberg, Pieter-Fokko van Loo, Rob Roovers, Robert Doornbos, Leo Price, Stefan Braam, Setareh van Driel, Lex Bakker, Ton Logtenberg, John de Kruif, Mark Throsby. Mechanism of action of MCLA-128, a humanized bispecific IgG1 antibody targeting the HER2:HER3 heterodimer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-261. doi:10.1158/1538-7445.AM2015-LB-261