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Introduction: MCLA-128 is as an ADCC-enhanced IgG1 bispecific antibody that targets the HER2:HER3 dimer and is currently being tested in Phase I/II clinical trials. MCLA-128 demonstrates an in vitro potency superior to other anti-HER2 and anti-HER3 antibodies in cells stimulated with high concentrations of heregulin (HRG) thereby overcoming one of the resistance mechanisms of current HER2 therapies. This study investigates the binding mode of MCLA-128 and proof of concept studies in HRG-driven tumor models. Methods: Alanine scanning shotgun mutagenesis was used to map the epitopes of MCLA-128 to HER2 and HER3. Fab fragments of MCLA-128 were crystallized with the soluble extracellular domains of HER2 and HER3. SAXS analysis on the HER2-HER3-MCLA-128 complex was performed to investigate the binding mode of the bispecific antibody in solution. Ligand-induced dimer specificity was investigated with PathHunter® heterodimerization assays. Bispecific anti-HER2xHER3 antibody and its parental anti-HER3 monoclonal antibody were labelled with 64Cu to compare their biodistribution profiles. The efficacy of MCLA-128 in HRG-driven systems was shown in vitro in MDA-MB-175 cells and in vivo in an orthotopic intracranial patient-derived xenograft (PDX) model originating from a breast cancer brain metastasis Results: The shotgun mutagenesis study identified that the bispecific antibody MCLA-128 binds amino acids T144, R166, R181 in HER2 domain I and R426 in HER3 domain III. Crystallographic studies confirmed the involvement of these critical residues and suggested that MCLA-128 locks the HER3 receptor in its ligand-unbound inactive confirmation. SAXS analysis suggests that the bispecific antibody MCLA-128 forms inter-dimer rather than intra-dimer interactions. In vitro, MCLA-128 specifically blocked HRG-induced signaling of HER2:HER3 but not HER2:HER4 heterodimers. Biodistribution of MCLA-128 in a xenograft model of breast cancer showed that the penetration of MCLA-128 in JIMT-1 HER2-amplified tumors is HER2-dependent despite the high affinity of the HER3 Fab arm for its receptor. MCLA-128 efficiently blocked tumor growth of the HRG-driven HER2 (1+) breast cancer cell line MDA-MB-175 in 3D in vitro. Treatment of orthotopically transplanted HER2-amplified breast cancer brain tumors in mice led to 100% survival with MCLA-128, in contrast to 38% and 0% survival in T-DM1 and vehicle treated mice respectively. Conclusion: MCLA-128 targets HER2-positive tumors via its HER2 arm and locks HER3 in an inactive confirmation. The potent anti-proliferative activity of MCLA-128 in vitro and in vivo supports the clinical development of this bispecific HER2xHER3 antibody in HRG-driven tumors. Citation Format: David Maussang-Detaille, Camilla de Nardis, Linda Hendriks, Carina Bartelink-Clements, Eric Rovers, Tristan Gallenne, Robert Doornbos, Lex Bakker, John de Kruif, Ton Logtenberg, Piet Gros, Cecile Geuijen, Mark Throsby. The binding mode of the bispecific anti-HER2xHER3 antibody MCLA-128 is responsible for its potent inhibition of HRG-driven tumorigenesis [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 33. doi:10.1158/1538-7445.AM2017-33

Although chemotherapy regimens induce initial remissions in most acute myeloid leukemia (AML) patients, their long term prognosis is very poor. Novel targeted therapies that effectively eradicate both AML blasts and their progenitors are needed.MCLA-117, a potent human CLEC12AxCD3 bispecific IgG antibody redirects patient's cytotoxic T cells to induce AML tumor lysis. CLEC12A is a myeloid differentiation antigen that is expressed on 90-95% of newly diagnosed and relapsed AML. Moreover, CLEC12A is selectively expressed on leukemic stem cells (LSCs) but not normal early hematopoietic progenitors, including hematopoietic stem cells (HSCs). MCLA-117 is a full length human bispecific IgG that incorporates CH2 region amino acid substitutions to abrogate Fcγ receptor and C1q interactions while retaining its binding to FcRn. MCLA-117 specifically binds to CLEC12A expressing myeloid cells and CD3 expressing T cells, as evaluated on healthy donor samples by flow cytometry. In line with the CLEC12A expression profile within the hematopoietic progenitor compartment, MCLA-117 did not bind the HSCs, nor the common myeloid progenitor cells. As determined by Surface Plasmon Resonance, MCLA-117 had an affinity of 3 nM for human CLEC12A and 177 nM for human CD3. The efficacy of MCLA-117 to induce T cell mediated lysis of CLEC12A+ tumor cells was evaluated in cytotoxicity assays. In co-cultures with resting healthy donor T cells and AML tumor cells MCLA-117 efficiently induced CLEC12A antigen dependent T cell activation, T cell proliferation and tumor target cell lysis. MCLA-117 induced tumor target lysis with an EC50 of 66±37 ng/mL. Upregulation of the activation CD69 marker on CD8 T cells was the most sensitive read-out for the activity of MCLA-117 (EC20 of 11±3 ng/mL). Therefore the MABEL (minimum anticipated biological effect level) concentration for MCLA-117 was defined as 10 ng/mL and is used to calculate a safe starting dose in a planned first in human study. MCLA-117 was able to activate and redirect AML patient-derived T cells to CLEC12APOS tumor cells as potently as that of healthy donor-derived T cells. The efficacy of MCLA-117 to induce lysis of AML blasts by autologous T cells in primary AML bone marrow (BM) samples with low T cell to AML blast ratios (E:T ratios of 1:7-1:80) was examined. AML patient BM samples taken at diagnosis were cultured in medium with low amounts of a cytokine cocktail to support in vitro survival of AML blasts. MCLA-117 induced up to 30-fold T cell expansion after 7-10 days. More importantly, MCLA-117 efficiently induced AML blast lysis (up to 88%) in 6/6 tested primary AML patient BM samples, even at very low effector to target ratios (see figure 1). The cytokine release potential was assessed by incubation of human whole blood and PBMC preparations with MCLA-117. Moderate to strong cytokine release in whole blood was observed (IFNγ, IL-6, IL-8, IL-10 and TNFα) after 24 hours at concentrations of 10,000 and 1,000 ng/mL MCLA-117. At concentration of 100 ng/mL and 10 ng/mL, moderate to low cytokine release and no cytokine release was observed, respectively. In PBMC preparations, moderate to strong IFNγ release was observed after 48 hours at MCLA-117 concentrations of 10,000, 1,000 and 100 ng/mL. At these concentrations, only low release of IL-2, IL-10 and TNFα and no release of IL-1β, IL-6 and IL-8 was observed. MCLA-117 may provide a novel treatment option for all subtypes of AML with highly specific targeting of myeloid blasts and progenitors and sparing the normal HSCs, as well as eradicating residual LSCs residing in the BM niche compartments. A first clinical study is planned to evaluate the safety and preliminary efficacy of MCLA-117 in adult AML patients. Disclosures Van Loo: Merus B.V.: Employment. Doornbos:Merus B.V.: Employment. Shamsili:Merus B.V.: Employment. Bakker:Merus B.V.: Employment.

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

560 Background: Amplification and dimerization of HER2 promotes growth and survival of malignant cells. Tumor responses to available therapeutic agents targeting HER2 are variable. Re-activation of...

With the discovery of a second type of T cells, those expressing T cell receptor (TCR) γδ, the question has arisen: “What contribution do these cells make to the immune system?” We review here how TCR diversity is generated at the molecular level and what this means for the capability of TCR αβ and TCR γδ to recognize a variety of antigenic peptides in the context of conventional and alternative presenting molecules. We will discuss how tissue distribution and repertoire selection of γδ T cells are in part developmentally regulated and how the repertoire may be further shaped by antigenic stimulation. Critical investigation of the causes and consequences of γδ T cell proliferation in human disease situations will hopefully provide insight into the normal functions of this elusive T cell population.