ICT01, AN ANTI-BTN3A MAB, AND NL-201, AN ALPHA-INDEPENDENT IL-2/IL-15 AGONIST, COMBINE TO ELICIT A POTENT ANTI-TUMOR RESPONSE BY SYNERGISTICALLY STIMULATING VG9VD2 T CELL ACTIVATION AND PROLIFERATION

Backgroundγ9δ2 T-cells are attractive mediators of cancer immunotherapy due to their strong cytolytic and pro-inflammatory activities and the positive correlation between tumor infiltration and good prognosis [1,2]. ICT01, a novel anti-BTN3A mAb activating γ9δ2 T-cells, is being evaluated in a Phase 1/2a clinical study (NCT04243499)[3,4]. Previous studies have shown that IL-2 (Proleukin®) promotes γ9δ2 T-cells expansion following ICT01 stimulation, which may be clinically useful given that γ9δ2 T-cells are normally <5% of total T-cells [5]. However, the severe toxicity of IL-2 has limited its widespread use. NL-201 is a de novo alpha-independent IL-2/IL-15 agonist that preferentially stimulates CD8 T and NK cell proliferation at low concentrations, enabling a potentially wider therapeutic index than IL-2, and is being evaluated in a Phase 1 clinical study (NCT04659629)[6,7]. Here, we explore the potential of ICT01 and NL-201 to synergistically stimulate the activation and proliferation of γ9δ2 T-cells.MethodsFlow cytometry was used to assess IL-2R signaling (pSTAT5), and γ9δ2 T-cell activation and expansion after in vitro culture of huPBMCs with ICT01, NL201 or the combination. Tumor cell killing activity was monitored upon co-culture of huPBMCs with tumor cell lines (Incucyte). In vivo pharmacology was performed in NCG mice engrafted with 20x106 huPBMCs and treated with ICT01 (1 mg/kg IV)±NL-201 (1, 3 or 10 µg/kg IV). Immune cells were phenotyped by flow cytometry in blood and organs collected at sacrifice (Day 16).ResultsNL-201 is ~100X more potent than IL-2 in triggering IL-2R signaling in γ9δ2 T-cells, without preferential activity on Tregs. NL-201 plus ICT01 induces synergistic expansion of γ9δ2 T-cells, approaching ~50% of T-cells after 8 days versus ~10% with single agents. In addition, the combination of NL-201 and ICT01 promotes γ9δ2 T-cell effector memory differentiation, in contrast to IL-2, which induces primarily central memory phenotype. Importantly, NL-201 enhances ICT01-mediated killing of cancer cells by γ9δ2 T-cells.In mice, a dose-dependent expansion of peripheral γ9δ2 T-cells from ~1–2% at baseline to up to 40% of T-cells was observed in the ICT01+NL-201 combination groups. Consistently, γ9δ2 T-cell number and frequency increase in spleen and lungs of the ICT01+NL-201 treated animals as compared to controls. Expanded γ9δ2 T-cells in the combination groups display an effector memory phenotype, confirming our in vitro results.ConclusionsThese results demonstrate the ability of the ICT01+NL-201 combination to synergistically trigger γ9δ2 T-cell activation, expansion and anti-tumor activity and support clinical evaluation of this combination as a novel therapeutic approach for cancer patients.ReferencesGentles, A. J. et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat Med 21, 938-945, doi:10.1038/nm.3909 (2015).Tosolini, M. et al. Assessment of tumor-infiltrating TCRVgamma9Vdelta2 gammadelta lymphocyte abundance by deconvolution of human cancers microarrays. Oncoimmunology 6, e1284723, doi:10.1080/2162402X.2017.1284723 (2017).Gassart, A. d. et al. 687 Enhancement of anti-tumor immunity by ICT01: a novel g9d2 T cell-activating antibody targeting butyrophilin-3A (BTN3A). Journal for ImmunoTherapy of Cancer 8, A412-A413, doi:10.1136/jitc-2020-SITC2020.0687 (2020).Marabelle, A. et al. 316 EVICTION Study: Preliminary results in solid tumor patients with ICT01, a first-in-class, gamma9 delta2 T cell activating antibody targeting butyrophilin-3A. Journal for ImmunoTherapy of Cancer 8, A194-A195, doi:10.1136/jitc-2020-SITC2020.0316 (2020).Gassart, A. d. et al. 442 ICT01, an anti-BTN3A mAb that activates Vg9Vd2 T cells, plus interleukin-2: a potent and promising combination for cancer immunotherapy. Journal for ImmunoTherapy of Cancer 8, A268-A269, doi:10.1136/jitc-2020-SITC2020.0442 (2020).Walkey, C., Swanson, R., Ulge, U., Silva Manzano, D. A. & Drachman, J. 576 NL-201, a de novo IL-2 and IL-15 agonist, demonstrates enhanced in vivo antitumor activity in combination with multiple cancer immunotherapies. Journal for ImmunoTherapy of Cancer 8, A346-A346, doi:10.1136/jitc-2020-SITC2020.0576 (2020).Walkey, C. D. et al. Abstract 4518: Pre-clinical development of NL-201: A de novo α-independent IL-2/IL-15 agonist. Cancer Research 80, 4518–4518, doi:10.1158/1538-7445.Am2020-4518 (2020).Ethics ApprovalAll procedures involving animals described in this study have been reviewed and approved by the local ethic committee (CELEAG) and the French Ministry of Research.