The activation of human Vδ2 γδ T cells by phosphoantigens (pAg) strictly depends on transmembrane butyrophilin (BTN) molecules, specifically BTN3A isoforms and BTN2A1. Several bacteria, including M. tuberculosis, produce potent pAg and thus trigger a strong activation of Vδ2 T cells. The antigen-specific activation of CD4 and regulatory (Treg) T cells can be monitored by the rapid upregulation of CD154 and CD137, respectively. We have previously established that CD137 is also rapidly upregulated on Vδ2 T cells upon stimulation with pAg. In the present study, we have used antagonistic anti-BTN3A/2A1 antibodies to dissect the pAg-dependent and pAg-independent activation of Vδ2 T cells by various microbes. While the activation of Vδ2 T cells by pAg and aminobisphosphonate zoledronate was completely blocked by anti-BTN3A/2A1 antibodies, only partial inhibition was observed for activation with M. tuberculosis and other bacteria as analyzed by CD137/CD154 upregulation and intracellular interferon-γ expression. Similarly, anti-TCR antibody 7A5 and Lck inhibitor emodin had only a minimal inhibitory effect on activation by bacteria but strongly reduced pAg activation of Vδ2 T cells. Further studies revealed a crucial role of IL-18 in the BTN/TCR-independent early activation of Vδ2 T cells by bacteria. Neutralizing anti-IL-18 antibodies and inflammasome inhibition did not affect pAg activation of Vδ2 T cells but strongly reduced their activation by bacteria. Our results identify a BTN/TCR-independent but IL-18 and inflammasome-dependent activation pathway of Vδ2 T cells, which might be relevant for the role of Vδ2 T cells during bacterial infections.

Tumor-associated macrophages (TAMs), often adopting an immunosuppressive M2-like phenotype, correlate with unfavorable cancer outcomes. Our investigation unveiled elevated expression of the butyrophilin (BTN)2A1 in M2-like TAMs across diverse cancer types. We developed anti-BTN2A1 monoclonal antibodies (mAbs), and notably, one clone demonstrated a robust inhibitory effect on M2-like macrophage differentiation, inducing a shift toward an M1-like phenotype both in vitro and ex vivo in TAMs from patients with cancer. Macrophages treated with this anti-BTN2A1 mAb exhibited enhanced support for T cell proliferation and interferon-gamma (IFNγ) secretion. Mechanistically, BTN2A1 engagement induced spleen tyrosine kinase (SYK) recruitment, leading to sequential SYK and extracellular signal-regulated kinase (ERK) phosphorylation. Inhibition of SYK or ERK phosphorylation abolished M2 reprogramming upon BTN2A1 engagement. Our findings, derived from an analysis of macrophages from healthy donors and human tumors, underscore the pivotal role of BTN2A1 in immunosuppressive macrophage differentiation and function, offering potential applications in cancer immunotherapy.

Background The NKG2D receptor binds to eight stress-induced ligands (NKG2DL): the major histocompatibility complex class I chain-related A and B (MICA/B) and the UL16 binding protein family (ULBP1–6). These ligands are absent from most normal tissues, but frequently expressed in various types of tumors, making NKG2D a promising tool for cancer immunotherapy. The binding affinity of NKG2D to all its ligands is not completely known, and it is likely that different ligands elicit distinct responses, moreover the high polymorphic nature of MICA/B implies different lytic consequences. Methods We created different chimeric antigen receptor (CAR) T-cells containing the full length human NKG2D fused to the CD3zeta signaling domain (as a type I or II protein), and assessed the interaction of the NKG2D CAR T-cells with: i) cells combining differential levels of multiple-NKG2DLs or ii) cell lines expressing single NGK2DLs. Results We first assessed the NKG2DLs expression in healthy and cancer tissues (from multiple solid tumor indications), where we could confirm expression was limited to cancer tissues (with no expression visible on normal healthy tissues), with differing staining patterns depending on the ligand. Next, we assessed the ability of CAR T-cells to bind to NKG2DLs and activate. Interestingly, while all CAR T-cells activated similarly, the levels of cytokine secretion was not similar between the NKG2DLs assessed, indicating that the NKG2DLs do lead to the same activation pathway but not to the same degree. Furthermore, in all instances the type I NKG2D secreted higher levels of cytokines in comparison to the type II. Next, we assessed cytolytic activity of NKG2D CAR T-cells against cancer cell lines expressing different NKG2DLs combination and could confirm a more pronounced role for MICA/B rather than ULBP1–6. Interestingle, even slight polymorphisms led to clear differences in both cytokine and lytic activity of NKG2D CAR T-cells. While, both type I/II led to potent cytolytic activity, cell persistence and proliferative capacity was clearly enhanced in the type I NKG2D. Conclusions NKG2D-receptor architecture plays a major role in NKG2D CAR T mediated activity. Type I based structure enhanced cellular persistence and proliferation in comparison to type II, independent of the NKG2DLs. Furthermore, while all different polymorphisms of the NKG2DLs led to CAR T activity, the levels of CAR T activity were not similar between the different conditions. Indicating that affinity does most likely plays a role in CAR T-cell mediated activity.

Allogeneic chimeric antigen receptor (CAR) T holds the promise of taking this therapeutic approach to broader patient populations while avoiding the intensive manufacturing demands of autologous cell products. One limitation to delivering an allogeneic CAR T is T-cell receptor (TCR) driven toxicity. In this work, the expression of a peptide to interfere with TCR signaling was assessed for the generation of allogeneic CAR T cells. The expression of a truncated CD3ζ peptide was shown to incorporate into the TCR complex and to result in blunted TCR responses. When coexpressed with a natural killer group 2D (NKG2D) CAR, the allogeneic T cells (called CYAD-101) failed to induce graft-versus-host disease in mouse models while maintaining antitumor activity driven by the CAR in vitro and in vivo. Two clinical grade discrete batches of CYAD-101 cells were produced of single donor apheresis resulting in 48 billion CAR T cells sufficient for the entire dose-escalation phase of the proposed clinical trial. The 2 batches showed high consistency producing a predominantly CD4+ T-cell population that displayed an effector/central memory phenotype with no evidence of exhaustion markers expression. These clinical grade CYAD-101 cells secreted cytokines and chemokines in response to ligands expressing target cells in vitro, demonstrating effector function through the CAR. Moreover, CYAD-101 cells failed to respond to TCR stimulation, indicating a lack of allogeneic potential. This bank of clinical grade, non-gene-edited, allogeneic CYAD-101 cells are used in the alloSHRINK clinical trial (NCT03692429).

Background ICT01, a novel, anti-BTN3A immunotherapeutic mAb for activating g9d2T cells, is currently evaluated in a Phase 1/2a clinical trial in patients with advanced-stage, relapsed/refractory cancer (NCT04243499, EVICTION). ICT01 indirectly activates g9d2 T cells that secrete inflammatory cytokines and migrate into tumors to coordinate antitumor immune responses. Therefore, the baseline number of g9d2 T effector cells constitutes a biomarker of interest and a potential selection criterion for target patients. Methods Full immunophenotyping (cell counts and activation state) was performed by flow cytometry on fresh blood collected pre- and on-treatment. Serum cytokines were monitored at baseline and post-treatment. Tumor biopsies were harvested at baseline and on Day 28, and multiplex IHC coupled with digital pathology was used to quantify g9d2T cell, CD8 T cell, NK cell, and T reg infiltration and activation state Results Baseline circulating g9d2 T cell count was highly variable in solid tumor patients enrolled in the monotherapy arm of EVICTION (median 6918 cell/mL, n=26). Melanoma and colorectal patients displayed respectively the highest (median 42277 cell/mL, n=3) and the lowest (median 3040 cell/mL, n=9) baseline number. During the dose escalation phase, g9d2 T cell activation (CD69+) and migration from the blood was observed 30 min post-ICT01 administration. Serum cytokine levels showed variability within ICT01 dose cohorts. IFNg, TNFa, IL-6 and IL-8 levels post-ICT01 dosing were ICT01 dose dependent and clearly related to baseline number of circulating g9d2 T cells. Activation of peripheral blood NK cells, granulocytes and CD8 T cells was observed post-dosing at ICT01 doses ≥7 mg, which was significantly correlated with baseline g9d2 T cell counts, but not with other immune subsets (Spearman r=0.51, 0.47 and 0.65 for CD69+NK, CD69+CD8 and PD-L1+granulocytes respectively, p<0.05, n=19). Baseline circulating g9d2 T cell count was positively correlated with gdTCR+ T cell density in baseline tumor biopsies (Spearman r=0.76, p=0.0086, n=11). Finally, a trend was observed between baseline g9d2 T cell counts and overall tumor immune cell infiltration and activation post-ICT01 treatment, with 4 patients (out of 13 with available biopsy pairs) with g9d2 T cell counts above the median displaying the highest tumor immune cell infiltration and activation. Conclusions These results suggest the utility of measuring baseline g9d2 T cells as part of the patient selection process for ICT01 clinical trials. Patient enrichment based on this biomarker will be tested in EVICTION expansion arms where a minimum baseline threshold of g9d2 T cells counts will be one of the eligibility criteria. Trial Registration NCT04243499 Ethics Approval The study has obtained Competent Authority and Ethics Committee approvals. Informed consent forms were obtained from all enrolled patients.

Background γ9δ2 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. Methods Flow 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). Results NL-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. Conclusions These 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. References Gentles, 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 Approval All procedures involving animals described in this study have been reviewed and approved by the local ethic committee (CELEAG) and the French Ministry of Research.

Gamma delta T (γδ T) cells are among the most potent cytotoxic lymphocytes. Activating anti–butyrophilin 3A (BTN3A) antibodies prime diverse tumor cell types to be killed by Vγ9Vδ2 T cells, the predominant γδ T cell subset in peripheral circulation, by mechanisms independent of tumor antigen–major histocompatibility complex (MHC) complexes. In this report, we describe the development of a humanized monoclonal antibody, ICT01, with subnanomolar affinity for the three isoforms of BTN3A. We demonstrate that ICT01-activated Vγ9Vδ2 T cells kill multiple tumor cell lines and primary tumor cells, but not normal healthy cells, in an efficient process requiring approximately 20% target occupancy. We show that ICT01 activity is dependent on BTN3A and BTN2A but independent of the phosphoantigen (pAg)–binding B30.2 domain. ICT01 delays the growth of hematologic and solid tumor xenografts and prolongs survival of NOD/SCID/IL2rγnull (NSG) mice adoptively transferred with human Vγ9Vδ2 T cells. In single- and multiple-dose safety studies in cynomolgus macaques that received up to 100 mg/kg once weekly, ICT01 was well tolerated. With respect to pharmacodynamic endpoints, ICT01 selectively activated Vγ9Vδ2 T cells without affecting other BTN3A-expressing lymphocytes such as αβ T or B cells. A first-in-human, phase 1/2a, open-label, clinical study of ICT01 was thus initiated in patients with advanced-stage solid tumors (EVICTION: NCT04243499; EudraCT: 2019-003847-31). Preliminary results show that ICT01 was well tolerated and pharmacodynamically active in the first patients. Digital pathology analysis of tumor biopsies of a patient with melanoma suggests that ICT01 may promote immune cell infiltration within the tumor microenvironment.

Background gdT-cells are innate-like lymphocytes described as potent killer of cancer cells whose infiltration into tumors is associated with a positive prognosis.1 2 This supports gd T-cells use in cancer immunotherapy. BTN3A, which belongs to the B7-subfamily of Ig proteins, is required for the recognition of malignant or infected cells by human g9d2 T-cells by sensing intracellular accumulation of phosphoantigens.3 ImCheck Therapeutics is developing ICT01, a humanized anti-BTN3A (IgG1, Fc-silenced), g9d2 T-cell-activating antibody for the treatment of patients with solid or hematologic tumors. Methods A complete IND-enabling program was conducted to characterize the preclinical activity and safety of ICT01. ICT01 effects on human and cynomolgus PBMCs were characterized in vitro using flow cytometry. ICT01-mediated killing activity of g9d2 T-cells was assessed using in vitro co-cultures with tumor and non-tumor cells. Immunocompromised mice bearing human tumors and adoptively transferred with human g9d2 T cells were used to assess ICT01 anti-tumor activity in vivo. The PK, PD and safety of intravenous ICT01 (0.1 to 100 mg/kg single- and repeated-dose) were evaluated in Cynomolgus monkeys. Results ICT01 selectively binds to all three BTN3A isoforms with high affinity (<10nM). When assayed in human and cynomolgus PBMCs in vitro, ICT01 promoted a robust and specific activation of g9d2 T-cells as shown by concentration dependent increase in cell surface CD69 and CD25 and cytokines secretion (IFNγ, TNFα). In co-culture experiments, ~20% of target occupancy on tumor cells is sufficient for maximal g9d2 T-cell degranulation (e.g. CD107a/b expression). ICT01-activated g9d2 T-cells continuously and serially kill a wide range of tumor cells in multi-day co-culture conditions. In contrast, non-tumoral BTN3A-expressing B cells, HUVEC and fibroblasts were unaffected. In mouse AML and ovarian cancer models, repeated injections of ICT01 delayed tumor growth and significantly prolonged animal survival. In primates, ICT01 exposure and target engagement was dose-dependent, with all tested doses producing a specific g9d2 T cell activation and trafficking out of the circulation within 1 hour. ICT01 administration was well tolerated with no safety signals observed at doses up to 25 mg/kg/week based on clinical, laboratory, and anatomic pathology parameters. Conclusions The combined in vitro and in vivo pharmacology data provide evidence that ICT01 is an attractive and novel therapeutic approach for enhancing the innate anti-tumor potential of g9d2 T-cells by activating BTN3A. Importantly, ICT01 did not affect healthy BTN3A-expressing cells, and NHP studies confirmed ICT01 safety with a wide therapeutic index. Therefore, ICT01 is being tested in the ongoing EVICTION trial (NCT04243499). Ethics Approval Pseudonymized samples isolated from healthy volunteers’ whole blood by ImCheck Therapeutics under the agreement n° 7173 between ImCheck Therapeutic SAS and EFS PACA (Etablissement Français du Sang Provence-Alpes-cote d’Azur) References Gentles AJ, Newman AM, Liu CL, et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nature Medicine 2015;21(8):938–945. Tosolini M, Pont F, Poupot M, et al. Assessment of tumor-infiltrating TCRVγ9Vδ2 γδ lymphocyte abundance by deconvolution of human cancers microarrays. OncoImmunology. 2017;6(3):e1284723. Harly C, Guillaume Y, Nedellec S, et al. Key implication of CD277/butyrophilin-3 (BTN3A) in cellular stress sensing by a major human γδ T-cell subset. Blood 2012;120(11):2269–2279.

Background gdT-cells are attractive targets for cancer immunotherapy given their strong cytolytic and pro-inflammatory cytokine secretion activities, and the association between tumor infiltration and positive prognosis.1 2 ImCheck Therapeutics is developing ICT01, an anti-human butyrophilin-3A (BTN3A/CD277) mAb specifically activating g9d2 T-cells in a phosphoantigen (pAg)-independent manner. ICT01 is currently in a Phase 1/2a study in solid and hematologic tumors (NCT04243499).IL-2 has been shown to expand g9d2 T-cells in vitro and in non-human primates in presence of pAgs.3 4 5 We wanted to characterize the proliferative effects of combining ICT01 with IL-2 on γ9δ2 T-cells as an approach to potentiate g9d2 T-cell mediated cancer immunotherapy. Methods g9d2 T-cell activation and expansion was assessed in vitro in human PBMCs treated with ICT01±IL-2, and in vivo, in the blood of immunocompromised NCG mice engrafted with 20 × 106 human PBMCs and treated with ICT01 (single IV dose, 5 mg/kg on Day 1) ±IL-2 (0.3MIU/kg IP on Day 1–4). A dose-ranging ICT01 (single IV dose, 1 or 5 mg/kg on Day 1)+IL-2 combination (1 MIU SC QD on Days 1–5) study was conducted in cynomolgus monkeys. Results In PBMCs cultures in vitro, ICT01 selectively activated g9d2 T-cells and IL-2 significantly enhanced ICT01-mediated g9d2 T-cell proliferation, this compartment reaching >50% of T-cells after 8 days of treatment versus ~10% with ICT01 alone. This was confirmed in vivo in mice models. Flow cytometry analysis of mice blood revealed a 5.5-fold increase in human g9d2 T-cell number in the combination groups compared to ICT01 or IL-2 alone treated animals, with g9d2 T-cell frequency reaching ~35% of the CD3+ T-cell compartment. In Cynomolgus, a specific expansion and activation of peripheral g9d2 T-cells from ~1–2% at baseline to up to 30% of T cells 7 days post ICT01 administration was observed. No ICT01 effect was observed on other immune cells. Histopathological examinations revealed a trend towards higher numbers of g9d2 T-cells in several organs in ICT01+IL-2 treated monkeys. There was no evidence for a systemic cytokine release syndrome at any time point. Adverse effects with variable severity were observed, most of them being reversible and commonly associated with IL-2 alone, and not reported in the IND-enabling GLP toxicity study with ICT01 monotherapy at doses up to 100 mg/kg. Conclusions These results demonstrate the ability of ICT01+IL-2 combination to trigger profound γ9δ2 T-cell activation and expansion, suggesting that the clinical combination of ICT01 with a lymphoproliferative cytokine (e.g., IL-2) may be a novel therapeutic approach for cancer patients. Ethics Approval Pseudonymized samples isolated from healthy volunteers: whole blood by ImCheck Therapeutics under the agreement n° 7173 between ImCheck Therapeutic SAS and EFS PACA (Etablissement Francais du Sang Provence-Alpes-cote d’Azur) References Gentles AJ, Newman AM, Liu CL, et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nature Medicine 2015;21(8):938–945. Tosolini M, Pont F, Poupot M, et al. Assessment of tumor-infiltrating TCRVγ9Vδ2 γδ lymphocyte abundance by deconvolution of human cancers microarrays. OncoImmunology 2017;6(3):e1284723. Nada MH, Wang H, Workalemahu G, Tanaka Y, Morita CT. Enhancing adoptive cancer immunotherapy with Vγ2Vδ2 T cells through pulse zoledronate stimulation. Journal for ImmunoTherapy of Cancer 2017;5(1):9. Sicard H, Ingoure S, Luciani B, et al. In Vivo Immunomanipulation of Vγ9Vδ2 T cells with a synthetic phosphoantigen in a preclinical nonhuman primate model. The Journal of Immunology 2005;175(8):5471–5480. Ali Z, Shao L, Halliday L, et al. Prolonged (E)-4-Hydroxy-3-Methyl-But-2-Enyl pyrophosphate-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vγ2Vδ2 T cells in macaques. The Journal of Immunology 2007;179(12):8287–8296.

Background: Anti-tumoral response of Vg9Vd2 T cells requires sensing of phosphoantigens accumulated in malignant cells through binding of butyrophilin 3A(BTN3A). Moreover, an unknown partner located in human Chr6 was shown to be mandatory to BTN3A-mediated Vg9Vd2 T cell activation in murine models. Here, we identified butyrophilin 2A (BTN2A), which is located to Chr6, as a requirement for BTN3A-mediated Vg9Vd2 T cell cytotoxicity against cancer cells. Methods: CRISPR-Cas9-mediated inactivation of BTN2A1/2A2 isoforms was performed in Daudi, K562 and HEK-293T cells. Vg9Vd2 T cells expanded from healthy PBMCs were co-cultured with wild-type or BTN2AKO cells +/- BrHPP (1 µM), HMBPP (0.1 µM) or zoledronate (45 µM), or anti-BTN2A mAb, and Vg9Vd2 T cell degranulation (%CD106ab+ cells), and intracellular TNFa and IFNg assessed after 4h. Mouse T cell hybridoma 53/4 expressing TCRVg9Vd2-MOP were co-cultured overnight with NIH3T3 murine fibroblasts transfected with BTN3A- and/or BTN2A-encoding plasmids +/-HMBPP(10 µM), or increasing doses of HMBPP or anti-BTN3 20.1 mAb. BTN2A transcript expression in normal vs. tumoral tissue was analyzed using GEPIA tool. Anti-BTN2A mAb staining was performed on human samples of primary AML, cervical and pancreatic carcinoma and assessed by flow cytometry. Results: Degranulation and intracellular IFNg/TNFa (n=6) were abolished in Vg9Vd2 T cells co-cultured with BTN2AKO Daudi, K562 and HEK-293T cells compared to wild-type, in all conditions tested including anti-BTN3 20.1. Murine cells do not express no BTN2A1 or BTN3A orthologs and are unable to activate human Vg9Vd2 T cells. Ectopic expression of BTN2A and BTN3A combination but neither BTN2A or BTN3A alone in murine NIH3T3 cells, allows triggering of IL-2 secretion in mouse 53/4-TCRVg9Vd2-MOP reporter cells in presence of HMBPP or 20.1 mAb in dose-dependent manner. Anti-BTN2A mAb was able to suppress Vg9Vd2 T cell degranulation/cytokine secretion against cancer cell lines and activation of mouse 53/4-TCRVg9Vd2-MOP reporter by BTN2A/BTN3A-expressing NIH3T3 in a dose-dependent manner. BTN2A transcript was significantly up-regulated in pancreatic, ovarian and cervical carcinoma vs. normal tissue. Extracellular BTN2A protein was detected in primary hematological and solid tumors. Conclusion: Here, we show that BTN2A is mandatory for BTN3A-mediated function in human Vg9Vd2 T cells. Moreover, concomitant BTN2A and BTN3A expression empowers murine T cells with activation through Vg9Vd2 TCR, opening new roads for mouse models of Vg9Vd2 T cell anti-tumoral responses. We describe an anti-BTN2A able to suppress Vg9Vd2 T cell function, and we show BTN2A expression in primary tumors. These results are relevant for understanding Vg9Vd2 T cell antitumoral immunity triggered by phosphoantigens and amino-bisphosphonates. Disclosures Olive: ImCheck Therapeutics: Consultancy, Equity Ownership, Patents & Royalties; GlaxoSmithKline: Patents & Royalties.

e15042Background: Most CAR T cell therapy relies on autologous cells with potential limitation in quality of the cells. Cells derived from an allogeneic healthy donor may be more consistent in quality but their use raises two issues: the elimination of the donor T cells due to the host versus graft (HvG) response, and graft versus host disease (GvHD) due to the recognition of non-self molecules expressed on recipient cells by the T Cell Receptor (TCR) of donor cells therefore leading to potential safety concerns for the recipient. HvG can be controlled by pre-conditioning of the patient. However, to avoid GvHD, TCR signalling of the donor T cells could be inhibited by means of the expression of a truncated form of CD3z called TIM (TCR inhibitory molecule). Theobjective of that study was to assess TIM functionality in human primary T cells in combination with an NKG2D-based CAR Methods: Peripheral blood mononuclear cells from healthy donors were transduced with the appropriate vectors with subsequent enric...

e15040 Background: The majority of CAR-T cell therapy studies use the patients’ own autologous cells which carry many potential restrictions related to manufacturing such as quantity and quality of cells. Sourcing cells from an allogeneic healthy donor avoids many of these manufacturing issues. However, the recognition of recipient HLA by the T Cell Receptor of the donor T cells drives a graft versus host (GvH) response leading to normal tissue damage thereby limiting the potential of allogenic T cell therapy. Blunting this response is critical for the successful development of a safe allogeneic CAR-T cell therapy. Hypothesis: Inhibition of TCR activity using shRNAs targeting the CD3 complex can reduce or eliminate TCR driven GvH activity. Objective: To identify candidate shRNA that can knockdown CD3 and achieve inhibition of TCR signaling in primary human T cells. Methods: Peripheral blood derived T cells from healthy donors were transduced with the appropriate vector then subjected to puromycin selection for further 48 hours after transduction. On day 8, cells were finally harvested and characterized. Results: There was a systematic decrease of CD3e or CD3z expression at the RNA level in cells transduced respectively with shRNA against CD3e or CD3z chains of the TCR complex in the Jurkat T cell line as well as in human primary T cells This also correlated with a systematic decrease of CD3e and TCRa/b expression at the protein level in cells transduced with either shRNA against CD3e or CD3z Most interestingly shRNA targeting of the CD3 complex drove reduced TCR signaling as determined by reduced levels of cytokine release upon mitogenic stimulation Conclusions: Targeting of the CD3 complex with shRNA reduced the level of CD3 expression in a Jurkat T cell line as well as in primary human T cells. This resulted in decreased in vitro responses of the engineered T cells during mitogenic stimulation, most likely due to impaired TCR assembly and cell surface expression. These studies support the concept of exploiting shRNA technology for allogenic CAR-T cell therapy that using a single vector, non-gene editing approach.

Chimeric Antigen Receptor (CAR) T cells expressing the fusion of the NKG2D protein with CD3ζ (NKG2D-CAR T Cells) acquire a specificity for stress-induced ligands expressed on hematological and solid cancers. However, these stress ligands are also transiently expressed by activated T cells implying that NKG2D-based T cells may undergo self-killing (fratricide) during cell manufacturing or during the freeze thaw cycle prior to infusion in patients. To avoid target-driven fratricide and enable the production of NKG2D-CAR T cells for clinical application, two distinct approaches were investigated. The first focused upon the inclusion of a Phosphoinositol-3-Kinase inhibitor (LY294002) into the production process. A second strategy involved the inclusion of antibody blockade of NKG2D itself. Both processes impacted T cell fratricide, albeit at different levels with the antibody process being the most effective in terms of cell yield. While both approaches generated comparable NKG2D-CAR T cells, there were subtle differences, for example in differentiation status, that were fine-tuned through the phasing of the inhibitor and antibody during culture in order to generate a highly potent NKG2D-CAR T cell product. By means of targeted inhibition of NKG2D expression or generic inhibition of enzyme function, target-driven CAR T fratricide can be overcome. These strategies have been incorporated into on-going clinical trials to enable a highly efficient and reproducible manufacturing process for NKG2D-CAR T cells.

Acute Myeloid Leukemia (AML) is the most common acute leukemia affecting adults characterized by the accumulation of immature myeloblasts in the marrow or peripheral blood. Natural Killer Group 2D (NKG2D) is an activating receptor expressed on Natural Killer (NK) cells and activated CD8+ T cells

Abstract Chimeric antigen receptor (CAR) protein are artificial protein created by the fusion of an extracellular domain targeting one or several cell surface antigens, a transmembrane domain and an intracellular domain responsible for signal initiation and transmission. First-generation CARs consist of an intracellular signaling domain, often a cytoplasmic tail of the CD3ζ chain. The second and third generation include the addition of one or two co-stimulation domains (e.g. cytoplasmic domain of CD28 and/or 4-1BB). NKG2D is a receptor present on the cell surface of NK cells, NK T cells and subgroups of CD8+ T cells. The receptor exists as a dimer that interacts with a co-adaptor protein called DAP10. Upon interaction of NKG2D with one of its ligand (MICA, MICB, and ULBP1-ULBP6), the DAP10 cytoplasmic tail will induce downstream signaling. DAP10 has a similar signaling motif to CD28 and similarly gets phosphorylated and recruits kinases. NKG2D ligands are expressed on a vast majority of solid and liquid tumors and are thus a very attractive target for CAR T cells. In this study, multiple NKG2D constructs were created and compared to one another. The first CAR construct tested (chNKG2D.1) consists of a fusion of full-length wild-type NKG2D and the cytoplasmic domain of CD3ζ. This construct is currently tested in clinical settings. This CAR benefits from the co-stimulatory signal provided by DAP10 and can therefore be considered as a second generation CAR. Next to this, a new chNKG2D was constructed by combining DAP10 overexpression (chNKG2D.1.DAP10). Secondly classic second-generation CARs were created by adding CD28 (chNKG2D.2.1) or 4-1BB (chNKG2D.2.2) co-stimulatory domains to the chNKG2D. Co-expression of DAP10 increased chNKG2D expression at the cell surface of both CD4 and CD8 T cells (although the increase was more pronounced in CD4 T cells). Furthermore, co-cultures of T cells expressing this construct with K562 cells led to similar IFN-γ levels irrelevant of the CD4/CD8 ratio. Other CD4 specific cytokines were highly increased when the ratio was in favor of CD4 positive T cells. Interestingly, the addition of the co-stimulatory domains to the construct (chNKG2D.2.1 and .2.2) did not lead to higher levels of IFN-γ or any of the CD4 cytokines. Next to cytokine release, cytolytic activity was also assessed which interestingly showed no difference in any of the conditions even when the CD4/CD8 cell ratio was different. In conclusion NKG2D CAR T cells only showed increased cytokine release when DAP10 was overexpressed, all other conditions did not lead to any changes when compared to the ‘NKG2D CAR T cells currently tested in the clinic. This implies that NKG2D is optimally costimulated through the DAP 10 co-signaling, and that this co-signaling is a least as potent as traditional CD28 or 4-1BB based co stimulation. Citation Format: Jennifer BOLSEE, Eytan BREMAN, Fanny HUBERTY, Benjamin VIOLLE, Jerome MARIJSSE, Céline JACQUES-HESPEL, Celine MARCHAND, Nancy RAMELOT, Thuy NGUYEN, Julien HOUSSA, David E. GILHAM, Sophie AGAUGUE. NKG2D as a chimeric antigen receptor - DAP 10 provides optimal co stimulation for NKG2D based CARs [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 3583.

Chimeric antigen receptors (CARs) are genetically engineered proteins that combine an extracellular antigen-specific recognition domain with one or several intracellular T-cell signaling domains. When expressed in T cells, these CARs specifically trigger T-cell activation upon antigen recognition. While the clinical proof of principle of CAR T-cell therapy has been established in hematological cancers, CAR T cells are only at the early stages of being explored to tackle solid cancers. This special report discusses the concept of exploiting natural killer cell receptors as an approach that could broaden the specificity of CAR T cells and potentially enhance the efficacy of this therapy against solid tumors. New data demonstrating feasibility of this approach in humans and supporting the ongoing clinical trial are also presented.

In immunotherapy of cancer, different vaccination strategies have been developed and evaluated in clinical trials, e.g. monoclonal antibodies, injection of DNA, peptides, proteins, autologous cells (tumoral cells, dendritic cells (DC) or T cells) processed in vitro or even direct injection of modified viral vectors. THERAVECTYS is a Paris-based, privately-owned, fully integrated discovery & clinical development biotech company, developping lentiviral vectors for human vaccination and immunotherapy applications. THERAVECTYS’ vaccine candidates based on a proprietary lentiviral vector technology enable a direct vaccination mechanism in patients by inducing a broad, intense and long-lasting T cell mediated response. Theravectys’ technology presents several advantages compared to these various vaccination approaches: -Our lentivectors encompasse up to 15kb DNA, the resulting polypeptide hence targeting a combination of different antigens which mobilizes different actors of the anti-tumor immune system-No selection of patients is needed according to HLA haplotype since epitopes are physiologically processed in vivo.-Unlike all other vaccination approaches, this technology allows the transduction of non-dividing cells such as DC, the most potent antigen-presenting cells (APC) of the immune system.-Break of immunotolerance: lentivectors not only provide sufficient amounts of antigens to DCs but also ensure an efficient processing and prolonged presentation of the antigens, an efficient maturation of DCs that possess all the stimulating abilities to induce an efficient activation of T cells-Non replicative and non persistent lentivectors-antigen expressing cells and transduced cells are eliminated by the immune response elicited after injection.-Direct injections in patients, without ex vivo process of autologous cells.-Different pseudotypes of lentivectors are developed to avoid specific immune reaction against the vector during boost injection(s).-Vaccination with lentiviral vectors are more potent and efficient than DNA or peptide or protein vaccination. The phase I/II clinical trial with our first vaccine candidate has been completed in HIV-infected patients. The vaccine is safe, as demonstrated by the clinical trial ongoing in 38 patients (no Serious Adverse Events have been reported during the trial). The interim analysis of the immunological data from the first two cohorts of patients performed by THERAVECTYS demonstrates the ability of the vaccine candidate to elicit multi-specific and poly-functional CD4+ and CD8+ cellular immune responses in vaccinated patients, even at the lowest dose. The interim analysis of this trial supports the potential of the lentiviral vector platform developed by THERAVECTYS for the future development of therapeutic vaccines and immunotherapies in oncology and infectious diseases. As a second indication, THERAVECTYS is developing an anti-HTLV-1 vaccine candidate to treat patients who have developed a virally-induced adult T cell leukemia. This vaccine candidate should enter into the clinics in mid-2015. Other indications in the pipeline are urogenital cancers, triple-negative breast cancer, EBV-induced nasopharyngeal cancers, HPV-induced cancers (cervix, oropharyngeal and anal cancers) and multiple myeloma.