Despite evidence of cancer immune-surveillance, which plays a key role in tumor rejection, cancer cells can escape immune recognition through different mechanisms. Thus, evasion to Natural killer (NK) cell-mediated anti-tumor activity is commonly described and is mediated by various mechanisms, mainly cancer cell-induced down-regulation of NK-activating receptors (NCRs, NKG2D, DNAM-1, and CD16) as well as up-regulation of inhibitory receptors (killer-cell immunoglobulin-like receptors, KIRs, NKG2A). Alterations of NK cells lead to an impaired recognition of tumor cells as well as a decreased ability to interact with immune cells. Alternatively, cancer cells downregulate expression of ligands for NK cell-activating receptors and up-regulate expression of the ligands for inhibitory receptors. A better knowledge of the extent and the mechanisms of these defects will allow developing pharmacological strategies to restore NK cell ability to recognize and lyse tumor cells. Combining conventional chemotherapy and immune modulation is a promising approach likely to improve clinical outcome in diverse neoplastic malignancies. Here, we overview experimental approaches as well as strategies already available in the clinics that restore NK cell functionality. Yet successful cancer therapies based on the manipulation of NK cell already have shown efficacy in the context of hematologic malignancies. Additionally, the ability of cytotoxic agents to increase susceptibility of tumors to NK cell lysis has been studied and may require improvement to maximize this effect. More recently, new strategies were developed to specifically restore NK cell phenotype or to stimulate NK cell functions. Overall, pharmacological immune modulation trends to be integrated in therapeutic strategies and should improve anti-tumor effects of conventional cancer therapy.

Given their recognized ability to kill acute myeloid leukemia (AML) blasts both in vitro and in vivo, Vg9Vd2 T cells are of growing interest in the design of new strategies of immunotherapy. We show that the Butyrophilin3A (BTN3A, CD277) subfamily is a critical determinant of Vg9Vd2 TCR-mediated recognition of human primary AML blasts ex vivo. Moreover, anti-BTN3A 20.1 agonist monoclonal antibodies (mAbs) can trigger BTN3A on AML blasts leading to further enhanced Vg9Vd2 T cell-mediated killing, but this mAb had no enhancing effect upon NK cell-mediated killing. We show that monocytic differentiation of primary AML blasts accounts for their AminoBisphosphonate (N-BP)-mediated sensitization to Vg9Vd2 T cells. In addition, anti-BTN3A 20.1 mAbs could specifically sensitize resistant blasts to Vg9Vd2 T cells lysis and overcome the poor effect of N-BP treatment on those blasts. We confirmed the enhancement of Vg9Vd2 T cells activity by anti-BTN3A 20.1 mAb using a human AML xenotransplantation mouse model. We showed that anti-BTN3A 20.1 mAb combined with Vg9Vd2 T cells immunotherapy could increase animal survival and decrease the leukemic burden in blood and bone marrow. These findings could be of great interest in the design of new immunotherapeutic strategies for treating AML.

Universal CAR T-cell therapies are poised to revolutionize cancer treatment and to improve patient outcomes. However, realizing these advantages in an allogeneic setting requires universal CAR T-cells that can kill target tumor cells, avoid depletion by the host immune system, and proliferate without attacking host tissues. Here, we describe the development of a novel immune-evasive universal CAR T-cells scaffold using precise TALEN-mediated gene editing and DNA matrices vectorized by recombinant adeno-associated virus 6. We simultaneously disrupt and repurpose the endogenous TRAC and B2M loci to generate TCRαβ- and HLA-ABC-deficient T-cells expressing the CAR construct and the NK-inhibitor named HLA-E. This highly efficient gene editing process enables the engineered T-cells to evade NK cell and alloresponsive T-cell attacks and extend their persistence and antitumor activity in the presence of cytotoxic levels of NK cell in vivo and in vitro, respectively. This scaffold could enable the broad use of universal CAR T-cells in allogeneic settings and holds great promise for clinical applications.

NKp46 is a major determinant of natural killer (NK) cell function and it is implicated in tumor immune surveillance in acute myeloid leukemia (AML). The purpose of this study was to investigate the prognostic significance of NKp46 expression in an independent cohort of patients with AML, and to investigate the impact of NKp46 on clinical outcome after allogeneic stem cell transplantation (allo-SCT).NKp46 expression was assessed at diagnosis on NK cells by flow cytometry (N = 180 patients). Clinical outcome was evaluated with regard to NKp46 expression. Patients with NKp46high phenotype at diagnosis had better progression-free survival (PFS) and overall survival (OS) than patients with NKp46low phenotype (74.3% vs. 46.6%, p = 0.014; 82.6% vs. 57.1%, p = 0.010, respectively). In multivariate analysis, high NKp46 was an independent factor for improved OS (HR = 0.409, p = 0.010) and PFS (HR = 0.335, p = 0.011). Subgroup analysis revealed that allo-SCT had a favorable impact on PFS in patients with NKp46high phenotype (p = 0.025). By contrast, allo-SCT did not impact PFS in patients with low NKp46 expression (p = 0.303).In conclusion, we validate the prognostic value of NKp46 expression at diagnosis in AML. However, the prognostic value of NKp46 expression is limited to patients treated with allo-SCT, thus suggesting that NKp46 status may be predictive for allo-SCT responsiveness.

Immunomodulatory drugs (IMiDs) are anticancer drugs with immunomodulatory, anti-angiogenesis, anti-proliferative, and pro-apoptotic properties. IMiDs are currently used for the treatment of multiple myeloma, myelodysplastic syndrome, and B-cell lymphoma; however, little is known about efficacy in acute myeloid leukemia (AML). We proposed in this study to investigate the relevance of IMiDs therapy for AML treatment. We evaluated the effect of IMiDs on primary AML blasts (n = 24), and the impact in natural killer (NK) cell-mediated immunosurveillance of AML. Using primary AML cells and an immunodeficient mouse leukemia xenograft model, we showed that IMiDs induce AML cell death in vitro and impair leukemia progression in vivo. In addition, treatment of AML blasts with IMiDs resulted in enhanced allogeneic NK cell anti-leukemia reactivity. Treatment by pomalidomide of AML blasts enhanced lysis, degranulation, and cytokine production by primary allogeneic NK cells. Furthermore, the treatment with lenalidomide of patients with myeloid malignancies resulted in NK cell phenotypic changes similar to those observed in vitro. IMiDs increased CD56 and decreased NKp30, NKp46, and KIR2D expression on NK cells. Finally, AML blasts treatment with IMiDs induced phenotypic alterations including downregulation of HLA-class I. The effect of pomalidomide was not correlated with cereblon expression and A/G polymorphism in AML cells. Our data revealed, a yet unobserved, dual effects on AML affecting both AML survival and their sensitivity to NK immunotherapy using IMiDs. Our study encourages continuing investigation for the use of IMiDs in AML, especially in combination with conventional therapy or immunotherapy strategies.

Clinical trials with SRC family kinases (SFKs) inhibitors used alone or in a combination with anti-CD20 monoclonal antibodies (mAbs) are currently underway in the treatment of B-cell tumors. However, molecular interactions between these therapeutics have not been studied so far. A transcriptional profiling of tumor cells incubated with SFKs inhibitors revealed strong downregulation of MS4A1 gene encoding CD20 antigen. In a panel of primary and established B-cell tumors we observed that SFKs inhibitors strongly affect CD20 expression at the transcriptional level, leading to inhibition of anti-CD20 mAbs binding and increased resistance of tumor cells to complement-dependent cytotoxicity. Activation of the AKT signaling pathway significantly protected cells from dasatinib-triggered CD20 downregulation. Additionally, SFKs inhibitors suppressed antibody-dependent cell-mediated cytotoxicity by direct inhibition of natural killer cells. Abrogation of antitumor activity of rituximab was also observed in vivo in a mouse model. Noteworthy, the effects of SFKs inhibitors on NK cell function are largely reversible. The results of our studies indicate that development of optimal combinations of novel treatment modalities with anti-CD20 mAbs should be preceded by detailed preclinical evaluation of their effects on target cells.

Siglec-9 is a MHC class I-independent inhibitory receptor expressed on NK and myeloid cells (including dendritic cells, monocytes and neutrophils). Its ligands are sialic acid-containing carbohydrates which are over-expressed on various tumor types compared to normal tissues (1). Sialylation of tumor cells is involved in tumor cell malignancy and is reported for decades as a mechanism of escape from immune surveillance (2). The loss of beta-2-microglobulin, an essential component of MHC class I antigen presentation, was recently described as a common mechanism of resistance to checkpoint blockade in clinical trials and revealed the need for MHC class I-independent therapies (3-4). Thus, Siglec-9-sialic acid interaction disruption may promote anti-tumor immunity independently of MHC class I expression by tumors. Here, we describe the discovery and characterization of first-in class anti-human Siglec-9 antibodies as new checkpoint blockade therapy in a wide range of cancers. Antibodies were discovered that efficiently block the interaction between Siglec-9 and its ligands. Epitope mapping revealed that antibodies bind to distinct epitopes on Siglec-9 near the sialic acid binding site. In vitro assays showed that they potently reverse inhibitory functions of Siglec-9 on NK cells leading to subsequent sialic acid-expressing tumor cell killing. Interestingly, Siglec-9 is enhanced on both CD4+ and CD8+ T cells from RCC, melanoma and NSCLC PBMC patients suggesting a putative additional role on adaptive immunity. Siglec-9 was also co-expressed with other inhibitory receptors on NK cells and combination with other immune checkpoint blockers in in vitro assays is ongoing. The antibodies displaying the most interesting features were successfully humanized. (1) Pathol Oncol Res. 2016 Jul;22(3):443-7 (2) Nature. 1968 Jun 29;218(5148):1254-5 (3) Nat Commun. 2017 Oct 26;8(1):1136 (4) N Engl J Med. 2016 Sep 1;375(9):819-29 Citation Format: Olivier Bénac, Marion Gaudin, Mélody Ors, Aude Le Roy, Hélène Rispaud Blanc, Caroline Soulas, Stéphanie Chanteux, Benjamin Rossi, Laurent Gauthier, Carine Paturel, Yannis Morel, Ivan Perrot, Stéphanie Cornen. Preclinical development of first-in-class antibodies targeting Siglec-9 immune checkpoint for cancer immunotherapy [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 2713.

Regulatory T cells (Treg) are an immunosuppressive subtype of CD4+ T cells essential for maintaining self-tolerance in physiological settings. Tregs also abundantly infiltrate inflamed tumor tissues, impeding the host’s antitumor immune response and contributing to tumor growth and metastasis. In breast cancers, subsets of Tregs express highly immunosuppressive effector phenotypes that favor tumorigenesis, progression, and resistance to immune-checkpoint inhibitor therapies. Tregs share phenotypic features with cytotoxic lymphocytes, rendering them difficult to inhibit without compromising productive anti-tumor immunity. In addition, systemic targeting of Tregs causes serious autoimmune adverse events (SAAE) in cancer patients. Hence, the identification of candidate targets or methodologies allowing the specific elimination of tumor antigen-specific Tregs, including tumor-infiltrating Tregs, is a prerequisite for developing efficient and safe combinatorial immunotherapeutic strategies in breast cancers. To date, numerous preclinical studies have demonstrated that specific targeting of breast tumor-infiltrating Tregs restores a competent antitumor immune response and improves responses to immune-checkpoint inhibitors such as PD-1/PD-L1 blockade. Herein, we discuss major candidate molecules for Treg-targeted therapeutic strategies in breast cancers, detailing the pros and cons of various approaches including monoclonal antibody (mAb)-mediated depletion, homeostasis destabilization, and functional blockade.