Novel Association of KLRC4-KLRK1 Gene Polymorphisms with Susceptibility and Progression of Antithyroid Drug-Induced Agranulocytosis.

OBJECTIVE: Antithyroid drug (ATD)-induced agranulocytosis (TIA) is the most serious adverse effect during ATD treatment of Graves' disease (GD). Previously, the MICA gene was reported to be associated with TIA. MICA protein is an important ligand for the NKG2D protein, which is encoded by the KLRK1 gene and KLRC4-KLRK1 read-through transcription. This study further investigated the association between KLRC4-KLRK1 gene polymorphisms and susceptibility to TIA. METHODS: Twenty-eight candidate single nucleotide polymorphisms (SNPs) on KLRC4-KLRK1 read-through transcription were evaluated by the iPLEX MassARRAY system in 209 GD control patients and 38 TIA cases. RESULTS: A significant association of rs2734565 polymorphism with TIA was found (p=0.02, OR=1.80, 95% CI=1.09-2.96). The haplotype C-A-A-C-G, including rs2734565-C, was associated with a significantly higher risk of TIA (p=4.79E-09, OR=8.361, 95% CI=3.737-18.707). In addition, the interval time from hyperthyroidism to agranulocytosis onset was shorter in patients carrying the rs2734565-C allele than in non-carrying groups (45.00 (14.00-6570.00) d vs. 1080.00 (30.00-3600.00) d, p=0.046), and the interval from ATD treatment to agranulocytosis onset was also shorter in patients carrying rs2734565-C allele (29.00 (13.00-75.00) d vs. 57.50 (21.00-240.00) d, p=0.023). CONCLUSIONS: The findings suggest that the KLRC4-KLRK1 gene polymorphism is associated with susceptibility and progression of ATD-induced agranulocytosis. Patients carrying the rs2734565-C allele had a higher susceptibility and faster onset time of TIA.

IL-21 collaborates with anti-TIGIT to restore NK cell function in chronic HBV infection.

Available therapies for chronic hepatitis B virus (HBV) infection are not satisfying, and interleukin-21 (IL-21) and checkpoint inhibitors are potential therapeutic options. However, the mechanism underlying IL-21 and checkpoint inhibitors in treating chronic HBV infection is unclear. To explore whether IL-21 and checkpoint inhibitors promote HBV clearance by modulating the function of natural killer (NK) cells, we measured the phenotypes and functions of NK cells in chronic HBV-infected patients and healthy controls on mRNA and protein levels. We found that chronic HBV infection disturbed the transcriptome of NK cells, including decreased expression of KLRK1, TIGIT, GZMA, PRF1, and increased expression of CD69. We also observed altered phenotypes and functions of NK cells in chronic HBV-infected patients, characterized by decreased NKG2D expression, increased TIGIT expression and impaired interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) production. Furthermore, these alterations cannot be restored by telbivudine treatment but can be partially restored by IL-21 and anti-TIGIT stimulation. IL-21 upregulated the expression of activating receptor CD16, CD69, and NKG2D on NK cells, enhanced IFN-γ production, cytolysis, and proliferation of NK cells, while anti-TIGIT promoted IFN-γ production in CD56dim subset exclusively in chronic HBV infected patients. Additionally, IL-21 was indispensable for anti-TIGIT in HBsAg clearance in mice bearing HBV. It enhanced IFN-γ production in splenic NK cells rather than intrahepatic NK cells, indicating a brand-new mechanism of IL-21 in HBV clearance when combined with anti-TIGIT. Overall, our findings contribute to the design of immunotherapy through enhancing the antiviral efficacy of NK cells in chronic HBV infection.

A Phase 2b, Randomised, Double-blind, Placebo-controlled, Parallel-arm, Multicenter Study Evaluating the Safety and Efficacy of Tesnatilimab in Patients with Moderately to Severely Active Crohn's Disease.

BACKGROUND AND AIMS: Tesnatilimab, a monoclonal antibody targeting NKG2D, was evaluated in Crohn's disease [CD] patients who had failed or were intolerant to biologic or conventional therapy. METHODS: TRIDENT was a phase 2b, two-part, randomised, double-blind, placebo-controlled, parallel-arm, multicenter study. In Part 1 [proof of concept], 145 patients who were biologic intolerant or refractory [Bio-IR] or had not failed biologic therapy [Bio-NF] were randomised in a 1:1 ratio to placebo subcutaneously [SC] or tesnatilimab 400 mg SC. In Part 2 [dose ranging], 243 Bio-IR and Bio-NF patients were randomised in a 1:1:1:1:1 ratio to placebo, tesnatilimab [50 mg, 150 mg, 400 mg], or intravenous infusion of ustekinumab ~6 mg/kg at Week 0 and 90 mg SC at Weeks 8 and 16. The primary endpoint was mean change from baseline in Crohn's Disease Activity Index [CDAI] at Week 8 [Part 1] and Week 12 [Part 2]. Clinical and endoscopic remission/response were evaluated. Efficacy analyses were also assessed by NKG2D and MICB single nucleotide polymorphism [SNP] status [SNP-positive means positive in at least one of two SNPs]. Safety events were summarised. RESULTS: In Part 1, mean change from baseline in CDAI score was significantly greater with tesnatilimab vs placebo at Week 8 [-103.6 vs -60.0; p < 0.01]. In Part 2, no dose-response signal was detected. Mean changes from baseline in CDAI at Week 12 were -93.2, -72.2, and -84.3 for low, middle, and high doses of tesnatilimab, respectively, vs -59.2 for placebo and -148.8 for ustekinumab. Similar reductions from baseline in CDAI score were observed in patients receiving tesnatilimab, regardless of SNP status. Clinical remission rates were greater with tesnatilimab than placebo in Parts 1 and 2, whereas endoscopic response rates were greater with tesnatilimab only in Part 1. No unexpected safety events occurred. CONCLUSIONS: Tesnatilimab was well tolerated. The efficacy of tesnatilimab in patients with CD was significant for the primary endpoint in Part 1; however, no dose-response signal was detected for the primary endpoint in Part 2. Based on these inconsistent findings, tesnatilimab was not considered an effective treatment for patients with CD and no further development is planned. CLINICALTRIALS.GOV IDENTIFIER: NCT02877134.

CYAD-01, an autologous NKG2D-based CAR T-cell therapy, in relapsed or refractory acute myeloid leukaemia and myelodysplastic syndromes or multiple myeloma (THINK): haematological cohorts of the dose escalation segment of a phase 1 trial.

BACKGROUND: CYAD-01 is an autologous chimeric antigen receptor (CAR) T-cell product based on the natural killer (NK) group 2D (NKG2D) receptor, which binds eight ligands that are overexpressed in a wide range of haematological malignancies but are largely absent on non-neoplastic cells. Initial clinical evaluation of a single infusion of CYAD-01 at a low dose in patients with relapsed or refractory acute myeloid leukaemia, myelodysplastic syndromes, and multiple myeloma supported the feasibility of the approach and prompted further evaluation of CYAD-01. The aim of the present study was to determine the safety and recommended phase 2 dosing of CYAD-01 administered without preconditioning or bridging chemotherapy. METHODS: The multicentre THINK study was an open-label, dose-escalation, phase 1 study for patients with relapsed or refractory acute myeloid leukaemia, myelodysplastic syndromes, or multiple myeloma, after at least one previous line of therapy. Patients were recruited from five hospitals in the USA and Belgium. The dose-escalation segment evaluated three dose levels: 3 × 108 (dose level one), 1 × 109 (dose level two), and 3 × 109 (dose level three) cells per infusion with a 3 + 3 Fibonacci study design using a schedule of three infusions at 2-week intervals followed by potential consolidation treatment consisting of three additional infusions. The occurrence of dose-limiting toxicities post-CYAD-01 infusion was assessed as the primary endpoint in the total treated patient population. The trial was registered with ClinicalTrials.gov, NCT03018405, and EudraCT, 2016-003312-12, and has been completed. FINDINGS: Between Feb 6, 2017, and Oct 9, 2018, 25 patients were registered in the haematological dose-escalation segment. Seven patients had manufacturing failure for insufficient yield and two had screening failure. 16 patients were treated with CYAD-01 (three with multiple myeloma and three with acute myeloid leukaemia at dose level one; three with acute myeloid leukaemia at dose level two; and six with acute myeloid leukaemia and one with myelodysplastic syndromes at dose level three). Median follow-up was 118 days (IQR 46-180). Seven patients (44%) had grade 3 or 4 treatment-related adverse events. In total, five patients (31%) had grade 3 or 4 cytokine release syndrome across all dose levels. One dose-limiting toxicity of cytokine release syndrome was reported at dose level three. No treatment-related deaths occurred, and the maximum tolerated dose was not reached. Three (25%) of 12 evaluable patients with relapsed or refractory acute myeloid leukaemia or myelodysplastic syndromes had an objective response. Among responders, two patients with acute myeloid leukaemia proceeded to allogeneic haematopoietic stem-cell transplantation (HSCT) after CYAD-01 treatment, with durable ongoing remissions (5 and 61 months). INTERPRETATION: Treatment with a multiple CYAD-01 infusion schedule without preconditioning is well tolerated and shows anti-leukaemic activity, although without durability outside of patients bridged to allogeneic HSCT. These phase 1 data support the proof-of-concept of targeting NKG2D ligands by CAR T-cell therapy. Further clinical studies with NKG2D-based CAR T-cells are warranted, potentially via combinatorial antigen targeted approaches, to improve anti-tumour activity. FUNDING: Celyad Oncology.

Tumor cell lysis and synergistically enhanced antibody-dependent cell-mediated cytotoxicity by NKG2D engagement with a bispecific immunoligand targeting the HER2 antigen.

Natural killer group 2 member D (NKG2D) plays an important role in the regulation of natural killer (NK) cell cytotoxicity in cancer immune surveillance. With the aim of redirecting NK cell cytotoxicity against tumors, the NKG2D ligand UL-16 binding protein 2 (ULBP2) was fused to a single-chain fragment variable (scFv) targeting the human epidermal growth factor receptor 2 (HER2). The resulting bispecific immunoligand ULBP2:HER2-scFv triggered NK cell-mediated killing of HER2-positive breast cancer cells in an antigen-dependent manner and required concomitant interaction with NKG2D and HER2 as revealed in antigen blocking experiments. The immunoligand induced tumor cell lysis dose-dependently and was effective at nanomolar concentrations. Of note, ULBP2:HER2-scFv sensitized tumor cells for antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, the immunoligand enhanced ADCC by cetuximab, a therapeutic antibody targeting the epidermal growth factor receptor (EGFR) synergistically. No significant improvements were obtained by combining cetuximab and anti-HER2 antibody trastuzumab. In conclusion, dual-dual targeting by combining IgG1 antibodies with antibody constructs targeting another tumor associated antigen and engaging NKG2D as a second NK cell trigger molecule may be promising. Thus, the immunoligand ULBP2:HER2-scFv may represent an attractive biological molecule to promote NK cell cytotoxicity against tumors and to boost ADCC.

NKG2D-CAR-transduced natural killer cells efficiently target multiple myeloma.

CAR-T-cell therapy against MM currently shows promising results, but usually with serious toxicities. CAR-NK cells may exert less toxicity when redirected against resistant myeloma cells. CARs can be designed through the use of receptors, such as NKG2D, which recognizes a wide range of ligands to provide broad target specificity. Here, we test this approach by analyzing the antitumor activity of activated and expanded NK cells (NKAE) and CD45RA- T cells from MM patients that were engineered to express an NKG2D-based CAR. NKAE cells were cultured with irradiated Clone9.mbIL21 cells. Then, cells were transduced with an NKG2D-4-1BB-CD3z-CAR. CAR-NKAE cells exhibited no evidence of genetic abnormalities. Although memory T cells were more stably transduced, CAR-NKAE cells exhibited greater in vitro cytotoxicity against MM cells, while showing minimal activity against healthy cells. In vivo, CAR-NKAE cells mediated highly efficient abrogation of MM growth, and 25% of the treated mice remained disease free. Overall, these results demonstrate that it is feasible to modify autologous NKAE cells from MM patients to safely express a NKG2D-CAR. Additionally, autologous CAR-NKAE cells display enhanced antimyeloma activity demonstrating that they could be an effective strategy against MM supporting the development of NKG2D-CAR-NK-cell therapy for MM.

Memory T Cells Expressing an NKG2D-CAR Efficiently Target Osteosarcoma Cells.

Purpose: NKG2D ligands (NKG2DL) are expressed on various tumor types and immunosuppressive cells within tumor microenvironments, providing suitable targets for cancer therapy. Various immune cells express NKG2D receptors, including natural killer (NK) cells and CD8+ T cells. Interactions between NKG2DL and NKG2D receptors are essential for NK-cell elimination of osteosarcoma tumor-initiating cells. In this report, we used NKG2D-NKG2DL interactions to optimize an immunotherapeutic strategy against osteosarcoma. We evaluated in vitro and in vivo the safety and cytotoxic capacity against osteosarcoma cells of CD45RA- memory T cells expressing an NKG2D-4-1BB-CD3z chimeric antigen receptor (CAR).Experimental Design: CD45RA- cells from healthy donors were transduced with NKG2D CARs containing 4-1BB and CD3z signaling domains. NKG2D CAR expression was analyzed by flow cytometry. In vitro cytotoxicity of NKG2D-CAR+ CD45RA- T cells against osteosarcoma was evaluated by performing conventional 4-hour europium-TDA release assays. For the in vivo orthotopic model, 531MII YFP-luc osteosarcoma cells were used as targets in NOD-scid IL2Rgnull mice.Results: Lentiviral transduction of NKG2D-4-1BB-CD3z markedly increased NKG2D surface expression in CD45RA- cells. Genetic stability was preserved in transduced cells. In vitro, NKG2D-CAR+ memory T cells showed significantly increased cytolytic activity than untransduced cells against osteosarcoma cell lines, while preserving the integrity of healthy cells. NKG2D-CAR+ memory T cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas untransduced T cells were ineffective.Conclusions: Our results demonstrate NKG2D-4-1BB-CD3z CAR-redirected memory T cells target NKG2DL-expressing osteosarcoma cells in vivo and in vitro and could be a promising immunotherapeutic approach for patients with osteosarcoma. Clin Cancer Res; 23(19); 5824-35. ©2017 AACR.

Targeting multiple types of tumors using NKG2D-coated iron oxide nanoparticles.

Iron oxide nanoparticles (IONPs) hold great potential for cancer therapy. Actively targeting IONPs to tumor cells can further increase therapeutic efficacy and decrease off-target side effects. To target tumor cells, a natural killer (NK) cell activating receptor, NKG2D, was utilized to develop pan-tumor targeting IONPs. NKG2D ligands are expressed on many tumor types and its ligands are not found on most normal tissues under steady state conditions. The data showed that mouse and human fragment crystallizable (Fc)-fusion NKG2D (Fc-NKG2D) coated IONPs (NKG2D/NPs) can target multiple NKG2D ligand positive tumor types in vitro in a dose dependent manner by magnetic cell sorting. Tumor targeting effect was robust even under a very low tumor cell to normal cell ratio and targeting efficiency correlated with NKG2D ligand expression level on tumor cells. Furthermore, the magnetic separation platform utilized to test NKG2D/NP specificity has the potential to be developed into high throughput screening strategies to identify ideal fusion proteins or antibodies for targeting IONPs. In conclusion, NKG2D/NPs can be used to target multiple tumor types and magnetic separation platform can facilitate the proof-of-concept phase of tumor targeting IONP development.

NKG2D CARs as cell therapy for cancer.

The NKG2D cell receptor and its ligands have attracted considerable interest as a potential strategy to attack tumor cells. NKG2D ligands are expressed on most types of tumors, and they demonstrate relative selectivity of ligand expression on tumor cells compared to healthy cells. Several different variants of NKG2D-based chimeric antigen receptors (CARs) have been developed, and extensive in vivo mechanistic studies performed demonstrated that cytotoxicity and cytokines are important for the efficacy NKG2D CAR adoptive T-cell therapy. NKG2D CARs target tumor cells, and they also target immunosuppressive cells within the tumor microenvironment. Under certain conditions, NKG2D ligand expression can be found on nontumor tissue, so potential off-tumor toxicity remains. In this article, we review the use of NKG2D as a basis for CAR targeting of tumors.

Normally occurring NKG2D+CD4+ T cells are immunosuppressive and inversely correlated with disease activity in juvenile-onset lupus.

The NKG2D receptor stimulates natural killer cell and T cell responses upon engagement of ligands associated with malignancies and certain autoimmune diseases. However, conditions of persistent NKG2D ligand expression can lead to immunosuppression. In cancer patients, tumor expression and shedding of the MHC class I-related chain A (MICA) ligand of NKG2D drives proliferative expansions of NKG2D(+)CD4(+) T cells that produce interleukin-10 (IL-10) and transforming growth factor-beta, as well as Fas ligand, which inhibits bystander T cell proliferation in vitro. Here, we show that increased frequencies of functionally equivalent NKG2D(+)CD4(+) T cells are inversely correlated with disease activity in juvenile-onset systemic lupus erythematosus (SLE), suggesting that these T cells may have regulatory effects. The NKG2D(+)CD4(+) T cells correspond to a normally occurring small CD4 T cell subset that is autoreactive, primed to produce IL-10, and clearly distinct from proinflammatory and cytolytic CD4 T cells with cytokine-induced NKG2D expression that occur in rheumatoid arthritis and Crohn's disease. As classical regulatory T cell functions are typically impaired in SLE, it may be clinically significant that the immunosuppressive NKG2D(+)CD4(+) T cells appear functionally uncompromised in this disease.

Chimeric NKG2D receptor-expressing T cells as an immunotherapy for multiple myeloma.

OBJECTIVE: Most myeloma tumor cells from patients express NKG2D ligands. We have reported the development of a chimeric NKG2D receptor (chNKG2D), which consists of the NKG2D receptor fused to the CD3zeta chain. T cells expressing this receptor kill and produce cytokines in response to NKG2D-ligand+ tumor cells. Therefore, we investigated whether human chNKG2D T cells respond against human myeloma cells. MATERIALS AND METHODS: ChNKG2D T cells were generated from healthy donors and myeloma patients. The effector phase of chNKG2D T cells was analyzed by cell-surface marker expression and human myeloma cell lines were tested for expression of NKG2D ligands. Lysis of myeloma cell lines and cytokine secretion by chNKG2D T cells was determined. ChNKG2D T cells grown in serum-free media, or cyropreserved, were assessed for effector cell functions. RESULTS: Myeloma cell lines expressed NKG2D ligands. ChNKG2D T cells from healthy donors and myeloma patients lysed myeloma cells, and secreted proinflammatory cytokines when cultured with myeloma cells or patient bone marrow, but not with peripheral blood mononuclear cells or normal bone marrow. Lysis of myeloma cells was dependent on chNKG2D T-cell expression of NKG2D and perforin. Additionally, chNKG2D T cells upregulated CD45RO, did not express CD57, and maintained expression of CD27, CD62L, and CCR7, indicating that the T cells were at an early effector stage. Finally, we showed that chNKG2D T cells generated with serum-free media, or when cryopreserved, maintained effector functions. CONCLUSION: ChNKG2D T cells respond to human myeloma cells and can be generated using clinically applicable cell culture techniques.