Expression Of NKG2DL Research Articles

SOLUBLE FORMS OF THE NKG2D RECEPTOR AND LIGAND NKG2DL1 IN PATIENTS WITH COLORECTAL CANCER

Relevance. Natural killer (NK) cells are considered to be effective cytotoxic cells of the tumor microenvironment, whose function is to effectively eliminate tumor cells. Their activation can occur when the NKG2D receptor interacts with the NKG2DL ligand expressed, for example, on tumor cells, which leads to the death of the latter. The aim of the study was a comparative study of the content of soluble forms of the sNKG2D receptor and its ligand sNKG2DL1 in the blood serum of healthy donors and patients with colorectal cancer (CRC), taking into account the clinical and morphological characteristics of the disease and prog-nosis. Material and methods. We examined 65 patients with CRC (32 women and 33 men), who were treated at the “National Medical Research Center of Oncology named after N.N. Blokhin” of the Ministry of Health of Russia aged (median 60 years) and 30 healthy donors (20 women and 10 men) aged (median 47 years). The clinical diagnosis in all patients was confirmed by the data of the morphological study of the tumor according to the Interna-tional Histological Classification of Tumors of the Digestive System (WHO, 2019), all had adenocarcinoma. The concentration of NKG2D and NKG2DL proteins was determined by ELISA in blood serum before treatment with Human B7-H3 Quantikine ELISA Kit reagents firm R&D (USA) in accordance with the manufacturer's instructions. The measurements were carried out on an automatic enzyme immunoassay analyzer BEP 2000 Advance (Siemens Healthcare Diagnostics, Germany). Statistical analysis of the obtained results was carried out using GraphPad Prizm v. 9. Overall survival analysis was performed by constructing survival curves using the Kaplan-Meier method. Results. In patients with colorectal cancer, a significant increase in the content of sNKG2DL in blood serum was found compared to the control group. sNKG2D concentrations did not differ between controls and CRC patients. No significant associations were found between the content of sNKG2D and sNKG2DL1 proteins in the blood serum of CRC patients and the main clinical and morphological characteristics of the disease. A decrease in the con-centration of sNKG2D was noted with the progression of the disease, and the highest content of sNKG2D was found in the localization of the tumor in the caecum. For the ligand sNKG2DL1, the opposite pattern was observed, namely, its lowest concentration in blood serum in tumors located in the caecum. For the ligand sNKG2DL1, the opposite pattern was observed, namely, its lowest concentration in blood serum in tumors located in the cae-cum. At the same time, the levels of sNKG2D and sNKG2DL1 in the control group directly and significantly correlated with each other (r=0.453; p=0.014), while no such pattern was found in the group of CRC patients (r=0.014; p=0.935). sNKG2DL1 concentrations are not a prognostic factor in CRC, while high levels of sNKG2D tended to lead to a favorable prognosis of the disease, which indirectly confirms the positive significance of tumor in-filtration by NK cells. Conclusions. The mechanisms of the relationship between CRC avoidance of immune surveillance and the expression of the sNKG2D receptor and the sNKG2DL ligand are discussed as one of the directions in the study of immune checkpoints. We believe that the effect of CRC immunotherapy can be achieved by increasing the expression of NKG2D on immune cells and inducing the expression of NKG2DL in tumor cells, which is the basis of the con-cept of this line of research.

Abstract 4166: Effects of cyproheptadine in eliciting natural killer cell-mediated immune response in bladder cancer

Abstract Bladder cancer is one of the most common occurring cancers in urothelial carcinoma. The previous study suggested that cancer cells undergo immunoevasion due to epigenetic silencing of NKG2DL, in which NKG2D-NKG2DL interaction induced natural killer (NK) cell-mediated cytotoxicity. We hypothesized that NK-mediated cytotoxicity can be reactivated by restoring the expression of NKG2DL on tumor cells. Our previous studies demonstrated that cyproheptadine (CPH) is a novel HDAC inhibitor exhibiting antitumor activity by cell cycle arrest and apoptosis. To understand the mechanism behind, RNA-seq showed an upregulation of ULBP2, a NKG2DL, in bladder cancer cells treated with CPH. Interestingly, upregulation of ULBP2, was accompanied with the enrichment of H3K27Ac and H3K4me3 at ULBP2 promoter, was observed in cells treated with CPH. Increased NK-specific lysis was observed in CPH-treated cells co-cultured with NK-92 cells. More importantly, treatment of CPH enhanced the efficacy of immune checkpoint inhibitor in a syngeneic mice bladder cancer model. In conclusion, our results suggested that CPH may be a novel HDAC inhibitor which may elicit immune response in NK-cell dependent manner. Citation Format: Himani Kumari, Guan-Ling Lin, Chih-Chieh Yeh, Ciao-Ni Chen, Yu-Ming Chuang, Wan-Hong Huang, Wen-Long Huang, Steven Lin, Michael W. Chan. Effects of cyproheptadine in eliciting natural killer cell-mediated immune response in bladder cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4166.

Valproic Acid Increased CAR T Cell Cytotoxicity Against Acute Myeloid Leukemia

Background The treatment paradigm of B cell malignancies has dramatically changed in recent years with the introduction of immunotherapy, especially CAR-T cell therapy. However, only limited efficacy was observed in AML with CAR-T cell therapy. Methods In this study, the molecular, clinical, and tumor immune microenvironments characteristics of AML patients in TCGA were performed on an online bio-information analysis website. The expressions of CD123 and CLL-1 on the AML cells from clinical samples were analyzed by flow cytometry. Then we constructed anti-CD123 CAR and CLL-1 CAR with different co-stimulation domains CD28 or 4-1BB and detected the anti-AML effects. To increase the efficacy of CAR-T cell therapy, we tested different combination strategies, such as checkpoint inhibitor PD-1/PD-L1 antibodies and histone deacetylase inhibitor (HDACi) in vivo and in vitro. RNA sequencing and western blot were performed to analyze the potential mechanism of valproic acid (VPA) in upregulating NKG2DL. Results The data suggested that CD123 and CLL-1 were highly expressed on AML leukemia cells (p=0.0031, p=0.0069). The proportion of various immune cells such as T cell subsets and NK cells in AML patients was significantly lower than that in healthy donors. The process of immune infiltration mainly stagnated in the recruitment and activation of immune cells or the tracking migration of immune cells. The immunosuppressive molecule PD-1 was negatively correlated with memory T cells, while the active molecules CD27, CD28, and NKG2D were positively correlated with CD8 + memory T cells. Both CD123 CAR and CLL-1 CAR displayed specific anti-AML effects. To improve the lysis effects of CAR-T cells, we combined CAR-T cell therapy with different agents. PD-1/PD-L1 antibody could only slightly improve the potency of CAR-T cell therapy (CD123 CAR-T 60.92±2.9087% VS 65.43±2.1893%, 60.92±2.9087% VS 67.43± 3.4973%, CLL-1 CAR-T 37.37±3.908% VS 41.89 ±5.1568%, 37.37±3.908% VS 42.84± 4.2635%). While, one of HDACi, valproic acid (VPA) could synergize with CAR-T cell therapy and significantly improve CAR-T cell potency against AML cells, especially in pre-treated with VPA for 48h ( CLL-1 CAR-T 34.97±0.3051% VS 88.167±1.5327% p<0.0001, CD123 CAR-T 26.87±2.7010% VS 82.56±3.086%, p<0.0001 in MV411, CLL-1 CAR-T 78.77±1.2061% VS 93.743±1.2333% p<0.0001, CD123 CAR-T 64.10±1.5130% VS 94.427±0.142%, p=0.0001 in THP-1). What's more, the anti-AML effects of combination therapy were also observed in the AML mice model. NCG mice were injected with 1×106 THP-1 cells that express firefly luciferase and GFP label (THP-1-Fluc) via tail vein, 1 week later mice were infused with VPA 0.5mM (iv, n=6), VPA 100mg/kg (ip, n=6), VPA 0.5mM (iv, twice a day, n=6) and PBS (iv, n=6), then CD123 CAR-T cells or untreated T cells were infused 48h later. The combination therapy prolonged the overall survival of mice when compared to single CAR-T cell therapy (median survival:180 days VS unfollowed). One of the possible mechanisms underlying we found was that NKG2D could upregulate on activated CD8+T cells, and VPA could upregulate NKG2D ligand expression in the AML cells which may contribute to NKG2D-mediated cytotoxicity of CAR-T cells against tumor cells. In addition, the expression of HDAC2 and H4 in THP-1 cells was significantly decreased after VPA treatment, suggesting that VPA may enhance the expression of NKG2DL by degrading HDAC2 expression. Conclusion CD123 and CLL-1 are promising targets for AML CAR-T cell therapy. A combination of VPA pre-treatment and CAR-T against AML exhibits synergic effects, which may provide a promising strategy in the clinical treatment of R/R AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Fatty acid metabolism predicts prognosis and NK cell immunosurveillance of acute myeloid leukemia patients

Cell metabolic reprogramming is a hallmark of tumor prognosis, and fatty acid metabolism (FAM) plays a crucial role in the tumor microenvironment (TME). However, the relationship between FAM, TME, and prognosis of acute myeloid leukemia (AML) patients remains elusive. We extracted the single-cell RNA sequencing (scRNA-Seq) and bulk transcriptome data of AML patients from the TCGA and GEO databases and assessed the relationship between FAM, TME, and AML patient prognosis. We also performed functional enrichment (FE) assay to evaluate the significance of FAM in anti-AML immunosurveillance. Our scRNA-Seq analysis revealed that the leukemic stem cell (LSC)-enriched population exhibited elevated levels of FAM-related genes. Using these FAM-related genes, we developed a prognostic model that accurately estimated AML patient outcome. FE analysis showed that FAM was strongly related to alterations of TME-based immunosurveillance in AML patients. More importantly, we demonstrated that FAM inhibition via pharmaceutical targeting of PLA2G4A, a highly expressed FAM gene in AML patients with poor prognosis, enhanced the NK cell-mediated immunosurveillance in leukemia cells. Leukemic stem cell (LSC)-enriched population exhibited elevated levels of FAM-related genes. We have successfully established the FAM formula that predicts AML patient prognosis and alterations in the TME-based immunosurveillance. We also found that PLA2G4A was a highly expressed FAM gene in AML patients with poor prognoses. Pharmaceutical targeting of PLA2G4A increased the expression of NKG2DL in leukemia cells in vitro and thus enhanced the NK cell-mediated immunosurveillance.

Romidepsin Enhances the Killing Ability of NKG2D-CAR-T Cells through Enhanced Expression of NKG2DL against Ovarian Cancer Cells

Background: Upregulating tumor cell targeting antigens and improving the cytotoxicity of chimeric antigen receptor T cell (CAR-T) are expected to facilitate better treatment efficacy for solid cancers represented by ovarian cancer. Methods: Killer cell lectin-like receptor subfamily K member 1 ligands (NKG2DL) are the target antigens for ovarian cancer. NKG2D-CAR-T cells were constructed for NKG2D ligand on the ovarian cancer cell surface. We used flow cytometry to evaluate the expression of NKG2DL on SKOV3 (a human ovarian cancer adenocarcinoma cell line). Innovatively, when combined with romidepsin to treat ovarian cancer cell SKOV3, to evaluate the killing ability of the combined strategy, we verified the cytotoxicity of CAR-T cells by lactate dehydrogenase (LDH) release test and determined the secretion of cytokines by enzyme-linked immuno sorbent assay (ELISA). Results: The results of flow cytometry showed effective activation of the NKG2D-CAR-T cells, and romidepsin treatment resulted in increased expression of NKG2DL on the surface of SKOV3. Cytotoxicity test showed that romidepsin could enhance the killing ability of NKG2D-CAR-T cells against ovarian cancer cells by regulating their NKG2DL expression (p &lt; 0.05). The killing effects and secretion of interferon-γ (IFN-γ) increased synchronously (p &lt; 0.05). Levels of interleukin-2 (IL-2) and Pore-forming protein (PFP) were statistically significant at a low target ratio but programmed cell death protein 1 (PD-1) remained unaffected (p ≥ 0.05). Conclusions: Enhancing the expression of tumor target antigen is a solution to improve the limited application of CAR-T cells in solid cancers.

C-Myc Targets HDAC3 to Suppress NKG2DL Expression and Innate Immune Response in N-Type SCLC through Histone Deacetylation.

Simple SummaryNatural killer group 2, member D ligand (NKG2DL) is the most relevant ligand of NK cells to perform immune surveillance and is rarely expressed in most small cell lung cancer (SCLC) with the unclear mechanism. This study aimed to investigate the mechanisms underlying the NKG2DL deficiency in C-MYC (MYC)-amplificated N-type SCLC (SCLC-N) with less immune infiltrate. Our data showed that c-Myc was the suppressor of NKG2DL in SCLC-N. Further, c-Myc suppressed the transcription of NKG2DL by recruiting HDAC3 to deacetylate H3K9ac at the promoter of MICA and MICB in SCLC-N and inhibited the cytotoxicity of NK cells. The above findings revealed the role of c-Myc/HDAC3 axis in the regulation of NKG2DL expression, supplying a new perception for comprehending the mechanism of SCLC-N immune escape, which was poorly understood and providing the therapeutic targets that SCLC-N may benefit from.SCLC is an aggressive malignancy with a very poor prognosis and limited effective therapeutic options. Despite the high tumor mutational burden, responses to immunotherapy are rare in SCLC patients, which may be due to the lack of immune surveillance. Here, we aimed to examine the role and mechanism of oncogene MYC in the regulation of NKG2DL, the most relevant NK-activating ligand in SCLC-N. Western Blotting, Immunofluorescence, flow cytometry, quantitative real-time PCR (qRT-PCR), Co-Immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), and Cytotoxicity assay were used on H2227 cells, H446 cells, and other SCLC cell lines, and we found that c-Myc negatively regulated NKG2DL expression in SCLC-N cells. Mechanistically, c-Myc recruited HDAC3 to deacetylate H3K9ac at the promoter regions of MICA and MICB, suppressing the MICA/B expression of SCLC-N cells and the cytotoxicity of NK cells. Treatment with selective HDAC3 inhibitor up-regulated the expression of NKG2DL on SCLC-N cells and increased the cytotoxicity of NK cells. Furthermore, analysis of the CCLE and Kaplan-Meier plotter data performed the negative correlation between MYC and NKG2DL in SCLC-N cells and the correlation with the prognosis of lung cancer patients. Collectively, the results provided the new insight into the role and mechanism of c-Myc/HDAC3 axis in NKG2DL expression and innate immune escape of SCLC-N, suggesting the potential target for SCLC-N immunotherapy.

P-007: Exploring a safety switch in NKG2D and BCMA CAR NK-92MI immunotherapy

P-007: Exploring a safety switch in NKG2D and BCMA CAR NK-92MI immunotherapy

Bispecific NKG2D-CD3 and NKG2D-CD16 fusion proteins for induction of NK and T cell reactivity against acute myeloid leukemia

BackgroundMonoclonal antibodies (mAbs) mediate their effects in great part by inducing ADCC of NK cells, and multiple efforts aim to increase this function by engineering mAbs optimized Fc-parts. Even more potent antitumor immunity can be induced by strategies to stimulate T cells with their profoundly higher effector potential. However, upon increased immunostimulatory potential, the necessity to target highly tumor-specific antigens becomes critically important to reduce side effects.MethodsWe here report on bispecific fusion proteins (BFP) that target ligands of the immunoreceptor NKG2D (NKG2DL), which are widely expressed on malignant cells but generally absent on healthy tissue. They consist of the extracellular domain of NKG2D as targeting moiety fused to Fab-fragments of CD3 (NKG2D-CD3) or CD16 (NKG2D-CD16) antibodies.ResultsNKG2D-CD16 displayed increased affinity to the FcγRIII on NK cells compared to engineered Fc-parts, which are contained in optimized mAbs that presently undergo clinical evaluation. In line, NKG2D-CD16 induced superior activation, degranulation, IFN-γ production and lysis of acute myeloid leukemia (AML) cell lines and patient AML cells. NKG2D-CD3 in turn potently stimulated T cells, and comparison of efficacy over time revealed that NKG2D-CD16 was superior upon short term application, while NKG2D-CD3 mediated overall more potent effects which manifested after longer times. This can be attributed to treatment-induced proliferation of T cells but not NK cells.ConclusionsTaken together, we here introduce novel “antibody-like” BFP that take advantage of the highly tumor-restricted expression of NKG2DL and potently activate the reactivity of NK cells or T cells for immunotherapy of AML.

Induction of NK and T Cell Immune Responses Against Leukemia Cells By Bispecific NKG2D-CD16 and -CD3 Fusion Proteins

Monoclonal antibodies (mAbs) have by now become an established tool in therapy of many malignancies. The interaction of a mAb's Fc portion with Fcγ receptors (FcγR) on immune effector cells is important for its efficacy, but often insufficient to potently induce antitumor immunity e.g. due to FcγR polymorphisms. Moreover, Fc parts of mAbs may bind to FcγRs expressed on non-cytotoxic cells (e.g., platelets and B cells) and interact with FcγRs that do not trigger cytotoxicity (e.g. CD16b on granulocytes). These shortcomings can be overcome by novel antibody formats like bispecific antibodies allowing for improved activation of a specific pool of effector cells. Here we report on the development and preclinical characterization of two bispecific fusion proteins that target ligands of the immunoreceptor NKG2D (NKG2DL) which are widely expressed on malignant cells but generally absent on healthy tissue. Our fusion proteins consist of the extracellular domain of NKG2D as targeting moiety for tumor-expressed NKG2DL fused to Fab-fragments of either an agonistic CD3 or CD16 antibody. Specific binding of these NKG2D-CD3 and NKG2D-CD16 constructs was confirmed using NKG2DL-transfectants with regard to their target arm and either NK cells or T cells with regard to their different effector parts. Dose titration assays revealed an increased affinity of NKG2D-CD16 to the FcγR on NK cells as compared to our previously described (e.g., Steinbacher et al, 2014) Fc-optimized NKG2D-IgG1 fusion protein, which was mirrored by a potently increased ability to induce lysis of NKG2DL-tranfectants and NKG2DL-positive primary acute myeloid leukemia (AML) cells by allogeneic NK cells. The novel NKG2D-CD3 construct in turn was found to potently activate allogeneic and autologous CD4+ and CD8+ T cells. Next we comparatively analyzed the efficacy of T cells and NK cells to lyse autologous leukemia cells upon treatment with NKG2D-CD3 and NKG2D-CD16, respectively, by using PBMC of AML patients (blast counts 30-70%) directly ex vivo in long term cytotoxicity assays. NKG2D-CD16 potently induced AML cell lysis, and this was, in line with their proliferative and higher effector potential, by far exceeded upon stimulation of T cells with NKG2D-CD3. Taken together, we here introduce novel "antibody-like" bispecific constructs that take advantage of the highly tumor-restricted expression of NKG2DL and potently activate the reactivity of NK or T cells for immunotherapy of leukemia. DisclosuresNo relevant conflicts of interest to declare.

Methylation of NKG2D ligands contributes to immune system evasion in acute myeloid leukemia.

Engagement of the activating receptor NKG2D (natural killer group 2 member D) with its ligands (NKG2DL) major histocompatibility complex class I related-A and -B (MICA/B), UL-16 binding protein families (ULBPs 1-6) is important to ensure the innate immunity to tumor cells. However, these cells have developed strategies to downregulate NKG2DL expression and avoid immune recognition. We demonstrate that DNA methylation can contribute to the absence of NKG2DL expression during tumor progression. We analyzed the DNA methylation profiles for each NKG2DL by pyrosequencing in acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), hepatocellular carcinoma (HC), breast cancer and colon cancer cell lines. High levels of DNA methylation for NKG2DL were found in some tumor cell lines, mainly in AML cells. This hypermethylation was correlated with the absence of transcription for NKG2DL. Higher DNA methylation levels for MICA, ULBP1 and ULBP2 were observed in AML patients (n=60) compared with healthy donors (n=25). However, no DNA methylation for NKG2DL was found in colon cancer patients (n=44). Treatment with demethylating agents (5-azacytidine and 5-aza-2'-deoxycytidine) restored the expression of NKG2DL on the cell surface of AML cells, leading to an enhanced recognition by NKG2D-expressing cells. Our data suggest that NKG2DL may be aberrantly silenced by DNA methylation as a consequence of tumor development in AML patients.

Fc-optimized NKG2D-Fc constructs induce NK cell antibody-dependent cellular cytotoxicity against breast cancer cells independently of HER2/neu expression status.

The ability of NK cells to mediate Ab-dependent cellular cytotoxicity (ADCC) largely contributes to the clinical success of antitumor Abs, including trastuzumab, which is approved for the treatment of breast cancer with HER2/neu overexpression. Notably, only ∼25% of breast cancer patients overexpress HER2/neu. Moreover, HER2/neu is expressed on healthy cells, and trastuzumab application is associated with side effects. In contrast, the ligands of the activating immunoreceptor NKG2D (NKG2DL) are selectively expressed on malignant cells. In this study, we took advantage of the tumor-associated expression of NKG2DL by using them as target Ags for NKG2D-IgG1 fusion proteins optimized by amino acid exchange S239D/I332E in their Fc part. Compared to constructs with wild-type Fc parts, fusion proteins carrying the S239D/I332E modification (NKG2D-Fc-ADCC) mediated highly enhanced degranulation, ADCC, and IFN-γ production of NK cells in response to breast cancer cells. NKG2D-Fc-ADCC substantially enhanced NK reactivity also against HER2/neu-low targets that were unaffected by trastuzumab, as both compounds mediated their immunostimulatory effects in strict dependence of target Ag expression levels. Thus, in line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to its highly increased affinity to CD16, NKG2D-Fc-ADCC potently enhances NK cell reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, NKG2D-Fc-ADCC may constitute an attractive means for immunotherapy especially of HER2/neu-low or -negative breast cancer.

Abstract 3652: Surface expression and shedding of NKG2D ligands by (metastasizing) tumor cells is altered by platelets leading to impaired NK cell immunosurveillance

Abstract NK cells play an important role in anti-tumor immunity. The mechanisms leading to NK cell activation are described by the ‘missing-self’ and ‘induced-self' hypotheses, implying that cells with low or absent expression of MHC class I and stress-induced expression of ligands for activating receptors such as NKG2D (NKG2DL), are targeted by NK cells. Besides the direct interaction with their target cells, NK activity is also influenced by various other hematopoietic cells. In mouse models, thrombocytopenia impairs metastasis, and this is reversed by additional depletion of NK cells. Hence, the knowledge of the underlying molecular mechanisms by which platelets influence NK cells is still fragmentary. We already showed that release of TGF-β by platelets upon their interaction with (metastasizing) tumor cells downmodulates the activating receptor NKG2D on NK cells (Kopp et al, Cancer Res. 2009; Placke, J Innate Immun. 2011). Moreover, platelets transfer ´healthy´ MHC class I to the tumor cell surface. Thus, platelets may facilitate metastasis by interfering with both, ‘induced and missing self' NK cell recognition. Here we provide evidence for a yet unknown mechanism by which platelets further impair NKG2D-mediated immunosurveillance. Tumor cells were incubated with platelets from healthy donors resulting in coating of tumor cells and activation of the platelets, or treated with platelet-derived soluble factors (releasate) obtained either by tumor cell-induced platelet activation (TCIPA) or the platelet agonist thrombin. Presence of platelet derived factors derived either from coating of tumor cells or contained in platelet releasate substantially reduced surface expression of NKG2DL on tumor cells. This was paralleled by enhanced levels of soluble NKG2DL in culture supernatants, indicating that platelet-derived factors mediate NKG2DL shedding from the tumor cell surface. Diminished NKG2DL surface expression resulted in diminished NKG2D-dependent natural cytotoxicity of NK cells, as revealed by blocking experiments using NKG2D antibody and NKG2DL-specific F(ab)2 fragments targeting the specifically modulated NKG2DL. Our data thus identify induction of NKG2DL shedding as novel mechanism by which interaction of platelets with metastasizing tumor cells impairs NK cell immunosurveillance. Citation Format: Stefanie Raab, Korbinian Nepomuk Kropp, Alexander Steinle, Gerd Klein, Hans-Georg Kopp, Helmut R. Salih. Surface expression and shedding of NKG2D ligands by (metastasizing) tumor cells is altered by platelets leading to impaired NK cell immunosurveillance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3652. doi:10.1158/1538-7445.AM2014-3652

MicroRNA-mediated down-regulation of NKG2D ligands contributes to glioma immune escape.

Malignant gliomas are intrinsic brain tumors with a dismal prognosis. They are well-adapted to hypoxic conditions and poorly immunogenic. NKG2D is one of the major activating receptors of natural killer (NK) cells and binds to several ligands (NKG2DL). Here we evaluated the impact of miRNA on the expression of NKG2DL in glioma cells including stem-like glioma cells. Three of the candidate miRNA predicted to target NKG2DL were expressed in various glioma cell lines as well as in glioblastomas in vivo: miR-20a, miR-93 and miR-106b. LNA inhibitor-mediated miRNA silencing up-regulated cell surface NKG2DL expression, which translated into increased susceptibility to NK cell-mediated lysis. This effect was reversed by neutralizing NKG2D antibodies, confirming that enhanced lysis upon miRNA silencing was mediated through the NKG2D system. Hypoxia, a hallmark of glioblastomas in vivo, down-regulated the expression of NKG2DL on glioma cells, associated with reduced susceptibility to NK cell-mediated lysis. This process, however, was not mediated through any of the examined miRNA. Accordingly, both hypoxia and the expression of miRNA targeting NKG2DL may contribute to the immune evasion of glioma cells at the level of the NKG2D recognition pathway. Targeting miRNA may therefore represent a novel approach to increase the immunogenicity of glioblastoma.

An Fc-optimized NKG2D-immunoglobulin G fusion protein for induction of natural killer cell reactivity against leukemia.

Recruitment of Fc-receptor-bearing effector cells, such as natural killer (NK) cells, is a feature critical for the therapeutic success of antitumor antibodies and can be improved by the modifications of an antibody's Fc part. The various ligands of the activating immunoreceptor NKG2D, NKG2DL) are selectively expressed on malignant cells including leukemia. We here took advantage of the tumor-associated expression of NKG2DL for targeting leukemic cells by NKG2D-immunoglobulin G (IgG)1 fusion proteins containing modified Fc parts. Compared to NKG2D-Fc containing a wild-type Fc part (NKG2D-Fc-WT), our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell Fc receptor, respectively. Functional analyses with allogenic as well as autologous NK cells and primary malignant cells of leukemia patients revealed that NKG2D-Fc-KO significantly reduced NK reactivity by blocking immunostimulatory NKG2D-NKG2DL interaction. NKG2D-Fc-WT already enhanced antileukemia reactivity by inducing antibody-dependent cellular cytotoxicity (ADCC) with NKG2D-Fc-ADCC mediating significantly stronger effects. Parallel application of NKG2D-Fc-ADCC with Rituximab caused additive effects in lymphoid leukemia. In line with the tumor-associated expression of NKG2DL, no NK cell ADCC against resting healthy blood cells was induced. Thus, NKG2D-Fc-ADCC potently enhances NK antileukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL and may constitute an attractive means for immunotherapy of leukemia.

An Fc-optimized NKG2D-Ig fusion protein for induction of NK cell reactivity against breast cancer.

3054 Background: The anti-tumor activity and clinical success of the monoclonal antibody trastuzumab, approved for treatment of HER2/neu-overexpressing breast cancer, is at least partially mediated by induction of antibody dependent cellular cytotoxicity (ADCC). However, only about 20% of patients show HER2/neu overexpression, and trastuzumab treatment is associated with side effects. The ligands of the activating immunoreceptor NKG2D (NKG2DL) are widely expressed on malignant cells, but generally absent on healthy tissue. We aimed to take advantage of this tumor-restricted expression by using NKG2DL as target-antigens on breast cancer cells. To this end we generated NKG2D-Ig fusion proteins with modified Fc moieties and studied their ability to induce NK cell anti-tumor reactivity. Methods: The Fc parts within the constructs were modified by amino acid exchange as previously described (Lazar 2006; Armour 1999). Direct effects on tumor cell viability as well as induction of NK cell activation, degranulation, cytotoxicity and IFN-γ release in cultures with breast cancer cell lines expressing different HER2/neu levels were determined. Results: Compared to NKG2D-Fc containing a wildtype Fc part (NKG2D-Fc-WT) or trastuzumab, our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell Fc receptor, respectively. In contrast to trastuzumab, no direct effect of the constructs on tumor cell viability was observed. In cultures of NK cells and breast cancer cells, NKG2D-Fc-KO significantly reduced NK reactivity due to blocking immunostimulatory NKG2D-NKG2DL interaction. NKG2D-Fc-WT substantially enhanced NK reactivity by induction of ADCC, while the effects of NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT and, in case of HER2/neu-low targets also that of Herceptin. Conclusions: Fc-engineered NKG2D-Ig fusion protein effectively target breast cancer cells for NK anti-tumor reactivity. Due to the tumor-restricted expression of NKG2DL, NKG2D-Fc-ADCC may constitute an attractive means for immunotherapy especially of HER2/neu-low or -negative breast cancer.

Induction of NK Cell Reactivity in Breast Cancer by Fc-Engineered NKG2D-Ig Fusion Proteins

AbstractAbstract 253NK cells are cytotoxic lymphocytes that play an important role in anti-tumor immunity. Their capability to mediate Fc-receptor dependent effector functions like antibody dependent cellular cytotoxicity (ADCC) largely contributes to the clinical success of anti-tumor antibodies like Herceptin (Trastuzumab®), which is approved for treatment of breast cancer displaying HER2/neu-overexpression. Notably, only about 20% of breast cancer patients show overexpression of HER2/neu. Moreover, this antigen is also expressed on healthy cells, and application of Herceptin is associated with side effects. In contrast, ligands of the activating immunoreceptor NKG2D (NKG2DL) are widely expressed on malignant cells, but generally absent on healthy tissues. We aimed to take advantage of the tumor-restricted expression of NKG2DL by using them as tumor-antigens for Fc-optimized NKG2D-Ig fusion proteins targeting breast cancer cells for NK cell ADCC and IFN-γ production. NKG2D-Ig fusion proteins with distinct modifications in their Fc portion were generated by amino acid exchange as previously described (Lazar 2006; Armour 1999). Compared to wildtype NKG2D-Fc (NKG2D-Fc-WT) or Herceptin, our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell FcγRIIIa receptor (CD16), respectively. This resulted in lacking (NKG2D-Fc-KO) or highly enhanced (NKG2D-Fc-ADCC) NK cell activation. In cultures of NK cells and breast cancer cells, NKG2D-Fc-KO significantly reduced NK cell reactivity due to blockade of NKG2DL-mediated activating signals, while NKG2D-Fc-WT substantially enhanced NK reactivity by induction of ADCC and cytokine production. Notably, the effect of our NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT and, in case of HER2/neu low targets, also that of Herceptin, resulting in potently enhanced NK anti-tumor reactivity. Together, our results demonstrate that Fc-engineered NKG2D-Fc-ADCC fusion proteins can effectively target NKG2DL-expressing cancer cells for NK anti-tumor reactivity. In line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to its highly increased affinity to CD16 NKG2D-Fc-ADCC potently enhances NK cell reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, NKG2D-Fc-ADCC may thus constitute an attractive means for immunotherapy, especially of HER2/neu-low or -negative breast cancer. Disclosures:No relevant conflicts of interest to declare.

Diagnostic and prognostic impact of serum‐soluble UL16‐binding protein 2 in lung cancer patients

UL16-binding protein 2 (ULBP2) is one of the ligands for NKG2D (NKG2DL). ULBP2 expression is induced in transformed cells and is recognized by immune effector cells via the activating NKG2D immunoreceptor. Soluble forms of NKG2DL have been reported in the serum of patients with several types of cancer. The present study investigated the diagnostic and prognostic significance of serum-soluble ULBP2 (sULBP2) in lung cancer patients. We used flow cytometry to evaluate the surface expression of NKG2DL by various lung cancer cells, while sULBP2 was measured using our original ELISA. In addition, the immunological effect of sULBP2 on peripheral blood mononuclear cells (PBMC) was examined by the (51) Cr release assay. We found that ULBP2 was highly expressed and that the sULBP2 level was elevated in supernatants of cultured non-small-cell lung cancer (NSCLC) cells as well as in the serum of NSCLC patients. ULBP2 levels were especially high in squamous cell carcinoma (SQ) patients. Clinical stage IIIB and IV NSCLC patients with a sULBP2 level ≥ 8.7 pg/mL showed significantly shorter survival than patients with sULBP2 <8.7 pg/mL. In multivariate analysis, a sULBP2 level ≥ 8.7 pg/mL (hazard ratio [HR], 2.13; P = 0.038) and clinical stage IV (HR, 2.65; P = 0.019) were independent determinants of a poor outcome. As a possible mechanism, we demonstrated that sULBP2 directly suppresses the cytolytic activity of PBMC. In conclusion, ULBP2 is the most significant NKG2DL for lung cancer, and sULBP2 is useful in the diagnosis of SQ and as a prognostic indicator for patients with advanced NSCLC.

Fc-Engineered NKG2D-IgG1 Fusion Proteins Target Leukemia Cells for Antibody-Dependent Cellular Cytotoxicity (ADCC) of NK Cells

Abstract 1537The capability of anti-tumor antibodies to recruit Fc-receptor (FcR) bearing effector cells like NK cells, a feature considered critical for therapeutic success, can be markedly improved by modifications of the human IgG1 part. At present, Fc-engineered antibodies targeting leukemia cells are yet not available. The various ligands of the NK cell-activating immunoreceptor NKG2D (NKG2DL) are generally absent on healthy cells but upregulated on malignant cells of various origins including leukemia. We aimed to take advantage of the tumor-restricted expression of NKG2DL by using them as target-antigens for Fc-optimized NKG2D-IgG1 fusion proteins targeting leukemia cells for antibody-dependent cellular cytotoxicity (ADCC) and IFN-g production of NK cells. NKG2D-IgG1 fusion proteins with distinct modifications in their Fc portion were generated as previously described (Lazar 2006; Armour 1999). Compared to wildtype NKG2D-Fc (NKG2D-Fc-WT), the mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell FcgRIIIa receptor but comparable binding to NKG2DL-expressing target cells. Functional analyses with allogenic NK cells and leukemia cell lines as well as primary leukemic cells of AML and CLL patients revealed that NKG2D-Fc-KO significantly (p<0.05, Mann-Whitney U test) reduced NK cytotoxicity and IFN-g production (about 20% and 30% reduction, respectively), which can be attributed to blockade of NKG2DL-mediated activating signals. Treatment with NKG2D-Fc-WT significantly (p<0.05, Mann-Whitney U test) enhanced NK reactivity (about 20% and 100% increase in cytotoxicity and cytokine production, respectively). The effects observed upon treatment with NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT resulting in at least doubled NK ADCC and IFN-g production compared to NKG2D-Fc-WT. When applied in combination with Rituximab in analyses with CLL cells, a clear additive effect resulting in a more than four-fold increase of ADCC and FcgRIIIa-induced IFN-g production was observed. The NKG2D-Fc fusion proteins did not induce NK reactivity against healthy blood cells, which is in line with the tumor-restricted expression of NKG2DL. Of note, treatment with NKG2D-Fc-ADCC also significantly (p<0.05, Mann-Whitney U test) enhanced reactivity (up to 70% increase) of NK cells against NKG2DL-positive AML and CLL cells among patient PBMC in an autologous setting. Together, our results demonstrate that Fc-engineered NKG2D-Fc-ADCC fusion proteins can effectively target NKG2DL-expressing leukemia cells for NK anti-tumor reactivity. In line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to their highly increased affinity to the FcgRIIIa receptor, NKG2D-Fc-ADCC potently enhances NK anti-leukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, Fc-modified NKG2D-Ig may thus constitute an attractive means for immunotherapy of leukemia. Disclosures:No relevant conflicts of interest to declare.

NKG2D and Its Ligands In Leukemia: Comprehensive Analysis of Expression, Release and Modulation of NK Cell Reactivity.

AbstractAbstract 1686NK cells play an important role in the immunosurveillance of leukemia. The mechanisms leading to activation of NK reactivity are described by the principles of ‘missing-self’ and ‘induced-self’ which imply that cells with low or absent expression of MHC class I ('missing-self') and/or stress-induced expression of ligands of activating NK receptors ('induced-self') are preferentially recognized and eliminated by NK cells. NKG2D is a prototypic activating NK receptor and plays an important role in tumor immunosurveillance, as (i) its ligands (NKG2DL) are usually not expressed on healthy cells and (ii) sufficient expression of NKG2DL renders cells susceptible to NK cell lysis despite expression of MHC class I. Tumor cells may, however, evade NKG2D-mediated immune surveillance via reduction of NKG2DL surface expression by shedding. This results in release of NKG2DL in soluble (s) form and causes reduction of NKG2D expression leading to systemically impaired NK cell reactivity. In leukemia, the relevance of NKG2D and its ligands is still controversially discussed. Here we comprehensively studied the expression and release of NKG2DL in adult leukemia patients and the consequences for NKG2D-mediated NK activation. Substantial (SFI>1.5) surface expression of at least one NKG2DL was detected in 114 (72%) of 158 investigated cases of patients with AML (n=80), ALL (n=15), CLL (n=53) and CML (n=10). In 26 (23%) and 46 (40%) of cases, surface expression of two or >three different NKG2DL, respectively, was observed. CML cells generally expressed NKG2DL at rather low levels. Among the other entities, preferential expression of MICA, ULBP1 and ULBP3 was observed in AML, ALL and CLL, respectively. Moreover, all 132 investigated patient sera (AML, 54; ALL, 20; CLL, 46; CML 11) contained significantly elevated (p<0.001, Mann Whitney U test) levels of at least one sNKG2DL compared to healthy controls with sMICA displaying a higher median and being most frequently detected as compared to the other sNKG2DL (MICA: median, 305pg/ml, range, 0–1530pg/ml, 95% above the ELISA detection limit; MICB: median, 117pg/ml, range, 0–1755pg/ml, 87% above the detection limit; ULBP1: median, 61pg/ml, range, 0–485pg/ml, 77% above the detection limit; ULBP2: median, 41pg/ml, range, 0–1416pg/ml, 62% above the detection limit; ULBP3: median, 47pg/ml, range, 0–1961pg/ml, 59% above the detection limit). Three patients (2%) displayed elevated levels of only one NKG2DL, while in 18 (14%) and 111 (84%) cases elevated levels of two or >three different sNKG2DL, respectively, were observed. Comparing the four leukemia entities, no relevant differences in serum levels overall and no clear association of a specific sNKG2DL with a certain leukemia type was observed. NK cells of leukemia patients (n=30) displayed significantly reduced NKG2D expression as compared to healthy controls (p<0.01, Mann Whitney U test), and exposure of healthy NK cells to NKG2DL-containing sera of leukemia patients led to significantly (p<0.01 Mann Whitney U test) diminished NKG2D surface levels. Analysis of further samples is ongoing. The functional relevance of NKG2D-NKG2DL interaction was confirmed in functional analyses with allogenic NK cells and NKG2DL-expressing primary leukemia cells, where we observed reduction of NK reactivity upon blocking either NKG2D or its ligands. Where leukemia cells expressed more than one NKG2DL, additive effects were observed upon blocking two or more different NKG2DL, which is in line with the notion that NK reactivity critically depends on expression levels of NKG2DL. Altogether, our data demonstrate that NKG2D-NKG2DL interaction is not only relevant for NK reactivity against epithelial tumors, but also can contribute to NK cell-mediated surveillance of leukemia. Disclosures:No relevant conflicts of interest to declare.

Drug-Induced, NKG2D-Dependent Sensitization of ALL Cell Lines to NK Recognition.

Abstract 2024Poster Board II-1The combination of tumor-sensitizing drugs with NK cell infusion is beginning to emerge as a novel anti-tumor therapy. A growing body of in vitro studies show that drugs such as proteosome inhibitors, histone deacetylase inhibitors and thiazolidinediones are able to sensitize tumor cells but not their healthy counterparts to NK-mediated lysis. Drug induced NK-sensitization has shown promise in acute myeloid leukemias but no studies have yet proven this principle in acute lymphoblastic leukemia (ALL); a tumor phenotype reported to be relatively NK-resistant. The mechanisms underlying sensitization have not been fully identified but up regulation of ligands for TRAIL and the NK activating receptor NKG2D: MICA MICB and the UL16-binding proteins, may have a role. We set out to explore ALL susceptibility to NK cytotoxicity and whether this could be modulated by drug treatment. In contrast to published data, untreated ALL cell lines were positive for surface expression of MICB and ULBP2. Median fluorescence intensity ratios (mean ± SD; n = 6) for MICB detection on the cell lines 697, NALM-6, BV173 and SEM were: 3.2 ± 0.9; 3.8 ± 1.3; 4.0 ± 0.5; 2.5 ± 0.9, respectively and for ULBP-2: 2.3 ± 0.4; 55 ± 4.9; 2.9 ± 0.2; 1.8 ± 0.4, respectively. NALM-6 was also positive for ULBP1 (3.3 ± 0.6) while all were negative for MICA and ULBP3. Susceptibility of untreated ALL lines to NK mediated killing was assessed by chromium release assay using an IL-2 stimulated primary NK cell line. At effector to target ratio 40:1, specific release was 2.3% with cell line 697, 12% with NALM-6, 36% with BV173 and 63% with SEM. These results correlated with CD107a exposure in a degranulation assay using IL-2 stimulated peripheral blood lymphocytes: specific degranulation (% CD107a+ target with effector minus %CD107a+ effector alone) was 0.68% (697), 7.1% (NALM-6), 10% (BV173) and 17% (SEM). There was no correlation between baseline expression of NKG2DL and susceptibility to NK killing. Bortezomib, sodium valproate and troglitazone were added to cell cultures at sub-IC50 doses for 48 hours and compared with equimolar vehicle controls. Surface NKG2DL expression was measured by flow cytometry. On NALM-6 troglitazone treatment increased ULBP1 MFI by 2.0 ± 0.33 fold compared with vehicle control and increased percentage of ULBP1 positive cells by 39.6% (paired t-test: p=0.063). Sodium valproate increased MICA expression by 2.91 ± 1.18 fold and percentage of MICA positive cells by 12.3% (p=0.0382). On BV173, sodium valproate treatment increased ULBP2 MFI by 1.55 ± 0.07 fold and percentage of ULBP2 positive cells by 8.6% (p=0.04). There were no significant ligand changes after drug treatment on cell line 697. No NKG2DL changes were seen after Bortezomib treatment on any cell line. The functional significance of NKG2DL changes was assessed by CD107a degranulation assay. NALM-6 treated for 48 hours with drugs yielded the following fold increases in specific degranulation of NK cells compared to NALM-6 vehicle controls: 5.02 ± 5.98 for Bortezomib (mean ± SD), 2.4 ± 0.67 for Troglitazone and 1.44 ± 0.13 for Valproate. Levels of NK degranulation with 697 were very low (<5%) and drug treatment had no effect. Finally, we demonstrated that sensitization of NALM-6 was at least partly dependent on NKG2DL recognition, since blocking antibody to NKG2D reduced CD107a exposure by all three drugs. Compared with controls, blocking reduced CD107a expression by 59 ± 12% for Bortezomib, 47 ± 1.1% for Troglitazone and 48 ±11% for valproate-treated cells. This result was unexpected for Bortezomib as no changes in surface NKG2DL expression were detected after drug treatment. However, we are investigating the possibility that Bortezomib may down-regulate HLA class I expression, thus reducing inhibitory signaling upon NALM-6/NK interaction and unmasking an activation pathway that signals through NKG2D. In conclusion, we found basal levels of expression of NKG2DL on ALL cell lines. There was no correlation between NKG2DL expression and susceptibility to NK lysis, although this was to be expected given that a wealth of other activating and inhibitory receptors that contribute to NK activation. Bortezimib, valproate and troglitazone induced NKG2DL expression and sensitization to NK recognition in a cell line-specific manner. These drugs may therefore be useful to augment conventional chemotherapy or immunotherapeutic approaches to ALL. Disclosures:No relevant conflicts of interest to declare.