Natural Killer Cell Line Research Articles

PHENOTYPIC AND FUNCTIONAL CHARACTERISTICS OF MICROVESICLES PRODUCED BY NATURAL KILLER CELLS

Natural killer (NK) cells are of special interest among a multitude of microvesicle (MV) source cells. NK cells are a lymphocyte subpopulation performing contact cytolysis of virus-infected cells and tumor cells. Each of the NK cell populations has a unique receptor repertoire on its surface and, thus, unique functions. During their contact with a target cell, the most common mechanism of cytolysis is an exocytosis of lytic granules. However, some indirect evidence suggests that MV with CD56 phenotype and leukocyte-derived MV with various phenotypes are present in the peripheral blood plasma.This research is aimed to study the phenotype, composition and cytotoxic activity of microvesicles produced by NK cells. The analysis of receptor expression showed that MV, as well as source cells of the NK-92 cell line, had a similar CD56 molecule expression profile. The expression profile in MV differs from the same in source cells by higher CD119 and CD11b expression and by lower CD18 expression. Culturing of NK-92 cells in the presence of PMA, IL-1β, TNFα, IFNγ resulted in alterations of cell phenotypes and MV. Immunoblots revealed a change of perforin and granzyme B (GrB) in MV. The analysis of the cytotoxic activity of NK-92 cells in a natural killer in vitro assay employing K562 target cells demonstrated that MV obtained from TNFα-activated cells of the NK-92 cell line increased the cytotoxicity of the same TNFα-activated NK-92 cells regarding cytotoxicity levels. This coincides with the previously revealed increased content of GrB in MV obtained from TNFα-activated cells of the NK-92 cell line. To sum up depending on the cytokine NK-92 cells produce MV that differ in their phenotype, composition and activity. Any changes in MV composition can result in changes in their functional activity: in particular, changes can increase the cytotoxic activity of NK cells of the NK-92 cell line. Thus, besides a well-known and proved way for GrB delivery to a target cell, we can suggest an additional way – the transportation of GrB within MV.

Efficient ADCC killing of meningioma by avelumab and a high-affinity natural killer cell line, haNK.

Meningiomas are the most common adult primary tumor of the central nervous system, but there are no known effective medical therapies for recurrent meningioma, particularly for World Health Organization grade II and III tumors. Meningiomas arise from the meninges, located outside the blood-brain barrier, and therefore may be directly targeted by antibody-mediated immunotherapy. We found that programmed cell death ligand 1 (PD-L1) was highly expressed in multiple human malignant meningioma cell lines and patient tumor samples. PD-L1 was targeted with the anti-PD-L1 antibody avelumab and directed natural killer cells to mediate antibody-dependent cellular cytotoxicity (ADCC) of PD-L1-expressing meningioma tumors both in vitro and in vivo. ADCC of meningioma cells was significantly increased in target cells that upregulated PD-L1 expression and, conversely, abrogated in tumor cells that were depleted of PD-L1. Additionally, the high-affinity natural killer cell line, haNK, outperformed healthy donor NK cells in meningioma ADCC. Together, these data support a clinical trial designed to target PD-L1 with avelumab and haNK cells, potentially offering a novel immunotherapeutic approach for patients with malignant meningioma.

1199P - MG1124, a novel CEACAM1-targeted monoclonal antibody, has therapeutic potential as a combination partner of PD-1 inhibitors in NSCLC patients

BackgroundCEACAM1 is a member of the CEA family expressed in cell membranes of T cells and NK cells as well as tumor cells. CEACAM1 overexpressed in tumor cells interacts with CEACAM1 expressed in tumor infiltrating lymphocytes (TIL) and suppresses TIL activity by sending an inhibitory signal to immune cells, which is an immune escape mechanism in cancer. Therefore, we developed MG1124, a monoclonal antibody that specifically binds to CEACAM1 but not to other CEA family members, thereby blocking tumor growth by triggering immune response. MethodsCEACAM1 specific binding of MG1124 was tested in CEACAM1 or other CEA family over-expressing cells. MG1124 was examined by in vitro tumor cell lysis assays using NK92, a human NK cell line. The anti-tumor efficacy of MG1124 alone or in combination with a PD-1 inhibitor was studied in a humanized mouse model engrafted with non-small cell lung cancer (NSCLC) patient-derived tumor xenografts (NSCLC hu-PDX models). ResultsAn anti-CEACAM1 antibody, MG1124 binds to CEACAM1 overexpressing Jurkat cells but not to other CEA family members and blocks ligands interaction in a dose-dependent manner. MG1124 increased NK92-mediated tumor cell lysis against cancer cells with strong CEACAM1 expression, but not to those with no or weak CEACAM1 expression. In a single mouse trial, MG1124 (tumor growth inhibition (TGI) 55%) was efficacious in the high CEACAM1 expressing NSCLC hu-PDX model as monotherapy. In addition, the combination therapy (TGI 42%) with a PD-1 inhibitor displayed enhanced antitumor activity than the anti-CEACAM1 monotherapy (TGI 17%) in a CEACAM1-low expression model as well as in a CEACAM1-high expression model (combination therapy TGI 63% vs monotherapy TGI 46%). Moreover, tumor tissues (n=150) of NSCLC patients showed extensive expression of CEACAM1, suggesting a potential therapeutic use of MG1124 in NSCLC patients. ConclusionsIn this study, the anti-CEACAM1 antibody MG1124 showed antitumor efficacy in human CEACAM1-expressing cancer cell lines and NSCLC hu-PDX xenograft models, and demonstrated potential as a targeted therapy for NSCLC, both as a single agent and in combination with a PD-1 inhibitor. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

358P - Complete responses in patients with second-line or greater metastatic triple negative breast cancer (TNBC) following first-in-human immunotherapy combining NK and T cell activation with off-the-shelf high-affinity CD16 NK cell line (haNK)

BackgroundTNBC is an aggressive subtype with limited treatment options. We hypothesize that effective response against TNBC requires a coordinated approach that orchestrates both the innate and adaptive immune system. We further hypothesize that by orchestrating the activation of the entire immune system, we could accomplish immunogenic cell death with durable responses in this disease. We describe a first-in-human novel combination immunotherapy protocol of chemoradiation, cytokine-induced NK and T cell activation, checkpoint inhibition, and off-the-shelf high-affinity NK cell infusion. MethodsWe instituted this novel immunotherapy protocol consisting of low-dose metronomic chemoradiation therapy, combined with a novel IgG1 Fc-engineered IL-15-complexed protein (NabFc-N803), adenoviral and yeast tumor-associated antigen vaccines (MUC1, brachyury, CEA), a PD-L1 checkpoint inhibitor, and off-the-shelf high-affinity NK cells (haNK). A phase 1b trial in pts with metastatic TNBC was initiated in 9 pts, 8 of whom had relapsed after two or more lines of prior therapy (3rd-line). All pts received this combination therapy instituted over a three-week cycle as outpatients. Safety was assessed and efficacy confirmed by CT. ResultsNine pts have been treated to date. All pts received at least 3 treatment cycles. 8 grade ≥3 treatment-related AEs were observed in 4 pts to date, of which 2 had haNK-related SAEs consisting of fever and fatigue. No pts experienced cytokine release syndrome. To date, early efficacy results are as follows: a disease control rate (CR+PR+SD) of 78% (7/9 pts); ORR (PR+CR) of 56% (5/9 pts). Two pts (22%; 2/9) achieved a complete response (CR). To date, 8 pts are alive, and the duration of response ranges from 2mo to over 12mo. 7 pts remain on study to date. ConclusionsThis first in human clinical trial of combination chemoradiation, cytokine fusion protein, checkpoint inhibitor, and NK cell therapy demonstrated a safe and tolerable immunotherapy protocol. Early efficacy data is encouraging with a 78% disease control rate, 56% ORR and 22% (2/9) CR in pts with metastatic TNBC, 2nd-line or greater. Clinical trial identificationNCT03387085. Legal entity responsible for the studyNantKwest, Inc. FundingNantKwest, Inc. DisclosureP. Soon-Shiong: Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: NantCell; Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: NantKwest; Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: NantHealth; Leadership role, Shareholder / Stockholder / Stock options, Officer / Board of Directors: NantBio; Shareholder / Stockholder / Stock options: Celgene. S. Rabizadeh: Leadership role, Officer / Board of Directors: NantBio; Leadership role, Officer / Board of Directors: NantCell. J.H. Lee: Leadership role, Officer / Board of Directors: NantKwest; Leadership role, Officer / Board of Directors: NantCell. L. Sender: Leadership role, Officer / Board of Directors: NantKwest. F. Jones: Leadership role, Full / Part-time employment: NantCell; Leadership role, Officer / Board of Directors: Etubics. K. Niazi: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: NantBio. T. Seery: Speaker Bureau / Expert testimony: Ipsen; Advisory / Consultancy, Speaker Bureau / Expert testimony: Bayer. A. Rock: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment: NantCell. All other authors have declared no conflicts of interest.

Magnetic Nanoparticles Attached to the NK Cell Surface for Tumor Targeting in Adoptive Transfer Therapies Does Not Affect Cellular Effector Functions.

Adoptive cell transfer therapy is currently one of the most promising approaches for cancer treatment. This therapy has some limitations, however, such as the dispersion of in vivo-administered cells, causing only a small proportion to reach the tumor. Nanotechnological approaches could offer a solution for this drawback, as they can increase cell retention and accumulation in a region of interest. In particular, strategies employing magnetic nanoparticles (MNPs) to improve targeting of adoptively transferred T or NK cells have been explored in mice. In vivo magnetic retention is reported using the human NK cell line NK-92MI transfected with MNPs. Primary NK cells are nonetheless highly resistant to transfection, and thus we explore in here the possibility of attaching the MNPs to the NK cell surface to overcome this issue, and examine whether this association would affect NK effector functions. We assessed the attachment of MNPs coated with different polymers to the NK cell surface, and found that APS-MNP attached more efficiently to the NK-92MI cell surface. In association with MNPs, these cells preserved their main functions, exhibiting a continued capacity to degranulate, conjugate with and lyse target cells, produce IFN-γ, and respond to chemotactic signals. MNP-loaded NK-92MI cells were also retained in an in vitro capillary flow system by applying an EMF. A similar analysis was carried out in primary NK cells, isolated from mice, and expanded in vitro. These primary murine NK cells also maintained their functionality intact after MNP treatment and were successfully retained in vitro. This work therefore provides further support for using MNPs in combination with EMFs to favor specific retention of functional NK cells in a region of interest, which may prove beneficial to adoptive cell-therapy protocols.

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

Recurrent inflammatory disease caused by a heterozygous mutation in CD48

Abstract 1434: The Costimulatory Signal Domains 4-1BB and CD3ζ Do Not Improve the Function of CD16A Chimeric Antigen Receptor Transduced NK Cells

Abstract Chimeric Antigen Receptors (CAR)-T cells are genetically modified to express an extracellular binding domain, transmembrane domain and intracellular signaling domain. The extracellular binding domain is usually derived from antibodies; however, it can be replaced with other ligand-receptor binding systems, such as with an Fc receptor. CD16A has been tested in second-generation 4-1BB-CD3ζ CAR T cells and NK cell lines (NK-92) and enhances tumor killing; however, it is not clear whether these cells provide superior tumor killing compared to cells modified to express CD16A alone. In this study, we designed three CAR constructs containing the extracellular binding domain of high affinity mutant CD16A (CD16A-V158) and intracellular signal through 4-1 BB-CD3ζ that differed in leader sequences and transmembrane domains. CAR1 contained the CD16A leader and CD16A transmembrane domain; CAR2 had the CD16A leader and CD8α transmembrane domain; CAR3 contained the CD8α leader and CD8α transmembrane domain. When transduced into Jurkat cells via lentivirus, CD16A V158 and all three CARs showed stable high surface expression of CD16A (> 70%). In contrast, NK-92 cells were only successfully transduced to express CD16 by CD16A V158 (29.8%), CAR2 (42.3%), and CAR3 (42.5%) with no expression of CD16A by CAR1. Transduced cells were sorted by FACs based on CD16A expression. All sorted cells showed >90% stable expression of the constructs for up to 42 days post sorting. We observed a much higher CD16 MFI in cells transduced with CAR2 and CAR3 compared with CD16A V158. Western blotting with anti-CD3ζ identified the full assembly of CARs. We then tested the function of the transduced cells by a Rituximab binding assay. Although there was no difference in binding to Rituximab at 0.01 and 0.1 mg/mL; CAR2 and CAR3 showed better binding at the 1 and 10 mg/mL. In a 4-hour coculture toxicity assay with 721.221 LCLs, CD16A V158 and CAR3 increased the target lysis by two-fold while CAR2 only showed a 50% increase. When cocultured with 721.221 HLA-E+ LCLs, a 721.221 subclone more resistant to killing by NK-92 cells, we observed similar tumor killing with CD16A V158 and CAR3 (killing doubled) in contrast to CAR2(killing increased by only 50%). We next tested against the B-cell line Raji and found that there was only a mild increase of target lysis with transduced NK-92 cells; however, the CD16A V158 and CAR3 still showed higher toxicity (around a 40% increase). In summary, CAR2 and CAR3 expressed a higher CD16 MFI in NK cells and demonstrated better binding to Rituximab at1 and 10 mg/mL compared to CD16A V158; however, CAR3 and CD16A V158 had similar killing of target cells that was higher than was observed with CAR2. Our data indicate that NK cells transduced to have a CAR structure combining CD16A with 4-1BB-CD3ζ have augmented ADCC, although this augmentation is not superior to NK cells that are simply transduced to express CD16A alone. Citation Format: Long Chen, Vicky Li, David Allan, Robert Reger, Elena Cherkasova, Stephanie Pierre, Stefan Barisic, David Granadier, Emily Levy, Giacomo Waller, Susan Doh, Mala Chakraborty, Kate Stringaris, Richard Childs. The Costimulatory Signal Domains 4-1BB and CD3ζ Do Not Improve the Function of CD16A Chimeric Antigen Receptor Transduced NK Cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1434.

Abstract LB-102: Extranodal natural killer/T cell lymphoma (ENKTL) exhibits an unprecedented degree of global DNA hypermethylation, providing a potent targeted therapy in vivo

Abstract Extranodal natural killer/T cell lymphoma (ENKTL) is a rare aggressive form of non-Hodgkin lymphoma that is uniformly EBV-positive. Despite the implementation of combinatorial chemotherapies, almost all patients with advanced ENKTL die of their disease. The rational identification of potential novel therapeutic targets in ENKTL would be greatly aided by a more complete understanding of its molecular pathogenesis. We have identified atypical NK cell populations in untreated ENKTL patient samples. These atypical NK cells consistently showed an undifferentiated immunophenotype reminiscent of immature NK developmental stages. As cellular differentiation depends on epigenetic modifications and EBV is known to disrupt epigenetic patterns, we performed genome-wide DNA methylation profiling of ENKTL tumors (n=31 patients) using Illumina MethylationEPIC BeadChip arrays. We uncovered an unprecedented degree of epigenetic dysregulation in ENKTL, primarily involving extensive DNA hypermethylation. In the majority of ENKTL cases, greater than 50% of all CpG islands were hypermethylated, thus representing the most hypermethylated tumor type identified relative to available pan-cancer data (TCGA). In order to explore strategies to target the aberrant DNA hypermethylation in ENKTL, we exposed ENKTL cell lines YT and NK-92 to the demethylating agent 5-azacytidine (5-aza). We observed an unusually high sensitivity of ENKTL cell lines to 5-aza when compared to various other hematologic cancer cell lines, including several EBV-positive lymphoma lines. Hypomethylating agents altered DNA methylation at gene promoters and led to re-expression of epigenetically-silenced tumor suppressor genes in ENKTL cells. We also found synergistic inhibition of in vitro cell growth when combining 5-aza with the standard-of-care chemotherapeutic agents oxaliplatin and gemcitabine. Finally, through engraftment of primary ENKTL patient tumor cells, we have established a patient-derived xenograft (PDX) ENKTL mouse model. In this model, treatment with 5-aza resulted in profound cytoreduction, phenotypic and molecular differentiation of ENKTL cells, as well as significantly prolonged survival. In summary, we report massive, widespread DNA hypermethylation in ENKTL that exceeds the degree of hypermethylation found in other profiled cancers, including other EBV-positive malignancies. We hypothesize that epigenetic dysregulation plays a central role in ENKTL and rational targeting with epigenetic therapies may provide therapeutic benefit to patients. Our ongoing studies aim to identify the optimal epigenetic therapy and potential synergy with chemotherapy in preclinical models, thus providing the basis for novel treatments for ENKTL. Citation Format: Christoph Weigel, Bethany L. Mundy-Bosse, Yue-Zhong Wu, Kathleen McConnell, Anjali Mishra, Michael A. Caligiuri, Robert A. Baiocchi, Yaso Natkunam, Pierluigi Porcu, Jonathan Brammer, Aharon G. Freud, Christopher C. Oakes. Extranodal natural killer/T cell lymphoma (ENKTL) exhibits an unprecedented degree of global DNA hypermethylation, providing a potent targeted therapy in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-102.

Abstract 3947: Mechanisms of action of a neoantigen-targeting antibody NEO-201

Abstract NEO-201 is a humanized IgG1 monoclonal antibody (mAb) that reacts to tumor-associated antigens (TAA) derived from pooled allogeneic colon tumor tissue extracts. NEO-201 recognizes tumor-associated variants of CEACAM family members. This mAb is remarkably tumor-specific in its staining profile and reacts to a wide range of human carcinoma cell lines by flow cytometry and tumor tissues by immunohistochemistry. NEO-201 exhibited both ADCC and complement-dependent cellular cytotoxicity (CDC) activity against human carcinoma cell in vitro and counteracted the growth of human pancreatic xenograft tumors in vivo. We investigated an additional mechanism of action of NEO-201 and to further confirm the rationale for clinical testing. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell-surface protein expressed by immune cells and tumor cells, and it can inhibit T cell function similar to PD-1 and CTLA-4. CEACAM1 is also a potent inhibitor of natural killer (NK) cell function. Binding between CEACAM1 on NK cells and CEACAM1 or CEACAM5 on tumor cells inhibits activation signaling by NKG2D, which prevents NK cell cytolysis and permits tumor cells to evade NK killing. This study was designed determine whether NEO-201 blocks the CEACAM1 inhibitory pathway to restore antitumor functionality to NK cells. In vitro assays using human tumor cell lines were conducted to identify CEACAM family members bound by NEO-201. Functional assays were conducted to assess the ability of NEO-201 to potentiate the in vitro killing of tumor cells by the NK cell line NK-92, which expresses CEACAM1 and lacks CD16 and the ability to mediate ADCC. NEO-201 was found to react with distinct variants of CEACAM5 and CEACAM6, but not with CEACAM1 or CEACAM8. Expression profiling revealed that various NEO-201+ cell lines expressed differing levels of the native forms of CEACAM5/6 vs. the NEO-201-reactive variant forms of these molecules. Functionally, NEO-201 treatment augmented the cytolytic activity of NK-92 cells against NEO-201+ tumor cells in proportion to their level of CEACAM5 expression (average increase of 2-fold), but not against NEO-201+ cells that only expressed CEACAM6. This study demonstrates that NEO-201 is capable of an additional function. This antibody can block the interaction between tumor cell CEACAM5 and NK cell CEACAM1 to reverse CEACAM1-dependent inhibition of NK cytotoxicity. Experiments are in progress to determine the involvement of NK cell CEACAM1 and/or other checkpoint pathways in this mechanism of action. These results suggest that NEO-201 may potentially reverse CEACAM1-dependent immunosuppression of NK cells in patients whose tumors express the NEO-201-reactive variant of CEACAM5. Citation Format: Philip Martin Arlen, Massimo Fantini, Justin David, Christina M. Annunziata, Kwong Y. Tsang, Pia Morelli. Mechanisms of action of a neoantigen-targeting antibody NEO-201 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3947.

Abstract 2319: Accumulation and anti-tumor effect of chimeric antigen receptor (CAR) NK cells in metastasis uveal melanoma

Abstract Introduction: Metastatic uveal melanoma (MUM) is resistant to currently available treatments and overall prognosis is poor. MUM is an “immune cold” tumor and immune checkpoint blockades have only a marginal effect on this tumor. To bring immune cells to the tumor site and change the tumor microenvironment, we developed Chimeric Antigen Receptor (CAR)-NK cells targeting high molecular weight melanoma associated antigen (HMW-MAA/CPSG4). Methods: NK92 cell line derived from NK cell lymphoma was obtained from the American Type Culture Collection (ATCC). NK92 cells were transfected with retroviral pBMN-I vector co-expressing Green Fluorescent Protein (GFP) and anti-HMW-MAA-CD28-CD3z CAR construct. The efficacy of CAR construct transfection was confirmed by expression of His-Tag and F(ab)2 by using BD FACS Celesta flow cytometry. MUM cell line, TJU-UM001, was established from a hepatic metastasis of a uveal melanoma patient in our institution. TJU-UM001 cells have approximately 37,000 HMW-MAA molecules on their surface and are sensitive to NK92 cell killing. Tdtomato-transfected TJU-UM001 cells (106 cells) were intra-hepatically implanted into NOD-scid IL2Rgammanull (NSG) mice. Once establishment of hepatic tumor was confirmed by IVIS 200 imaging system (PerkinElmer) at Red Fluorescent Protein (RFP) excitation 535nm, 3 x 106 CAR-NK92 cells or NK92 cells without CAR constructs were injected into the spleen. Accumulations of GFP+ NK92 cells in the TJU-UM001 tumors in the liver were analyzed under fluorescence microscopy. In a separate experiment, 107 TJU-UM001 cells were subcutaneously implanted into NSG mice. The size of subcutaneous tumor was measured by Caliper. Once tumor became palpable (7-10 mm), 3 x 106 CAR- or non-CAR NK92 cells were directly injected to the MUM tumors. Forty thousand units of human IL-2 were intra-peritoneally injected into mouse once daily for 4 consecutive days after NK92 cell injections. Results: There were expressions of both His-tag and F(ab)2 on CAR-GFP NK92 cells. GFP and CD54 (ICAM-1) were expressed on 95% and 90% of CAR-GFP NK92 cells, respectively. There was no cross-expression of GFP or human CD54 on mouse cells. Therefore, GFP and CD54 were confirmed to be reliable markers for tracking CAR-GFP NK92 cells in mice. Both CAR- and non-CAR NK92 cells exhibited suppression of TJU-UM001 cell proliferation in in vitro assay. Direct injections of both CAR- and non-CAR NK92 cells showed anti-tumor growth effects on subcutaneously implanted TJU-UM001 tumors. More importantly, via splenic injection, CAR-NK92 cells accumulated selectively in the MUM tumors in the liver, while non-CAR NK92 cells were randomly distributed to normal liver tissue and the MUM tumors. These results support the rationale for hepatic arterial infusion of CAR-NK cells as treatment for uveal melanoma patients with hepatic metastasis. Citation Format: Bao Quoc Lam, Takahito Sugase, Mizue Terai, Meggie Danielson, Nadezhda Anikeyeva, Melissa A. Wilson, Yuri Sykulev, Takami Sato. Accumulation and anti-tumor effect of chimeric antigen receptor (CAR) NK cells in metastasis uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2319.

Abstract 4977: Dynamic transcriptional programs controlling single NK cell killing dynamics

Abstract NK cells have a vast heterogeneity in function and phenotype in vivo and their healthy function is critical in the prevention of nascent cancers. NK cell-based immunotherapies, such as adoptive NK cell therapy, are emerging and greater insights into the mechanism of NK cell activation could increase their efficacy. In vitro cytotoxicity assays have revealed that a small subset of NK cells kill the majority of cancer target cells, yet little is known about the dynamic state or triggers of these highly cytotoxic cells. Here, we present methodology for discovery of the molecular precursors and drivers of single NK cell killing events. We study the NK-92 cell line both as a model of primary NK cells and to gain direct insights for improving NK-92 based adoptive cell therapies. In our method, we first performed single cell RNA sequencing on NK-92 cells seeded sparsely on a monolayer of HeLa cancer cells along with a control NK-92 cell population. Clustering cells by principal component analysis revealed groups of NK cells that upregulated genes associated with NK cell cytotoxicity in the HeLa cell activated sample that are absent in the control group. Landmark genes associated with the cytotoxic NK cell clusters included PRF1, GZMK, and ITGB2. Utilizing live cell imaging, we identified categories of dynamic functional phenotypes in NK-92 cells exposed to a HeLa cell monolayer including non-motile and non-killing, highly motile but non-killing, single killing, serial killing, and exhausted. In order to link the systems-level single cell gene expression data to functional NK cell behaviors, we next performed high content live cell imaging of NK-92 cells bearing cas9-based endogenous genetic reporters as they surveilled and killed HeLa cells. Utilizing a custom analysis algorithm, we tracked the motility and killing activity of individual NK-92 cells, and correlated this activity to the dynamic gene expression. Next, we aim to identify the causality and sequence of transcriptional changes associated with NK cell killing and extend our results to the analysis of the critical transcription factor networks governing dynamic NK cell functional phenotypes. We note that the single cell functional discovery strategy that we have shown can be applied more broadly to other model systems of the cancer microenvironment. Citation Format: Matthew S. Hall, Joseph T. Decker, Emanuelle I. Grody, Rachel B. Blaisdell, Jacqueline S. Jeruss, Lonnie D. Shea. Dynamic transcriptional programs controlling single NK cell killing dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4977.

Abstract 3265: NKTR-255, a polymer-conjugated IL-15 enhances anti-tumor NK cell responses and synergizes with monoclonal antibodies to provide long-term survival in human lymphoma model

Abstract Background: IL-15 is a cytokine that activates and provides survival benefit to NK cells. Exploiting the therapeutic value of native IL-15 has been challenging due to its unfavorable pharmacokinetic properties and tolerability. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. NKTR-255 has potential for providing an enhanced immunotherapeutic effect when combined with monoclonal antibodies that mediate tumor killing by antibody dependent cellular cytotoxicity (ADCC). Here we investigate the pharmacological properties of NKTR-255 on NK cells and the therapeutic effect of NKTR-255 when combined with tumor-directed monoclonal antibodies in a B cell lymphoma model. Methods: KHYG-1 cells (human NK cell line) were used to measure phosphorylated STAT5 (pSTAT5) and cell proliferation. Human PBMCs were stimulated with NKTR-255 and/or daratumumab for in vitro NK cell characterization. In the cytotoxic assay, mice received single IV doses of 0.3 mg/kg of NKTR-255 and splenic NK cells were co-cultured with YAC-1 cells (mouse T lymphoma cell line) to measure cytotoxic function. In the Daudi lymphoma model, 1x107 Daudi cells were inoculated IV on Day 0. NKTR-255 (0.03, 0.1 or 0.3 mg/kg SC) was administered on Days 14, 21 and 28 and antibody treatment was administered on Day 14 (daratumumab 0.5 mg/kg IP) or on Days 14 and 17 (rituximab 40 mg/kg IP). Survival rate was determined by onset of hindlimb paralysis as a surrogate parameter. Results: NKTR-255 dose-dependently induced pSTAT5 and proliferation in KHYG-1 cells (EC50 values for pSTAT5: 0.2 ng/ml, proliferation: 5 ng/ml). NKTR-255 also resulted in enhanced pSTAT5 and NKG2D surface expression on human primary NK cells. In addition NKTR-255 increased NK cell degranulation in co-culture experiments with U266 cells (myeloma cell line) with/without daratumumab (anti-human CD38 antibody), as determined by enhanced CD107a surface expression. In vivo pretreatment with NKTR-255 resulted in sustained cytotoxic function of NK cells in both ex vivo and in vivo studies. Finally, NKTR-255 synergistically provided long-term survival benefit in a dose-dependent manner when administered with daratumumab or rituximab (anti-human CD20 antibody) in the Daudi B cell lymphoma model. Conclusions: NKTR-255 is an immune stimulator of NK cells that provides a dose-dependent effect in the proliferation and activation of NK cells. This property of NKTR-255, when administered with daratumumab or rituximab, translates into enhanced therapeutic efficacies of the antibodies in a B cell lymphoma model. These results indicate that combining NKTR-255 with a tumor-directed antibody having an ADCC mechanism can provide a synergistic effect for treating cancers. Citation Format: Takahiro Miyazaki, Saul Kivimäe, Rhoneil Pena, Peiwen Kuo, Marlene Hennessy, Murali Addepalli, Neha Dixit, Wildaliz Nieves, Sara Sheibani, Mekhala Maiti, Laurie VanderVeen, Joanna Wilczek, Loui Madakamutil, Jonathan Zalevsky. NKTR-255, a polymer-conjugated IL-15 enhances anti-tumor NK cell responses and synergizes with monoclonal antibodies to provide long-term survival in human lymphoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3265.

CD25 and Protein Phosphatase 2A Cooperate to Enhance IL-2R Signaling in Human Regulatory T Cells.

Low-dose IL-2 therapy is a direct approach to boost regulatory T cells (Tregs) and promote immune tolerance in autoimmune patients. However, the mechanisms responsible for selective response of Tregs to low-dose IL-2 is not fully understood. In this study we directly assessed the contribution of CD25 and protein phosphatase 2A (PP2A) in promoting IL-2R signaling in Tregs. IL-2-induced tyrosine phosphorylation of STAT5 (pSTAT5) was proportional to CD25 levels on human CD4+ T cells and YT human NK cell line, directly demonstrating that CD25 promotes IL-2R signaling. Overexpression of the PP2A catalytic subunit (PP2Ac) by lentiviral transduction in human Tregs increased the level of IL-2R subunits and promoted tyrosine phosphorylation of Jak3 and STAT5. Interestingly, increased expression of CD25 only partially accounted for this enhanced activation of pSTAT5, indicating that PP2A promotes IL-2R signaling through multiple mechanisms. Consistent with these findings, knockdown of PP2Ac in human Tregs and impaired PP2Ac activity in mouse Tregs significantly reduced IL-2-dependent STAT5 activation. In contrast, overexpression or knockdown of PP2Ac in human T effector cells did not affect IL-2-dependent pSTAT5 activation. Overexpression of PP2Ac in human Tregs also increased the expressions of proteins related to survival, activation, and immunosuppressive function, and upregulated several IL-2-regulated genes. Collectively, these findings suggest that CD25 and PP2A cooperatively enhance the responsiveness of Tregs to IL-2, which provide potential therapeutic targets for low-dose IL-2 therapy.

Abstract 2387: NKG2A-antagonizing monoclonal antibodies identified through phage display

Abstract The inhibitory receptor NKG2A-CD94 is mainly expressed on NK cells and some T cell and mediates an inhibitory signal through association with a non-classical MHC class I molecule HLA-E, which is commonly over-expressed in human cancers. Targeting NKG2A with monoclonal antibody holds potentials in immunotherapy of cancer. However, proteins with homologous sequences such as NKG2C/CD94 or NKG2E/CD94 complexes generally deliver an activating signal upon ligand binding. NKG2A-specific antagonizing antibodies would be needed to further explore therapeutic potentials of targeting NKG2A for immunotherapy of cancer. We have developed and characterized panels of monoclonal antibodies (mAbs) that specifically recognize NKG2A. Balb/c or SJL mice had been immunized with recombinant NKG2A/CD94 protein, recombinant plasmids encoding hNKG2A or stable cell line with ectopic expression of hNKG2A. Splenocytes from such immunized animals were collected for construction of phage-displayed antibody library in a single chain variable fragment (scFv) format. The NKG2A-specific antibodies, that had no binding to NKG2C, NKG2E or CD94, were isolated through multiple rounds of phage panning and ELISA-based screening. Such antibodies also block HLA-E ligand binding and enhanced cytotoxicity of NK cell lines and primary NK cells on cancer cells. In summary, isolation and characterization of a panel of NKG2A-specific antagonizing antibodies enable us to further determine the feasibility of NKG2A-targeted immunotherapy of cancer. Citation Format: Jing Gao, Lili Hu, Ruirui Sui, Cuicui Guo, Lina Wang, Teddy Yang, Jack Han, Qing Duan, Louis Liu. NKG2A-antagonizing monoclonal antibodies identified through phage display [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2387.

Differential Immunomodulatory Effects of Human Bone Marrow-Derived Mesenchymal Stromal Cells on Natural Killer Cells.

Mesenchymal stromal cells (MSCs) modulate immune responses through cell contact-dependent or paracrine mechanisms and are themselves known to have low immunogenicity. Given the increasing use of both natural killer (NK) cells and MSCs in cell-based therapies, we investigated the interaction between the two cell types using the NK cell lines, KHYG-1 and NK-92, and human bone marrow-derived MSCs. NK lines were cocultured with MSCs, either directly or in a transwell system, and the effects on proliferation, interferon-gamma (IFN-γ) production, and cytolytic activity of NK cells were analyzed. Cytotoxicity was measured in a 4 h chromium release assay. MSCs did not affect the proliferation of NK cell lines but reduced IFN-γ production by KHYG-1, but not NK-92, when cocultured directly at 10:1 NK:MSC ratio. MSCs suppressed K562 lysis by both KHYG-1 and NK-92 cells in contact-free transwell cocultures but only reduced cytotoxicity of KHYG-1 and not NK-92 cells when cells were in direct contact in coculture. Immunosuppressive effects of MSCs were mediated by indoleamine-2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) secreted by MSCs and were abrogated in the presence of IDO and PGE2 inhibitors. In the presence of MSCs, granule polarization was suppressed and induced respectively, in KHYG-1 and NK-92. Consistent with this, MSCs were susceptible to lysis by NK-92 but not KHYG-1. These studies indicate the differential crosstalk between MSCs and two highly cytotoxic NK lines and may be important when designing future cell therapy protocols with these two cell types.

CD56 expression in breast cancer induces sensitivity to natural killer-mediated cytotoxicity by enhancing the formation of cytotoxic immunological synapse

We examined the potential value of the natural killer (NK) cell line; NK-92, as immunotherapy tool for breast cancer (BC) treatment and searched for biomarker(s) of sensitivity to NK-92-mediated cytotoxicity. The cytotoxic activity of NK-92 cells towards one breast precancerous and nine BC cell lines was analyzed using calcein-AM and degranulation assays. The molecules associated with NK-92-responsiveness were determined by differential gene expression analysis using RNA-sequencing and validated by RT-PCR, immunostaining and flow cytometry. NK-target interactions and immunological synapse formation were assessed by fluorescence microscopy. Potential biomarker expression was determined by IHC in 99 patient-derived BC tissues and 10 normal mammary epithelial tissues. Most (8/9) BC cell lines were resistant while only one BC and the precancerous cell lines were effectively killed by NK-92 lymphocytes. NK-92-sensitive target cells specifically expressed CD56, which ectopic expression in CD56-negative BC cells induced their sensitivity to NK-92-mediated killing, suggesting that CD56 is not only a biomarker of responsiveness but actively regulates NK function. CD56 adhesion molecules which are also expressed on NK cells accumulate at the immunological synapse enhancing NK-target interactions, cytotoxic granzyme B transfer from NK-92 to CD56-expressing target cells and induction of caspase 3 activation in targets. Interestingly, CD56 expression was found to be reduced in breast tumor tissues (36%) with strong inter- and intratumoral heterogeneity in comparison to normal breast tissues (80%). CD56 is a potential predictive biomarker for BC responsiveness to NK-92-cell based immunotherapy and loss of CD56 expression might be a mechanism of escape from NK-immunity.

PF439 EXPLORING THE THERAPEUTIC POTENTIAL OF T‐CELL‐RECEPTOR REDIRECTED CD3+ NATURAL KILLER CELLS FOR ADOPTIVE CELLULAR THERAPY TO ACUTE MYELOID LEUKEMIA

Background:Adoptive cellular therapy (ACT) with redirected T cells expressing a chimeric antigen receptor (CAR) or transgenic T‐cell‐receptor (tTCR) has revolutionized cellular immunotherapy to hematological neoplasia, in particular to acute lymphoid leukemia, and also shows great promise as therapy for solid tumors. While CARs can only detect fully cell surface expressed target structures, TCR‐mediated recognition is not limited to surface antigens, but covers processed tumor neoantigens derived from the whole proteome. However, mispairing of transgenic and endogenous TCRs and restriction to patient‐derived, autologous T lymphocytes with variable “fitness” and T cell subsets due to individual health conditions and age of the patient exemplify the current limitations encountered in TCR‐redirected ACT. The natural killer (NK) cell line NK92 elicits lytic activity comparable to T cells, has been approved by the FDA for ACT and shown not to cause graft‐vs‐host disease. Since NK cell redirection is currently limited to CARs, NK92 cells engineered to express a CD3/TCR or CD3/CD8/TCR complex might evolve as an attractive, standardized cellular source for off‐the‐shelf TCR‐based ACT.Aims:Thus, the goal of this study was to explore NK92‐CD3+ and NK92‐CD3+CD8+ variants redirected to acute myeloid leukemia (AML) by expression of different AML‐reactive TCRs for antileukemic immunity in vitro and in vivo.Methods:NK92 cells expressing the γδεζ‐chains of the CD3 complex (NK92CD3+) were kindly provided by Dr. C. Wölfel (III. Dept. of Medicine) and further genetically engineered to express different human CD8 isoforms. Upon retroviral transfer (RT) of 3 different codon‐optimized and murinized TCRs obtained from AML‐specific CD8+CTL clones into NK92‐CD3+cells TCR expression was examined by flow cytometry (FC). NK92CD3+TCR+as well as NK92CD3+CD8+TCR+cells (together referred to as NK92TCR+) were enriched using antibiotic selection and immunomagnetic cell sorting (MACS®). Biological reactivity of redirected NK92TCR+cells to patient‐derived (primary) AML blasts was tested in vitro by IFN‐γ ELISPOT and 51Cr‐release assays. Comparable expression‐profiling of checkpoint molecules on NK92 and NK92TCR+cells such as e.g. CD80/86, PD1/PDL1, NKG2A, TIM3, TIGIT and HLA‐E was performed by FC. A patient‐derived AML‐xenograft (PDX) model in NSG mice has been already established.Results:Following RT and enrichment >80% of NK92‐CD3+cells expressed a given TCR, and >90% of NK92CD3+CD8+TCR+cells could be obtained. Upon coculture with different primary AML samples and corresponding EBV‐LCL blasts as controls NK92CD3+TCR+ elicited TCR dependent IFN–γ release and exhibited cytolytic activity to some but not all AML samples. CD8 coexpression slightly increased reactivity as measured by IFN‐γ release. Interestingly, as TCR‐redirected NK92 antileukemic responses appeared to be inhibited to some but not all AML samples we found high expression of HLA‐E on these AML blasts and of NGK2A on NK92TCR+ cells. Current blocking studies to examine whether a HLA‐E/NKG2A interaction might impair NK92TCR+ mediated AML reactivity are ongoing and will be presented. Moreover, first preliminary in vivo experiments of adoptively transferred NK92TCR+ cells into a NSG‐AML PDX model indicated biological activity but further studies are in progress to evaluate this point in more detail.Summary/Conclusion:These studies demonstrate that the established and FDA approved NK92 cell line can be redirected to elicit TCR‐mediated antitumoral immunity to AML. NK92TCR+ cells might thus represent a promising universal tool for an ‘off‐the‐shelf’ ACT product

Discovery of a novel natural killer cell line with distinct immunostimulatory and proliferative potential as an alternative platform for cancer immunotherapy

BackgroundHuman natural killer (NK) cell lines serve as an attractive source for adoptive immunotherapy, but NK-92 remains the only cell line being assessed in the clinic. Here, we established a novel NK cell line, NK101, from a patient with extra-nodal natural killer/T-cell lymphoma and examined its phenotypic, genomic and functional characteristics.MethodsSingle cell suspensions from lymphoma tissue were expanded with anti-NKp46/anti-CD2-coated beads in the presence of IL-2. A continuously growing CD56+ cell clone was selected and designated as NK101. Flow cytometry and RNA sequencing were used to characterize phenotypic and genomic features of NK101. In vitro cytotoxicity and IFN-γ/TNF-α secretion were measured by flow cytometry-based cytotoxicity assay and enzyme-linked immunosorbent assay, respectively, after direct co-culture with tumor cells. Immunomodulatory potential of NK101 was assessed in an indirect co-culture system using conditioned medium. Finally, in vivo antitumor efficacy was evaluated in an immunocompetent, syngeneic 4T1 mammary tumor model.ResultsNK101 displayed features of CD56dimCD62L+ intermediate stage NK subset with the potential to simultaneously act as a cytokine producer and a cytotoxic effector. Comparative analysis of NK101 and NK-92 revealed that NK101 expressed lower levels of perforin and granzyme B that correlated with weaker cytotoxicity, but produced higher levels of pro-inflammatory cytokines including IFN-γ and TNF-α. Contrarily, NK-92 produced greater amounts of anti-inflammatory cytokines, IL-1 receptor antagonist and IL-10. Genome-wide analysis revealed that genes associated with positive regulation of leukocyte proliferation were overexpressed in NK101, while those with opposite function were highly enriched in NK-92. The consequence of such expressional and functional discrepancies was well-represented in (i) indirect co-culture system where conditioned medium derived from NK101 induced greater proliferation of human peripheral blood mononuclear cells and (ii) immunocompetent 4T1 tumor model where peritumoral injections of NK101 displayed stronger anti-tumor activities by inducing higher tumor-specific immune responses. In a manufacturing context, NK101 not only required shorter recovery time after thawing, but also exhibited faster growth profile than NK-92, yielding more than 200-fold higher cell numbers after 20-day culture.ConclusionNK101 is a unique NK cell line bearing strong immunostimulatory potential and substantial scalability, providing an attractive source for adoptive cancer immunotherapy.

Engineering antigen-specific NK cell lines against the melanoma-associated antigen tyrosinase via TCR gene transfer.

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T-lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL-TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off-target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/β chains, along with CD3 subunits, enables the functional expression of an antigen-specific TCR complex on NK cell lines NK-92 and YTS, demonstrated by using a TCR against the HLA-A2-restricted tyrosinase-derived melanoma epitope, Tyr368-377 . Most importantly, the introduction of a TCR complex to NK cell lines enables MHC-restricted, antigen-specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of "mispairing" in TCR gene therapy.

The role of oxidative stress in NK-cell based cancer immunotherapy

Immunooncology gained great attention in cancer treatment over the last few years. Adoptive cell therapy is a type of immunotherapy that uses cytotoxic lymphocytes or NK cells. NK cells precisely recognize and kill cancer cells without affecting healthy cells. These properties make NK cells an attractive strategy with potential therapeutic applications. NK cells can be further modified with chimeric antigen receptors (CAR). Currently, several dozen clinical trials with NK cells are registered for cancer treatment, including several CAR-NK cells or NK-92 cell line modified with CAR. Despite the undeniable success of immunotherapy in recent years, many concerns still remain unresolved. One of the challenges for the effective immunotherapy is the immunosuppressive tumor environment. Numerous studies indicate that persistent oxidative stress, an important element associated with cancer, inhibits NK cell activity. However, the exact mechanisms responsible for protecting NK cells against suppressive oxidative stress are not identified. Therefore, in my doctoral dissertation co-financed by the Scientific Polpharma Foundation doctoral scholarship I focused on investigating and identifying mechanisms protecting NK cells against oxidative stress. The obtained results indicate that NK cells are the most sensitive to the oxidative stress, when compared withother immune cells. Moreover, inhibition of PRDX1-TXN1-TXNRD1 system impairs NK cell effector functions, while the increase of PRDX1 significantly improves the survival of NK cells and CAR-NK cells in the presence of H<sub>2</sub>O<sub>2</sub>, resulting in improved effectiveness of NK cells under the oxidative stress conditions.