Human Peripheral Blood NK Cells Research Articles

Abstract 1326: Pre-clinical development of ARB011: A CDH17 targeting allogeneic nonviral RNA-based “Flash” CAR-NK therapy for gastrointestinal cancer

Abstract Background: Treatment for advanced gastrointestinal (GI) cancers has remained challenging, therefore, novel immunotherapies using chimeric antigen receptor (CAR)-T cells are being developed as a new hope for the patients. However, despite the clinical successes in hematologic malignancies, the overall safety and efficacy against solid tumors of CAR-T cells have yet to be established, and the manufacturing of autologous CAR-T cells requires lengthy cell expansion and viral transduction processes. In view of these technical challenges, we have developed ARB011, an RNA-based allogeneic CAR-NK therapy targeting GI cancers expressing cadherin-17 (CDH17), a promising therapeutic target with its overexpression associated with poor prognosis in patients with GI cancers. The use of allogeneic NK cells would mitigate the risk of graft-versus-host disease. Moreover, engineering NK cells with nonviral CAR-encoding mRNA would provide a rapid large-scale manufacturing platform with minimal risk of abnormal genetic alteration mediated by viral integration. Methods: To generate the ARB011 CAR-NK cells, ex vivo expanded human peripheral blood NK (Magicell-NK, Medigen, Taiwan) cells were electroporated with mRNA encoding a humanized anti-CDH17 scFv fused with transactivation domains. Flow cytometry, in vitro cytotoxicity assay, and cytokine release assay were performed to evaluate the expression and functionality of ARB011. To examine the developability of ARB011 as an off-the-shelf cell product, cryopreservation and large-scale electroporation were also studied. Results: Flow cytometry analysis revealed efficient electroporation with a high level of CDH17 CAR expression (>80%) and optimal CAR-NK cell viability (60%-80%). Following electroporation, cytotoxicity of ARB011 was tested against gastric (AGS and NCI-N87), colorectal (DLD-1 and T84), and liver (MHCC-97H) cancer cells by co-culturing at 1:1 and 2:1 effector/target ratio for 12-24h. ARB011 showed significant cytotoxicity against CDH17+ but not CDH17- cancer cells. The CDH17-sepcific cytotoxicity was accompanied with elevated levels of CD107a or LAMP-1 expression on CAR-NK cells and IFN-γ and TNF-α releases. Interestingly, cryopreserved ARB011 maintained optimal cell viability, CAR expression, and potent CDH17 target-specific cytolytic activity. Lastly, large-scale electroporation showed the establishment of up-scaling process of ARB011 production for clinical application. Conclusions: Overall, the findings suggest that ARB011 could be a novel off-the-shelf, CAR-NK therapy that warrants further studies in animal models and in clinical trials. Citation Format: Jaydeep Roy, Nga Tin Lin, Kronos Chow, Chih Ya Yang, James C.L. Lin, John M. Luk, Kwong F. Wong. Pre-clinical development of ARB011: A CDH17 targeting allogeneic nonviral RNA-based “Flash” CAR-NK therapy for gastrointestinal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1326.

CD19-Specific CAR NK Cells Coexpressing IL-21 Exhibit Superior Expansion and Antitumor Activity Against CD19-Postive Lymphoma

Background Chimeric antigen receptor (CAR) expressing NK cells induce robust antitumor responses in patients with hematologic malignancies but have limited persistent without cytokines supplement. IL-15 and IL-21 promote NK cell expansion, survival, and possess both overlapping and distinctive functions. CAR NK cells engineered to express IL-15 have shown long-term persistence and potent antitumor activity, however, the pleiotropic effects of IL-21 on CAR NK cell-mediated tumor rejection are lacking. Therefore, we compared the therapeutic potency of CD19-specific human NK cells that constitutively express IL-15 or IL-21. Methods The codon-optimized minigene encoding cytokine IL-21 or IL-15, linked with CD19-CAR containing 4-1BB costimulatory domain by a P2A sequence were synthesized and sub-cloned into the pSFG retroviral vector yielding the BBz.IL21 or BBz.IL15 retroviral construct. To produce CD19-CAR retrovirus, 293T/17 cells were transfected with a combination of plasmids containing CAR constructs, RDF plasmid and PegPam3 plasmid. Human peripheral blood NK cells isolated from healthy volunteer donors were stimulated with CD16/CD137 mAb and expanded with 500 IU/ml IL-2 (PeproTech) for 5 days before transduced with CAR retrovirus in plates coated with RetroNectin. The expression of CAR, phenotype, degranulation, cytokine production, and cytotoxicity of CAR NK cells were analyzed on day 3 post transduction. To measure IL-15 and IL-21 production, supernatants were collected from CAR NK cells cultured in the absence of IL-2 for 24h and evaluated by ELISA. Results After transduction, all constructs were stably expressed by NK cells, with construct containing IL-21 (71.23 ± 4.05 %, n = 3) demonstrating higher transduction efficiency compared with the BBz.IL15 (61.13 ± 3.43 %), but slightly lower than the BBz construct (79.63 ± 3.83 %). High levels of IL-21 were found in the supernatant of BBz.IL21 NK cells after resting 24h (3464 ± 199 pg/ml), while no detectable IL-21 was observed on BBz and BBz.IL15 NK cells. NK cells expressing BBz.IL15 produced significantly more IL-15 than BBz.IL21 and BBz NK cells as baseline (BBz.IL15: 213 ± 14 pg/ml, BBz.IL21: 61.86± 4 pg/ml, BBz: 60.56 ± 11 pg/ml). Given that IL-15 or IL-21 could potentially induce antigen-independent NK cell proliferation, we compared the ability of each NK-cell group to maintain growth in the presence of IL-2. The expansion of CAR NK cells expressing IL-21 was significantly higher than that of CAR NK cells expressing IL-15, while the number of BBz.IL15 NK cells and BBz NK cells were comparable (fold expansion in 5 days: BBz.IL21: 26.6 fold, BBz.IL15: 15.9 fold, BBz: 17.2 fold), suggesting a synergistic effect of IL-21 and IL-2 on CAR NK cell expansion. BBz.IL21 NK cells shared a similar immunophenotypic profile to BBz.IL15 NK cells, as the expression of inhibitory receptors PD1, LAG3, TIM3 and NKG2A, as well as activation receptors NKG2D and CD69 were comparable. CAR engagement by CD19-postive Raji cells specifically induced cytokines production by CD19-CAR NK cells but not by control NK cells. IL-21 coexpression significantly enhanced IFN-γ and TNF-α production compared with CAR NK cells as well as CAR NK cells expressing IL-15 (BBz.IL21 vs. BBz.IL15, P < 0.05; BBz.IL21 vs. BBz, P < 0.05; BBz.IL15 vs. BBz, P > 0.05). Moreover, a higher percentage of degranulated NK cells was observed on BBz.IL21 NK cells compared to BBz.IL15 NK cells and BBz NK cells. To confirm this observation, NK cells were cocultured with Raji cells at various E:T ratios for 6h. CAR NK cells expressing IL-21 showed superior cytotoxicity, compared to CAR NK cells expressing IL-15. Conclusion CD19-CAR NK cells engineered to express IL-21 gene produced high levels of IL-21 during cultivation and enabled greater ex vivo expansion of CAR NK cells compared with CAR NK cells expressing IL-15. IL-21 coexpression induced a significant increase in IFN-γ, TNF-α production and degranulation compared with IL-15 coexpression, resulting in enhanced antitumor activity of CD19-CAR NK cells against CD19-postive lymphoma.

Disruption of SOCS1 Promotes the Antitumor Activity of CD19-Specific CAR NK Cells Coexpressing IL-15

Background Chimeric antigen receptor (CAR) expressing NK cells exhibit robust antitumor responses in patients with hematologic malignancies but have limited persistent without cytokines supplement. Interleukin-15 (IL-15) promotes NK cells expansion and survival, while IL-15 signaling is often inhibited by the suppressor of cytokine signaling (SOCS) proteins (CIS, SOCS1-7), which is rapidly induced in response to IL-15 and negatively regulates cytokine signaling through the JAK/STAT pathway in NK cells. We hypothesized that SOCS1 would be a logical checkpoint to target in human NK cells to increase their persistence and thereby enhance their antitumor potency. Here, we devised a strategy that couples targeting the SOCS1 protein, with CD19-specific human NK cells that constitutively express IL-15. Method Firstly, we evaluated the SOCS1 mRNA expression levels on NK cells after cultured with or without IL-15 for 6 or 24 hours by reverse transcription polymerase chain reaction (RT-PCR). The retroviral vector encoding CAR19.4-1BB-zeta-2A-IL-15 (named BBz.IL-15) and the production of transient retroviral supernatants have been previously described. Human peripheral blood NK cells isolated from healthy volunteer donors were stimulated with CD16/CD137 mAb and expanded with 500 IU/ml IL-2 (PeproTech) for 5 days before transduced with CAR retrovirus in plates coated with RetroNectin. CAR expression on NK cells was determined by flow cytometry, and IL-15 secretion was measured by ELISA. Knockout (KO) of SOCS1 by CRISPR/Cas9 using the Neon transfection system was performed on day 3 post transduction in both non-transduced NK cells (NT-NK) and CAR-transduced NK cells (CAR-NK). To assess KO efficiency, we used polymerase chain reaction (PCR) gel electrophoresis, western blot, and Sanger sequencing. The degranulation, cytokine production, and cytotoxicity of CAR NK cells were analyzed on day 7 post electroporation. Results The expression of SOCS1 on NK cells was significantly induced within 6 hours of IL-15 treatment. Our approach for combined retroviral transduction with the anti-CD19 CAR and RNP-mediated gene editing of SOCS1 showed that surface expression of the CAR was maintained on NK cells in culture, with construct containing BBz.IL15 (37.18 ± 11.71%) demonstrating comparable transduction efficiency compared with the BBz construct (45.48 ± 10.54%) (P＞0.05), and the efficiency of SOCS1 KO was high in both the nontransduced (NT) control and CAR-expressing NK cells by TIDE and remained stable over time. When tested against CD19-postive Raji cells, SOCS1 KO BBz.IL-15 NK cells produced more IFN-γ and TFN-α, and displayed greater degranulation (CD107a) against their targets than did their BBz.IL-15 NK controls (BBz.IL-15 SOCS1 KO vs BBz.IL-15 control: 15.75±2.05% vs 7.93±0.47% for IFN-γ, P=0.032; 6.27±1.27% vs 1.88±0.26% for TFN-α, P=0.04; 23.25±2.33% vs 12.35±2.33% for CD107a, P=0.042). However, there was no significant difference between the SOCS1 KO BBz. or NT NK cells and their respective BBz. and NT controls regarding IFN-γ and TFN-α production and degranulation (BBz.SOCS1 KO vs BBz.control: 16.95±5.30% vs 16.85±8.70% for IFN-γ, P＞0.05; 6.83±2.98% vs 4.06±2.47% for TFN-α, P＞0.05; 26.65±7.14% vs 25.8±2.12% for CD107a, P＞0.05) (NT SOCS1 KO vs NT control: 21.75±6.58% vs 7.5±2.18% for IFN-γ, P＞0.05; 7.23±3.46% vs 1.58±0.03% for TFN-α, P＞0.05; 15.05±3.46% vs 6.84±2.15% for CD107a, P＞0.05). To confirm this observation, NK cells were cocultured with Raji cells at various E:T ratios for 6h. SOCS1 KO NK cells could kill the tumors more efficiently in comparison to the control. Proliferation data showed the SOCS1 KO NK cells can grow faster than the control group. Conclusion In conclusion, the results demonstrate that SOCS1 might be a potential target for genetic modification to improve activity of CAR- NK cells.

Development of allogenic nonviral RNA-based CAR-NK therapy targeting CDH17 in relapsed/refractory gastrointestinal cancer.

e14539 Background: Gastrointestinal (GI) cancers are commonly fatal after metastasis. The incidence and mortality of GI tumors are high, and the benefits of existing therapies are limited. Cadherin-17 (CDH17) is highly prevalent and specific to GI cancers which has potential for the development of novel treatments for GI cancers. Cell-based immunotherapy using genetically engineered autologous Chimeric Antigen Receptors T (CAR-T) cells has been clinically efficacious for treating hematological malignancies. However, its manufacturing is extremely expensive and lengthy with ~10% failure production rate. Equipping NK cells with CAR may represent a safer and equally effective approach for use in solid tumors as an alternative to autologous CAR-T-cell therapy. Nonviral RNA-based expression of CAR vector in NK cells will provide immediate and multiple accessibility to cancer patients, lower risk of abnormal genetic alteration mediated by viral integration, and potentially limit side effects. This paper demonstrates the development of RNA-CAR-NK targeting CDH17-positive GI cancers. Methods: Ex vivo expanded human peripheral blood NK (Magicell-NK, Medigen, Taiwan) cells were used in this study. To generate anti-CDH17 (C7) CAR-NK cells, mRNA containing a novel CDH17-specific single-chain variable fragment and the 4-1BB costimulatory domain was electroporated to Magicell-NK cell. Flow cytometry analysis was performed to evaluate the cell viability and the expression level of CAR and cytotoxic surface markers of the CAR-NK cells. CDH17 specific cytolytic activity against AGS gastric cancer cell line and the concentration of cytokines released by CAR-NK cells were measured by in vitro cytotoxicity assay and cytokine release assay, respectively. Results: Our results demonstrated efficient electroporation with high-level C7 CAR expression ( > 80%) and optimal CAR-NK cell viability (~60%). Interestingly, 12 h co-culture of C7 CAR-NK cells with AGS gastric cancer cells at 1:1 and 2:1 effector/target (E:T) ratio showed a significantly enhanced antitumor response of C7 CAR-NK cells on AGS-CDH17+ cells but not on AGS-CDH17- cells, indicating CDH17 target-specific cytolytic activity. Moreover, we observed an elevated level of cytokines such as INF-γ and TNF-α released by C7 CAR-NK cells only in co-culture with AGS-CDH17+ cells, confirming the mechanisms behind the CAR-mediated cytotoxicity. Furthermore, these results were supported by an increased level of surface CD107a or LAMP-1 expression in C7 CAR-NK cells during the 12 h of co-culture with AGS-CDH17+ cells at 1:1 and 2:1 E:T ratio. Conclusions: Our findings suggest that a non-viral based CDH17 CAR-NK cell therapy could be a potential allogeneic, off-the-shelf CAR-NK therapy candidate in GI cancer and support the rationale of further investigating it in vivo and clinical trials.

The co-inhibitory receptor TIGIT regulates NK cell function and is upregulated in human intrahepatic CD56bright NK cells.

The crosstalk between NK cells and their surrounding environment is enabled through activating and inhibitory receptors, which tightly control NK cell activity. The co-inhibitory receptor TIGIT decreases NK cell cytotoxicity and is involved in NK cell exhaustion, but has also been associated with liver regeneration, highlighting that the contribution of human intrahepatic CD56bright NK cells in regulating tissue homeostasis remains incompletely understood. A targeted single-cell mRNA analysis revealed distinct transcriptional differences between matched human peripheral blood and intrahepatic CD56bright NK cells. Multiparameter flow cytometry identified a cluster of intrahepatic NK cells with overlapping high expression of CD56, CD69, CXCR6, TIGIT and CD96. Intrahepatic CD56bright NK cells also expressed significantly higher protein surface levels of TIGIT, and significantly lower levels of DNAM-1 compared to matched peripheral blood CD56bright NK cells. TIGIT+ CD56bright NK cells showed diminished degranulation and TNF-α production following stimulation. Co-incubation of peripheral blood CD56bright NK cells with human hepatoma cells or primary human hepatocyte organoids resulted in migration of NK cells into hepatocyte organoids and upregulation of TIGIT and downregulation of DNAM-1 expression, in line with the phenotype of intrahepatic CD56bright NK cells. Intrahepatic CD56bright NK cells represent a transcriptionally, phenotypically, and functionally distinct population of NK cells that expresses higher levels of TIGIT and lower levels of DNAM-1 than matched peripheral blood CD56bright NK cells. Increased expression of inhibitory receptors by NK cells within the liver environment can contribute to tissue homeostasis and reduction of liver inflammation.

Engineering CD70-Directed CAR-NK Cells for the Treatment of Hematological and Solid Malignancies

Advances in cellular immunotherapy have led to multiple FDA approvals for autologous CAR-T cell therapies in acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphomas (NHL), and multiple myeloma (MM). While effective, autologous CAR-T therapies are limited by safety concerns, lack of scalability for patient derived starting material, and long vein-to-vein timelines. Allogeneic CAR-NK cell therapies have the potential to overcome these limitations by providing an off-the-shelf product capable of delivering clinical benefit without the safety and manufacturing challenges associated with CAR-T therapy. CAR-NK cell therapies are particularly attractive in AML as the inherent graft-versus-leukemia activity of NK cells can be effectively augmented by a CAR directed to an AML expressed antigen. CD70 expression is associated with several indications, including AML, NHL, and renal cell carcinoma (RCC), and it is an attractive target for CAR therapy in AML since it is highly expressed on leukemic stem cells and blasts and is absent in normal bone marrow hematopoietic stem cells. 1 While aberrant expression of CD70 is associated with several solid and hematological indications, its expression in normal tissue is restricted to immune cells including T, B, DC, and NK cells. 2 Here we demonstrate that CD70 is not expressed in resting peripheral blood NK cells but is strongly upregulated in response to NK cell activation by engineered feeder cells. Introduction of CARs targeting CD70 into activated NK cells leads to substantial reduction of NK cell expansion due to fratricide. While CD70 is expressed in activated NK cells, knockout (KO) of CD70 by CRISPR/Cas9 editing does not inhibit NK cell expansion nor impair endogenous cytotoxicity against K562 target cells. Using the non-viral TcBuster™ Transposon System (Bio-Techne), we were able to deliver transposons containing a CD70 CAR and an IL15 expression cassette while simultaneously knocking out CD70 by CRISPR/Cas9 in primary human peripheral blood NK cells. This single-step process resulted in >70% CAR integration/expression and >80% knockout of CD70. The resulting CD70 knockout CAR-NK cells were resistant to fratricide and expanded comparably to mock-engineered NK cells following feeder cell activation. The IL15 expression cassette enabled enhanced persistence of CAR-NK cells in vitro without exogenous cytokine support. In functional assays, CD70 KO NK cells engineered with the CD70 CAR and IL15 expression cassette mediated cytotoxicity against multiple CD70-positive tumor cell lines, expressed the degranulation marker CD107a (LAMP1), and expressed the cytokines IFNγ and TNFα. Overall, the results demonstrate the potential for targeting CD70 with CAR-NK cell therapy for the treatment of AML, RCC, and other CD70-positive malignancies while overcoming the risk posed by fratricide by engineering with a non-viral transposon delivery system in combination with CRISPR/Cas9 editing.1 Perna et al. 2017, Cancer Cell. 32:506-519.2 McEarchern et al. 2008, Clin Cancer Res. 14(23):7763-7772. DisclosuresChoi: Unum Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Walsh: Obsidian Therapeutics: Ended employment in the past 24 months. Khamhoung: Rubius Therapeutics, Inc.: Ended employment in the past 24 months. Johnson: Celsius Therapeutics: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Franco: KSQ Therapeutics: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months. Swiech: Agenus: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months; Unum Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Richardson: Novartis Pharma: Current equity holder in publicly-traded company; Obsidian Therapeutics: Current holder of stock options in a privately-held company, Ended employment in the past 24 months.

Incorporation of a Novel CD16-Specific Single-Domain Antibody into Multispecific Natural Killer Cell Engagers With Potent ADCC.

Multispecific antibodies that bridge immune effector and tumor cells have shown promising preclinical and clinical efficacies. Here, we isolated and characterized novel llama single-domain antibodies (sdAbs) against CD16. One sdAb, NRC-sdAb048, bound recombinant human and cynomolgus monkey CD16 ectodomains with equivalent affinity (KD: 1 nM) but did not recognize murine CD16. Binding was similar for human CD16a expressed on NK cells and CD16b (NA2) expressed on neutrophils but dramatically weaker (KD: ∼6 μM) for the CD16b (NA1) allotype. The sdAb stained primary human peripheral blood NK cells. Irrespective of fusion orientation and linker length, bispecific sdAb-sdAb and sdAb-scFv dimers (anti-CD16/EGFR, anti-CD16/HER2, and anti-CD16/CD19) retained full binding affinity for each target, coengaged both antigens simultaneously, elicited ADCC against target antigen-expressing tumor cells in a reporter bioassay, and triggered target-specific activation and degranulation of primary NK cells as measured via interferon-γ and CD107a expression. These molecules may have applications in cancer immunotherapy.

Serum- and feeder-free culture expansion of human peripheral blood NK cells

Abstract Natural killer (NK) cells are critical effectors of innate immunity that secrete proinflammatory cytokines and kill tumor cells and virus-infected cells. The ability to expand human NK cells in culture provides a ready source of cells for disease modeling, development of immunotherapies, and basic research. We have developed a culture system that supports the expansion of primary NK cells in the absence of feeder cells and serum. NK cells were enriched from peripheral blood using EasySep™ negative selection and cultured in serum-free medium with interleukin-2 in plates coated with a NK cell ‘activation coating matrix’ for 14 days. Cells were passaged with medium changes on days 7 and 11, and were harvested on day 14 for assessment or functional characterization. The average frequency of CD56+CD3− NK cells was 92% (range 80 – 97%, n=16) with an average expansion of 121-fold (range 10 – 274). On average 79% (48–92%) of expanded NK cells expressed CD16. The ability of expanded NK cells to produce interferon-γ (IFN-γ) and to degranulate were tested after stimulation with phorbol 12-myristate 13-acetate (PMA) + ionomycin or co-culture with K562 cells. As detected by intracellular flow cytometry, the average frequency of IFN-γ+ NK cells was 56% (range 41 – 63%, n = 4) and 40% (range 12 – 55%) when stimulated by PMA/ionomycin or K562 cells, respectively. Similarly, the average frequency of degranulated NK cells as detected by surface expression of CD107a, a lysosomal-associated membrane protein, was 69% (range 35 – 84%, n = 4) and 53% (range 18 – 75%), respectively. These results show that NK cells can be expanded and stimulated under serum- and feeder-free conditions to generate large numbers of functional NK cells for basic and translational research.

Abstract CT053: Use of CRISPR knock-out screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy

Abstract Natural killer (NK) cells play a key role in tumor immune surveillance by their ability to recognize and kill both hematological and solid tumor cells. NK cell-based immunotherapy is emerging as a powerful treatment against certain malignancies. However, intrinsic and extrinsic mechanisms can lead tumor cells to develop resistance mechanisms to escape NK cell-mediated killing, including up or down-regulation of genes involved in the interaction between tumor and immune cells. Here, we use a “two cell type” (TCT) screening system to identify novel molecular targets regulating NK cell-mediated killing of glioblastoma stem cells (GSC), meningioma cells, as well as head and neck squamous cells carcinomas (HNSCC). By using a whole genome-scale CRISPR-Cas9 library, we induced mutations in tumor cells to mimic loss of function mutations involved in resistance or sensitivity to NK cells. After a round of selection with human peripheral blood NK cells, we profiled the genes that impaired the killing activity of NK cells or increased the sensitivity of tumor cells to NK cells. The most common genes lost in GSC and meningioma cells that lead to increased sensitivity to NK cells are involved in the ER-phagosome pathway, antigen presentation and cellular localization. We then used GSC to validate our top genetic hits identified by the screening. One gene of interest, CHMP2A is a component of ESCRT-III, a complex involved in multi vesicular bodies and exosomes formation known to be involved in intercellular communications, mediating numerous physiological processes among which immunology and tumor biology. We confirmed that impaired expression of CHMP2A in GSC lines increases their sensitivity to NK cell-mediated killing. RNA sequencing demonstrates knock-out (KO) of CHMP2A leads to increased expression of immune response genes, including certain chemokines. Indeed, NK cells demonstrated increased migration towards KO GSC corresponding to higher concentrations of CXCL10, CXCL12 and IL-8 in GSC-KO conditioned media. Since HNSCC are among the most highly immune-infiltrated cancer types, including a high degree of infiltrating NK cells, we next tested CHMP2A-KO-Cal27 cells as a model of HNSCC. Again, we observed an increased sensitivity to NK cells, higher levels of CXCL10 and increased migration of NK cells, demonstrating these mechanisms regulate NK cell-mediated killing of diverse tumor types. Additionally, in vivo studies using a Cal27 xenograft model and adoptive transfer of human NK cells in immunodeficient mice demonstrate increased NK cell-mediated killing of CHMP2A-KO-Cal27 cells compared to wild type Cal27 cells. Together, these studies demonstrate the utility of a TCT screen to identify new mechanisms that mediate resistance of diverse tumor types to NK cell-mediated killing and allow us to now identify novel strategies to further improve this immunotherapeutic approach. Citation Format: Davide Bernareggi, Qi Xie, Briana Prager, Li Xiqing, Silvio Gutkind, Dan S. Kaufman. Use of CRISPR knock-out screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT053.

Abstract PR03: Use of CRISPR knockout screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy

Abstract Natural killer (NK) cells play a key role in tumor immune surveillance by their ability to recognize and kill both hematologic and solid tumor cells. NK cell-based immunotherapy is emerging as a powerful treatment against certain malignancies. However, intrinsic and extrinsic mechanisms can lead tumor cells to develop resistance mechanisms to escape NK cell-mediated killing, including up- or downregulation of genes involved in the interaction between tumor and immune cells. Here, we use a “two cell type” (TCT) screening system to identify novel molecular targets regulating NK cell-mediated killing of glioblastoma stem cells (GSC), meningioma cells, as well as head and neck squamous cell carcinomas (HNSCC). By using a whole genome-scale CRISPR-Cas9 library, we induced mutations in tumor cells to mimic loss of function mutations involved in resistance or sensitivity to NK cells. After a round of selection with human peripheral blood NK cells, we profiled the genes that impaired the killing activity of NK cells or increased the sensitivity of tumor cells to NK cells. The most common genes lost in GSC and meningioma cells that lead to increased sensitivity to NK cells are involved in the ER-phagosome pathway, antigen presentation, and cellular localization. We then used GSC to validate our top genetic hits identified by the screening. One gene of interest, CHMP2A, is a component of ESCRT-III, a complex involved in multivesicular bodies and exosomes formation known to be involved in intercellular communications, mediating numerous physiologic processes among which are immunology and tumor biology. We confirmed that impaired expression of CHMP2A in GSC lines increases their sensitivity to NK cell-mediated killing. RNA sequencing demonstrates knockout (KO) of CHMP2A leads to increased expression of immune response genes, including certain chemokines. Indeed, NK cells demonstrated increased migration towards KO GSC corresponding to higher concentrations of CXCL10, CXCL12, and IL-8 in GSC-KO conditioned media. Since HNSCC are among the most highly immune-infiltrated cancer types, including a high degree of infiltrating NK cells, we next tested CHMP2A-KO-Cal27 cells as a model of HNSCC. Again, we observed an increased sensitivity to NK cells, higher levels of CXCL10, and increased migration of NK cells, demonstrating these mechanisms regulate NK cell-mediated killing of diverse tumor types. Additionally, in vivo studies using a Cal27 xenograft model and adoptive transfer of human NK cells in immunodeficient mice demonstrate increased NK cell-mediated killing of CHMP2A-KO-Cal27 cells compared to wild-type Cal27 cells. Together, these studies demonstrate the utility of a TCT screen to identify new mechanisms that mediate resistance of diverse tumor types to NK cell-mediated killing and allow us to now identify novel strategies to further improve this immunotherapeutic approach. This abstract is also being presented as Poster A25. Citation Format: Davide Bernareggi, Qi Xie, Briana Prager, Li Xiqing, Silvio Gutkind, Dan S. Kaufman. Use of CRISPR knockout screen to identify genes that regulate tumor cell sensitivity to NK cell-based immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR03.

Trophoblast-derived CXCL12 promotes CD56bright CD82- CD29+ NK cell enrichment in the decidua.

Decidual natural killer (dNK) cells play key roles in maternal-fetal immune regulation, trophoblast invasion, and vascular remodeling, and most dNK cell populations are CD56bright CD16- NK cells. However, the enrichment and redistribution of dNK cells in the local decidua have not been clarified yet. A total of 45 women with normal pregnancies and 8 unexplained recurrent spontaneous abortion (RSA) patients were included. We isolated primary human dNK (n=53) and peripheral blood NK (pNK) cells (n=5) from specimen and analyzed CD56, CD82, and CD29 by flow cytometry (FCM). We assessed their adhesion ability by cell counts of NK cells adhered to decidual stromal cells (DSCs) in a co-culture system. We found that RSA patients had more CD56dim dNK cells with lower CD82 and higher CD29 than women with normal pregnancies. There were negative correlations of CD82 to CD29 on CD56dim and CD56+ dNK cells. In normal pregnancies, dNK cells had lower CD82 and higher CD29 expression with a stronger adhesion ability than pNK cells. Blocking CD82 on dNK cells increased the adhesive ability and CD29 expression, while blocking CD29 decreased the adhesive ability. Co-culturing dNK cells with trophoblast cells decreased CD82 expression and increased the adhesive ability of dNK cells and the percentage of CD56bright NK cells, while blocking trophoblast-derived CXCL12 increased CD82 expression, decreased CD29 expression, and impaired the adhesive ability of NK cells. Trophoblast cells enhance the adhesive ability of NK cells to DSCs via the CXCL12/CD82/CD29 signaling pathway and contribute to CD56bright NK cell enrichment in the uterus.

Influenza A Virus Infection Induces Hyperresponsiveness in Human Lung Tissue-Resident and Peripheral Blood NK Cells

NK cells in the human lung respond to influenza A virus- (IAV-) infected target cells. However, the detailed functional capacity of human lung and peripheral blood NK cells remains to be determined in IAV and other respiratory viral infections. Here, we investigated the effects of IAV infection on human lung and peripheral blood NK cells in vitro and ex vivo following clinical infection. IAV infection of lung- and peripheral blood-derived mononuclear cells in vitro induced NK cell hyperresponsiveness to K562 target cells, including increased degranulation and cytokine production particularly in the CD56brightCD16− subset of NK cells. Furthermore, lung CD16− NK cells showed increased IAV-mediated but target cell-independent activation compared to CD16+ lung NK cells or total NK cells in peripheral blood. IAV infection rendered peripheral blood NK cells responsive toward the normally NK cell-resistant lung epithelial cell line A549, indicating that NK cell activation during IAV infection could contribute to killing of surrounding non-infected epithelial cells. In vivo, peripheral blood CD56dimCD16+ and CD56brightCD16− NK cells were primed during acute IAV infection, and a small subset of CD16−CD49a+CXCR3+ NK cells could be identified, with CD49a and CXCR3 potentially promoting homing to and tissue-retention in the lung during acute infection. Together, we show that IAV respiratory viral infections prime otherwise hyporesponsive lung NK cells, indicating that both CD16+ and CD16− NK cells including CD16−CD49a+ tissue-resident NK cells could contribute to host immunity but possibly also tissue damage in clinical IAV infection.

Hypoxia Modifies the Transcriptome of Human NK Cells, Modulates Their Immunoregulatory Profile, and Influences NK Cell Subset Migration.

Hypoxia, which characterizes most tumor tissues, can alter the function of different immune cell types, favoring tumor escape mechanisms. In this study, we show that hypoxia profoundly acts on NK cells by influencing their transcriptome, affecting their immunoregulatory functions, and changing the chemotactic responses of different NK cell subsets. Exposure of human peripheral blood NK cells to hypoxia for 16 or 96 h caused significant changes in the expression of 729 or 1,100 genes, respectively. Gene Set Enrichment Analysis demonstrated that these changes followed a consensus hypoxia transcriptional profile. As assessed by Gene Ontology annotation, hypoxia-targeted genes were implicated in several biological processes: metabolism, cell cycle, differentiation, apoptosis, cell stress, and cytoskeleton organization. The hypoxic transcriptome also showed changes in genes with immunological relevance including those coding for proinflammatory cytokines, chemokines, and chemokine-receptors. Quantitative RT-PCR analysis confirmed the modulation of several immune-related genes, prompting further immunophenotypic and functional studies. Multiplex ELISA demonstrated that hypoxia could variably reduce NK cell ability to release IFNγ, TNFα, GM-CSF, CCL3, and CCL5 following PMA+Ionomycin or IL15+IL18 stimulation, while it poorly affected the response to IL12+IL18. Cytofluorimetric analysis showed that hypoxia could influence NK chemokine receptor pattern by sustaining the expression of CCR7 and CXCR4. Remarkably, this effect occurred selectively (CCR7) or preferentially (CXCR4) on CD56bright NK cells, which indeed showed higher chemotaxis to CCL19, CCL21, or CXCL12. Collectively, our data suggest that the hypoxic environment may profoundly influence the nature of the NK cell infiltrate and its effects on immune-mediated responses within tumor tissues.

Proteome analysis of human CD56neg NK cells reveals a homogeneous phenotype surprisingly similar to CD56dim NK cells.

NK cells lacking CD56 (CD56neg ) were first identified in chronic HIV-1 infection. However, CD56neg NK cells also exist in healthy individuals, albeit in significantly lower numbers. Here, we provide an extensive proteomic characterisation of human CD56neg peripheral blood NK cells of healthy donors and compare them to their CD56dim and CD56bright counterparts. Unbiased large-scale surface receptor profiling clustered CD56neg cells as part of the main NK cell compartment and indicated an overall CD56dim -like phenotype. Total proteome analyses of CD56neg NK cells further confirmed their similarity with CD56dim NK cells, and revealed a complete cytolytic inventory with high levels of perforin and granzyme H and M. In the present study, twelve proteins discriminated CD56neg NK cells from CD56dim NK cells with nine up-regulated and three down-regulated proteins in the CD56neg NK cell population. Those proteins were functionally related to lytic granule composition and transport, interaction with the extracellular matrix, DNA transcription or repair, and proliferation. Corroborating these results, CD56neg NK cells showed modest cytotoxicity, degranulation, and IFN-ɣ secretion as compared to CD56dim NK cells. In conclusion, CD56neg NK cells constitute functionally competent cells sharing many features of bona fide CD56dim NK cells in healthy individuals, but with some distinct characteristics.

Induced Human Decidual NK-Like Cells Improve Utero-Placental Perfusion in Mice.

Decidual NK (dNK) cells, a distinct type of NK cell, are thought to regulate uterine spiral artery remodeling, a process that allows for increased blood delivery to the fetal-placental unit. Impairment of uterine spiral artery remodeling is associated with decreased placental perfusion, increased uterine artery resistance, and obstetric complications such as preeclampsia and intrauterine growth restriction. Ex vivo manipulation of human peripheral blood NK (pNK) cells by a combination of hypoxia, TGFß-1 and 5-aza-2’-deoxycytidine yields cells with phenotypic and in vitro functional similarities to dNK cells, called idNK cells. Here, gene expression profiling shows that CD56Bright idNK cells derived ex vivo from human pNK cells, and to a lesser extent CD56Dim idNK cells, are enriched in the gene expression signature that distinguishes dNK cells from pNK cells. When injected into immunocompromised pregnant mice with elevated uterine artery resistance, idNK cells homed to the uterus and reduced the uterine artery resistance index, suggesting improved placental perfusion.

MicroRNA-30c promotes natural killer cell cytotoxicity via up-regulating the expression level of NKG2D

AimsNatural killer (NK) cells play critical roles in antitumor immunity. Our previous study showed that over-expression of miR-30c-1* enhanced NKL cell cytotoxicity through up-regulation of tumor necrosis factor-α via directly targeting transcription factor homeobox containing 1. MiR-30c, the complimentary microRNA of miR-30c-1*, has been found to exert regulatory effect on T cell function. However, the effect of miR-30c on NK cells is unknown. Therefore, this study aimed to investigate whether miR-30c could play a role to enhance NK cell activation and cytotoxicity. Main methodsChemosynthesis exogenous miR-30c mimics and miR-30c inhibitor were transfected into NKL cells and isolated human peripheral blood NK cells, respectively. The expression levels of NK group 2, member D (NKG2D), CD107a and FasL on cell surface and cytotoxic ability of miRNAs transfected NKL cells against SMMC-7721 cells were evaluated. Key findingsMiR-30c could increase the expression of NKG2D and CD107a on NKL cells, and enhance cytotoxic ability of NKL cells to kill SMMC-7721 cells. Moreover, miR-30c could up-regulate the expression of FasL on both NKL cells and human peripheral blood NK cells. However, the peripheral blood NK cells from only four in ten healthy donors appeared high expression levels of NKG2D and CD107a after miR-30c transfection. SignificanceMiR-30c could promote the cytotoxicity of NKL cells in vitro by up-regulating the expression levels of NKG2D, CD107a and FasL. However, the effect of miR-30c on ex vivo NK cells from different human individuals is diverse, indicating that miR-30c may play complicate and fine adjustment in immune system.

TNFα Augments Cytokine-Induced NK Cell IFNγ Production through TNFR2

NK cells play a central role in innate immunity, acting directly through cell-mediated cytotoxicity and by secreting cytokines. TNFα activation of TNFR2 enhances NK cell cytotoxicity, but its effects on the other essential function of NK cells - cytokine production, for which IFNγ is paramount - are poorly defined. We identify the expression of both TNFα receptors on human peripheral blood NK cells (TNFR2 > TNFR1) and show that TNFα significantly augments IFNγ production from IL-2-/IL-12-treated NK cells in vitro, an effect mimicked by a TNFR2 agonistic antibody. TNFα also enhanced murine NK cell IFNγ production via TNFR2 in vitro. In a mouse model characterized by the hepatic recruitment and activation of NK cells, TNFR2 also regulated NK cell IFNγ production in vivo. Specifically, in this model, after activation of an innate immune response, hepatic numbers of TNFR2-expressing and IFNγ-producing NK cells were both significantly increased; however, the frequency of IFNγ-producing hepatic NK cells was significantly reduced in TNFR2-deficient mice. We delineate an important role for TNFα, acting through TNFR2, in augmenting cytokine-induced NK cell IFNγ production in vivo and in vitro, an effect with significant potential implications for the regulation of innate and adaptive immune responses.

MicroRNA transcriptomes of distinct human NK cell populations identify miR-362-5p as an essential regulator of NK cell function.

Natural killer (NK) cells are critical effectors in the immune response against malignancy and infection, and microRNAs (miRNAs) play important roles in NK cell biology. Here we examined miRNA profiles of human NK cells from different cell compartments (peripheral blood, cord blood, and uterine deciduas) and of NKT and T cells from peripheral blood, and we identified a novel miRNA, miR-362-5p, that is highly expressed in human peripheral blood NK (pNK) cells. We also demonstrated that CYLD, a negative regulator of NF-κB signaling, was a target of miR-362-5p in NK cells. Furthermore, we showed that the over-expression of miR-362-5p enhanced the expression of IFN-γ, perforin, granzyme-B, and CD107a in human primary NK cells, and we found that silencing CYLD with a small interfering RNA (siRNA) mirrored the effect of miR-362-5p over-expression. In contrast, the inhibition of miR-362-5p had the opposite effect in NK cells, which was abrogated by CYLD siRNA, suggesting that miR-362-5p promotes NK-cell function, at least in part, by the down-regulation of CYLD. These results provide a resource for studying the roles of miRNAs in human NK cell biology and contribute to a better understanding of the physiologic significance of miRNAs in the regulation of NK cell function.

Preliminary Results of a Phase 1/2 Clinical Trial of Cndo-109-Activated Allogeneic Natural Killer Cells in High Risk Acute Myelogenous Leukemia Patients in First Complete Remission

BACKGROUNDWe previously reported that resting human peripheral blood NK cells can be primed to kill NK-resistant tumor cells by co-incubation with a lysate of the leukemia cell line CTV-1. CNDO-109 is a clinical-grade CTV-1 lysate that primes NK cells ex-vivo to kill NK-resistant acute myeloid leukemia (AML) cells. CNDO-109-activated NK cells can be cryopreserved and remain primed when thawed. Incubation or treatment with IL-2 is not required. We report preliminary safety, outcome and NK chimerism data from an ongoing Phase 1/2 transitional clinical trial of CNDO-109-NK cells.METHODSA 3x3 dose escalation phase 1 trial was opened in 2013 for patients with AML in 1st CR with high-risk disease and no conventional treatment options. Patients were given preparative chemotherapy of cyclophosphamide and fludarabine on Study Days -6 to -2, followed on day 0 by a single dose of CNDO-109-activated NK cells at the following doses; cohort 1 = 3×105, cohort 2 = 1×106, cohort 3 = up to 3×106 cells/kg recipient body weight. The MTD will set the dose for the transitional phase 2 trial.CNDO-109-NK cells were generated from a single apheresis collection from HLA-haploidentical related donors. NK cells were isolated with anti-CD56 microbeads (CliniMACS, Miltenyi Biotec) and co-incubated with CNDO-109 lysate (Coronado Biosciences) overnight under cGMP conditions. After lysate removal cells were cryopreserved in dosed aliquots and released for infusion. Exogenous IL-2 was not used. Quality control testing of the final product included NK purity (>50%), viability (> 70%), potency, sterility, mycoplasma and endotoxin. Residual T cell contamination (<104 cells/kg patient body weight) was a lot release safety criterion. Patients were assessed for safety and efficacy whilst expansion, proliferation and persistence of donor NK cells were assayed by molecular-based chimerism techniques, and by flow cytometry, where informative markers were available. Pre- and post-treatment samples from cohort 3 patients are being tested for flow cytometric and molecular MRD (University of Washington - Walter, R.B.2013 and NextGene Sequencing).RESULTSSeven eligible patients have been enrolled. All products met lot release and contained activated NK cells as determined by killing of NK resistant Raji cells as well as increased expression of CD69 & CD25: TableBaseline(Resting NK Cells)Activated NK CellsChangep-valueRaji Cell Lysis15.2%+/- 10.0%29.7% +/- 13.9%0.008CD69+ Expression5.9% +/- 2.9%42.6% +/- 12.5%0.008CD25+ Expression3.1% +/- 1.8%5.8% +/- 1.9%0.008No infusional toxicity, adverse events attributed to NK therapy, GvHD nor deaths have been reported. As expected, all patients experienced transient myelosuppression (approx. 2 weeks). 3 patients suffered early relapse post-treatment (2 in cohort 1, 1 in cohort 2; average time to relapse from CR1 for these 3 patients was 104 days).In 5 of 7 evaluable patients, persistence of donor activated NK cells was observed from Day +7 post-infusion (chimerism = 1%-84%) to as late as day +56 in one patient. Flow cytometric comparison of donor NK cells and patient NK cells in the same sample by selective gating on the mismatched HLA allele showed that circulating donor NK cells typically differed from recipient NK cell in higher expression of NKG2A (e.g. 63.5% vs 2.7%), CD57 (e.g. 80.5% vs 13.2%) and CD69 (e.g. 11.6% vs 2.52%) suggesting a more normal, mature and activated phenotype than the endogenous host NK population.Even after loss of circulating donor primed NK cells, 2 of the 3 patients tested showed persistence of low levels of activated autologous NK cells (~10-20% of circulating NK) exceeding the numbers circulating pre-CNDO-109 NK treatment, out to Day +56, suggesting that the therapy may induce endogenous NK activation to enhance the patients’ innate immunity to AML.CONCLUSIONSThis establishes proof of concept that CNDO-109-NK can persist transiently in patients with lasting microchimerism for > 1 month and can induce activation of endogenous NK cells in patients treated without cytokine administration. To date 4 of the 7 patients enrolled remain relapse free (max. = 410 days post CR1 date). These results highlight the potential of CNDO-109-NK cells in the treatment of AML and other malignancies. DisclosuresMiller:Coronado Biosciences: Consultancy. Hillman:Coronado Biosciences: Employment. Silver:Coronado Biosciences: Employment. Szarek:Coronado Biosciences: Consultancy. Lowdell:Coronado Biosciences: Consultancy, Patents & Royalties. Gorelik:Coronado Biosciences: Employment. Rowinsky:Coronado Biosciences: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Lirilumab Enhances Anti-Tumor Efficacy of Elotuzumab

Tumor cells that express reduced levels of Major Histocompatibility Complex (MHC) class I molecules may be recognized and killed by Natural Killer cells (NK cells), through a process known as “missing self” recognition. In humans, this is controlled by inhibitory receptors such as Killer Immunoglobulin-like Receptors (KIR) that recognize Human Leukocyte Antigen (HLA)-A, -B or –C. Engagement of KIR by HLA molecules results in inhibitory signaling that reduces NK cell-mediated natural killing and antibody-dependent cellular cytotoxicity (ADCC). Hence, antibodies that block interactions between inhibitory KIR and their HLA ligands are being evaluated as an anti-cancer therapeutic strategy. The anti-KIR2DL1/2/3-specific monoclonal antibody, lirilumab (BMS-986015 / IPH2102), is a fully human IgG4 that blocks binding of KIR to HLA-C, and is being developed for treating hematologic malignancies and solid tumors.Elotuzumab (BMS901608 / HuLuc63) is a humanized IgG1 anti-SLAMF7 (signaling lymphocyte activation molecule family member 7, CS-1) being developed for the treatment of Multiple Myeloma (MM). SLAMF7 is a cell surface glycoprotein highly expressed in myelomatous cells and only at low levels on normal cells. NK cell-mediated ADCC is one of the main mechanisms of action of elotuzumab, but ADCC is negatively regulated by KIR checkpoint receptors. Thus a combination of lirilumab and elotuzumab has strong scientific rationale. The aim of the present study was to assess whether lirilumab would enhance elotuzumab anti-MM activity in vitro with human peripheral blood NK cells and MM cell lines, and in vivo in a newly developed xenogenic mouse model.Two MM cell lines (OPM-2 and U266B1) were identified that express both HLA-C and SLAMF7. These MM cells were capable of activating peripheral blood NK cells from healthy donors in vitro, as assessed by three different endpoints (CD107 mobilization on NK cells surface and intracellular production of the cytokines IFN-g and TNF- a); each of these responses were significantly enhanced, in a dose-dependent manner, by both lirilumab and elotuzumab independently. Moreover, the elotuzumab-mediated functional activation of KIR2D+ NK cells could be further enhanced by the addition of increasing doses of lirilumab. The best combinatorial effect was observed in response to MM cells expressing low densities of SLAMF7. These data suggest that lirilumab treatment may increase the therapeutic efficacy of elotuzumab, particularly in MM patients with low SLAMF7 expression. In these experiments, it was not possible to clearly identify the impact of Fc receptor genotype or HLA-C genotype on the NK cell responses.To assess the therapeutic efficacy of lirilumab and elotuzumab in vivo, we generated a novel strain of double-transgenic mice expressing human KIR2DL3 as well as its ligand, HLA-cw3, on a Rag1-/- background (KIR-cw3-tgRAG mice), to allow engraftment of human MM tumor cells expressing SLAMF7. The OPM-2 MM cell line was subcutaneously engrafted in these mice and when high tumor volumes were reached, mice were treated with lirilumab, elotuzumab or a combination of both. As monotherapy, each of monoclonal antibody had some therapeutic effect while the combination of both resulted in a significantly stronger anti-tumor effect and increased survival of the mice. Median survival of mice treated with huIgG control was 38 days, 41 days with lirilumab, 42 days with elotuzumab and 51 days with both mAbs in combination (10 mice per group). In conclusion, we demonstrate that blockade of KIR checkpoint receptors with lirilumab was able to augment elotuzumab mediated ADCC in vitro and synergized with elotuzumab to mediate potent anti-MM activity in vivo. Taken together, these data provide a rationale for clinical trials to test combination treatment of lirilumab and elotuzumab in MM patients. DisclosuresSola:InnatePharma: Employment, Equity Ownership. Blery:Innate Pharma: Employment, Equity Ownership. Bonnafous:Innate Pharma: Employment, Equity Ownership. Bonnet:Innate Pharma: Employment, Equity Ownership. Fuseri:Innate Pharma: Employment, Equity Ownership. Graziano:Bristol-Myers Squibb: Employment; Bristol-Myers Squibb: Equity Ownership. Morel:Innate Pharma: Employment, Equity Ownership. André:Innate Pharma: Employment, Equity Ownership.