CD38 Knockout Primary NK Cells to Prevent "Fratricide" and Boost Daratumumab Activity

Multiple myeloma (MM) is a plasma cell neoplasm typically characterized by high and uniform CD38 expression. Daratumumab (DARA), a humanized monoclonal antibody targeting CD38 has dramatically improved the outcome of patients with refractory MM, but relapse is inevitable in most cases. DARA eliminates MM cells through several mechanisms including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis. Mechanisms of resistance to DARA include downregulated CD38 expression on target MM cells, impaired complement directed cytotoxicity via upregulation of complement inhibitory proteins (CD55, CD59), and impaired ADCC via DARA-induced NK cell "fratricide". As it relates to "fratricide", restoring ADCC by DARA-resistant NK cells has been proposed to bolster the therapeutic effect of DARA. Towards this goal, iPSC-derived CD38 knockout (CD38KO) NK cells have shown proof-of-concept for this approach but present major challenges towards clinical translation. Here, we used Cas9-RNP to generate CD38KO primary NK cells and characterized their resistance to DARA-induced "fratricide" and restoration of ADCC, but also assessed the impact of CD38 deletion on NK cell signaling and metabolism. First, primary NK cells were enriched from peripheral blood of healthy donors and expanded on feeder cells expressing mbIL-21 and 4-1BBL (PLoS One. 2012;7(1):e30264). Cas9/gRNA complexes targeting CD38 were electroporated into NK cells (J Vis Exp. 2018 Jun 14;(136)). Flow cytometric analysis revealed CD38KO efficiency was 81.9±6.9% (mean±SD, n=5). CD38KO cells were further purified to ≥ 95% purity using magnetic cell separation system. Whole genome sequencing identified no off-target mutations. CD38KO-NK cells alone showed no conjugation in the presence of DARA (Figure 1A) and no DARA-induced fratricide (Figure 1B), in stark contrast with wild type NK (WT-NK) cells. Consistent with these results, treatment of NSG mice with DARA had no impact on persistence of CD38KO-NK cells, whereas WT-NK cells were completely eliminated (NK cell frequency in peripheral blood 7 days after inoculation; WT-NK NO DARA 12.11±5.15%, KO-NK NO Dara 17.05±0.98%, WT-NK + Dara 0.21±0.08%, KO-NK + Dara 16.85±3.61%, mean±SD, n=4, p<0.01, ANOVA) (Figure 1C). To examine the functional status of CD38KO-NK cells, we used 6 MM cell lines with various CD38 expression levels (LP-1, RPMI8226, H929, MM.1s, OPM-2, and KMS-11) as well as three CD38+CD138+ primary MM samples isolated from bone marrow of newly diagnosed or relapsed patients. CD38KO-NK cells exhibited enhanced ADCC activity compared to WT-NK cells across all cell lines and primary samples tested in 4h and 24h cytotoxicity assay (Figure 1D). This was even observed in MM cells that express low levels of CD38, suggesting their potential efficacy in patients with relapsed disease after DARA (ADCC at E/T 5:1; MM.1s 7.8±1.8%(WT) vs 21.5±0.4%(KO), KMS-11 -3.5±0.6%(WT) vs 11.1±0.4%(KO), mean±SD). The CD38KO-NK cells preserved similar expansion potency as WT-NK counter parts. Next, to reveal any effect of manipulating CD38 expression on NK cells cytotoxicity and metabolism, we performed RNA sequencing analysis, which showed higher expression of several cytotoxic genes such as IFNG and GzmB in CD38-KO NK cells, and higher expression of glucose transporter genes and GAPDH, suggesting a favorable metabolic profile of these cells. Consistent with these data, we observed that CD38KO-NK cells have elevated capacity to take up glucose as measured by 2-NBDG glucose uptake assay (p=0.02, student t test). Taken together, these findings provide the proof-of-concept that CD38KO-NK cells generated from primary NK cells augment therapeutic effect of DARA and could be promising for adoptive immune cellular therapy targeting MM. Y.N. and M.N.K. contributed equally to this study. D.A.L. and G.G. contributed equally to this study. Figure 1 Disclosures Ali: Celgene: Research Funding; Poseida: Research Funding. Lee:Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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CD38low Natural Killer Cells Transiently Expressing CD16F158V m-RNA Potentiates the Therapeutic Activity of Daratumumab Against Multiple Myeloma with Minimal Effector NK Cell Fratricide

Blood ◽

10.1182/blood-2018-99-118926 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3199-3199 ◽

Cited By ~ 2

Author(s):

Subhashis Sarkar ◽

Sachin Chauhan ◽

Arwen Stikvoort ◽

Alessandro Natoni ◽

John Daly ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Advisory Committees ◽

Cd38 Expression ◽

Rpmi 8226

Abstract Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (moAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells mediating tumour immunosurveillance in vivo. NK cells also play an important role during moAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their Fcγ RIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring high affinity variant of CD16 harbouring a single point polymorphism (F158V), and this variant has been linked to improved ADCC. However, the contribution of NK cells to the efficacy of Daratumumab remains debatable as clinical data clearly indicate rapid depletion of CD38high peripheral blood NK cells in patients upon Daratumumab administration. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach, on CD38low NK cells could significantly enhance therapeutic efficacy of Daratumumab in MM patients. In the present study, we investigate the optimal NK cell platform for generating CD38low CD16F158V NK cells which can be administered as an "off-the-shelf"cell therapy product to target both CD38high and CD38low expressing MM patients in combination with Daratumumab. Methods: MM cell lines (n=5) (MM.1S, RPMI-8226, JJN3, H929, and U266) and NK cells (n=3) (primary expanded, NK-92, and KHYG1) were immunophenotyped for CD38 expression. CD16F158V coding m-RNA transcripts were synthesized using in-vitro transcription (IVT). CD16F158V expression was determined by flow cytometry over a period of 120 hours (n=5). 24-hours post electroporation, CD16F158V expressing KHYG1 cells were co-cultured with MM cell lines (n=4; RPMI-8226, JJN3, H929, and U266) either alone or in combination with Daratumumab in a 14-hour assay. Daratumumab induced NK cell fratricide and cytokine production (IFN-γ and TNF-α) were investigated at an E:T ratio of 1:1 in a 14-hour assay (n=3). CD38+CD138+ primary MM cells from newly diagnosed or relapsed-refractory MM patients were isolated by positive selection (n=5), and co-cultured with mock electroporated or CD16F158V m-RNA electroporated KHYG1 cells. CD16F158V KHYG1 were also co-cultured with primary MM cells from Daratumumab relapsed-refractory (RR) patients. Results: MM cell lines were classified as CD38hi (RPMI-8226, H929), and CD38lo (JJN3, U266) based on immunophenotyping (n=4). KHYG1 NK cell line had significantly lower CD38 expression as compared to primary expanded NK cells and NK-92 cell line (Figure 1a). KHYG1 electroporated with CD16F158V m-RNA expressed CD16 over a period of 120-hours post-transfection (n=5) (Figure 1b). CD16F158V KHYG1 in-combination with Daratumumab were significantly more cytotoxic towards both CD38hi and CD38lo MM cell lines as compared to CD16F158V KHYG1 alone at multiple E:T ratios (n=4) (Figure 1c, 1d). More importantly, Daratumumab had no significant effect on the viability of CD38low CD16F158V KHYG1. Moreover, CD16F158V KHYG1 in combination with Daratumumab produced significantly higher levels of IFN-γ (p=0.01) upon co-culture with CD38hi H929 cell line as compared to co-culture with mock KHYG1 and Daratumumab. The combination of CD16F158V KHYG1 with Daratumumab was also significantly more cytotoxic to primary MM cell ex vivo as compared to mock KHYG1 with Daratumumab at E:T ratio of 0.5:1 (p=0.01), 1:1 (p=0.005), 2.5:1 (p=0.003) and 5:1 (p=0.004) (Figure 1e). Preliminary data (n=2) also suggests that CD16F158V expressing KHYG1 can eliminate 15-17% of primary MM cells from Daratumumab RR patients ex vivo. Analysis of more Daratumumab RR samples are currently ongoing. Conclusions: Our study provides the proof-of-concept for combination therapy of Daratumumab with "off-the-shelf" CD38low NK cells transiently expressing CD16F158V for treatment of MM. Notably, this approach was effective against MM cell lines even with low CD38 expression (JJN3) and primary MM cells cultured ex vivo. Moreover, the enhanced cytokine production by CD16F158V KHYG1 cells has the potential to improve immunosurveillance and stimulate adaptive immune responses in vivo. Disclosures Sarkar: Onkimmune: Research Funding. Chauhan:Onkimmune: Research Funding. Stikvoort:Onkimmune: Research Funding. Mutis:Genmab: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding. O'Dwyer:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Glycomimetics: Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Targeting CD38high Acute Myeloid Leukaemia with "Affinity Optimized" Chimeric Antigen Receptor and Membrane Bound TRAIL Expressing Natural Killer Cells

Blood ◽

10.1182/blood-2019-128605 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5536-5536 ◽

Cited By ~ 1

Author(s):

Emma Nolan ◽

Arwen Stikvoort ◽

Mark Gurney ◽

Nutsa Burduli ◽

Lucy Kirkham-McCarthy ◽

...

Keyword(s):

Cell Surface ◽

Nk Cells ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees ◽

Cytotoxicity Assays ◽

Cd38 Expression ◽

Membrane Bound

Introduction: Chimeric Antigen Receptor (CAR) based cellular-immunotherapies have demonstrated significant clinical efficacy in haematological malignancies. However, the progress of cellular-immunotherapy for the treatment of Acute Myeloid Leukaemia (AML) has failed to gain momentum due to the lack of targetable tumour specific antigens. CD38 is a transmembrane glycoprotein expressed in lymphoid and myeloid cells with high expression in plasma B-cells, and is a well validated target for anti-CD38 therapy in Myeloma. A recent study has furthermore shown that a proportion of AML patients express CD38 on their leukemic blasts. TNF-related apoptosis-inducing ligand (TRAIL) receptor DR4 is another targetable antigen which has been shown to be expressed in 70% of AML patients. In this study, we investigate the therapeutic efficacy of "affinity-optimized" variant(s) of CD38 CAR and membrane bound TRAIL on NK-cell based platforms which can target AML blasts with high expression of CD38 (CD38high AML). The CAR variant is a CAR which binds with lower affinity to CD38 expressed on healthy immune cells such as CD38positive NK cells, while targeting CD38high AML. The membrane bound TRAIL variant (TRAIL4c9) is a mutant which binds with higher affinity to TRAIL-DR4 on AML cells, whilst avoiding binding to decoy receptors. We hypothesize that genetically modifying NK cells to express "affinity optimized" CD38 CARand/or TRAIL4c9 can effectively eliminate CD38high AML cells. Methods: AML cell lines THP-1, U937, and KG1a were immunophenotyped for CD38 and TRAIL-DR4 expression. Retrovirally transduced CD38 CAR-KHYG1 NK cells were used as immune effector cells and were co-cultured with AML cell lines in cytotoxicity assays. CD38low AML cell line KG1a was pre-treated with 10nM all-trans-retinoic acid (ATRA) to upregulate CD38 expression and were subsequently co-cultured with CD38 CAR-KHYG1 in cytotoxicity assays. CD38 CAR-KHYG1 was also co-cultured with n=4 patient derived AML cells in cytotoxicity assays. Using Maxcyte GT electroporation system primary donor derived IL-2 activated NK cells were either mock electroporated, or electroporated with TRAIL4c9 m-RNA orCD38 CAR m-RNA and subsequently co-cultured with THP-1 or ATRA pre-treated KG1a in a cytotoxicity assay. Expression of pro-apoptotic, anti-apoptotic and ligands for checkpoint inhibitory receptors was analysed by immunoblotting or flowcytometry. Results: Based on immunophenotyping, we classified AML cell lines as CD38high (THP-1), CD38moderate (U937) and CD38low (KG1a). CD38 CAR-KHYG1 was significantly more cytotoxic than MOCK KHYG1 against CD38high THP-1, at E:T ratios of 2.5:1, 5:1 and 10:1. CD38 CAR-KHYG1 were also more cytotoxic than MOCK KHYG1 against CD38moderate U937 at multiple E:T ratios; albeit the increase in cytotoxicity was at a much lower level in comparison to THP-1 (Fig 1a). Pre-treatment of CD38low KG1a cells with 10nM ATRA upregulated the cell surface expression of CD38, which were subsequently eliminated by CD38 CAR KHYG1 at E:T ratios of 2.5:1, 5:1 and 10:1. KG1a was intrinsically resistant to NK cells as compared to THP-1 and U937 (Fig 1b). This could partly be explained by the high intracellular expression of Bcl-xL, and higher cell surface expression of Nectin-1 and Sialic acid which are the ligands for checkpoint inhibitory receptors CD96 and Siglec-7/9 respectively on NK cell (Fig 1c). CD38 CAR-KHYG1 mounted a potent cytotoxic response against primary CD45intermediate AML blasts (n=4 patients) at multiple E:T ratios, and the extent of CAR induced cytotoxicity correlated with the cell surface CD38 expression on the primary AML blasts (R2=0.87) (Fig 1d,e). TRAIL4c9 or CD38 CAR m-RNA electroporated primary donor-derived NK cells were also potent in eliminating THP-1 and ATRA pre-treated KG1a at multiple E:T ratios (Fig 1f). This demonstrates the potential of therapeutically treating AML patients, with high CD38 expression, with a combination of NK cells expressing "affinity-optimized" CD38 CAR and membrane bound TRAIL variant. Conclusion: The study demonstrates the therapeutic potential of an "affinity-optimized" CD38 CAR NK cell-based therapy, which can potentially be combined with membrane bound TRAIL expressing NK cells to target CD38high AML. In patients with CD38low expressing AML blasts, patients could be pre-treated with ATRA followed by the combination therapy of CD38 CAR and TRAIL expressing NK cells. Disclosures Stikvoort: Onkimmune Ltd., Ireland: Research Funding. Kirkham-McCarthy:Onkimmune Ltd., Ireland: Research Funding. Van De Donk:Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Mutis:Celgene: Research Funding; Janssen Pharmaceuticals: Research Funding; Amgen: Research Funding; BMS: Research Funding; Novartis: Research Funding; Aduro: Research Funding; Onkimmune: Research Funding. Sarkar:Onkimmune: Research Funding. O'Dwyer:Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; GlycoMimetics Inc: Research Funding; AbbVie: Consultancy; BMS: Research Funding.

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CD38 deletion of human primary NK cells eliminates daratumumab-induced fratricide and boosts their effector activity

Blood ◽

10.1182/blood.2020006200 ◽

2020 ◽

Vol 136 (21) ◽

pp. 2416-2427 ◽

Cited By ~ 2

Author(s):

Meisam Naeimi Kararoudi ◽

Yuya Nagai ◽

Ezgi Elmas ◽

Marcelo de Souza Fernandes Pereira ◽

Syed Abbas Ali ◽

...

Keyword(s):

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Ex Vivo ◽

Human Monoclonal Antibody ◽

Metabolic Reprogramming ◽

Target Cells ◽

Plasma Cell Neoplasm ◽

Cd38 Expression ◽

The Impact

Abstract Multiple myeloma (MM) is a plasma cell neoplasm that commonly expresses CD38. Daratumumab (DARA), a human monoclonal antibody targeting CD38, has significantly improved the outcome of patients with relapsed or refractory MM, but the response is transient in most cases. Putative mechanisms of suboptimal efficacy of DARA include downregulation of CD38 expression and overexpression of complement inhibitory proteins on MM target cells as well as DARA-induced depletion of CD38high natural killer (NK) cells resulting in crippled antibody-dependent cellular cytotoxicity (ADCC). Here, we tested whether maintaining NK cell function during DARA therapy could maximize DARA-mediated ADCC against MM cells and deepen the response. We used the CRISPR/Cas9 system to delete CD38 (CD38KO) in ex vivo expanded peripheral blood NK cells. These CD38KO NK cells were completely resistant to DARA-induced fratricide, showed superior persistence in immune-deficient mice pretreated with DARA, and enhanced ADCC activity against CD38-expressing MM cell lines and primary MM cells. In addition, transcriptomic and cellular metabolic analysis demonstrated that CD38KO NK cells have unique metabolic reprogramming with higher mitochondrial respiratory capacity. Finally, we evaluated the impact of exposure to all-trans retinoic acid (ATRA) on wild-type NK and CD38KO NK cell function and highlighted potential benefits and drawbacks of combining ATRA with DARA in patients with MM. Taken together, these findings provide proof of concept that adoptive immunotherapy using ex vivo expanded CD38KO NK cells has the potential to boost DARA activity in MM.

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CSL362: A Monoclonal Antibody to Human Interleukin-3 Receptor (CD123), Optimized for NK Cell-Mediated Cytotoxicity of AML Stem Cells

Blood ◽

10.1182/blood.v120.21.3598.3598 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3598-3598 ◽

Cited By ~ 4

Author(s):

Samantha J. Busfield ◽

Mark Biondo ◽

Mae Wong ◽

Hayley S. Ramshaw ◽

Erwin M Lee ◽

...

Keyword(s):

Stem Cells ◽

Monoclonal Antibody ◽

Mouse Model ◽

Nk Cells ◽

Peripheral Blood ◽

Research Funding ◽

Nk Cell ◽

Target Cells ◽

Cell Leukemia ◽

Interleukin 3

Abstract Abstract 3598 The interleukin-3 receptor alpha chain (IL-3Rα/CD123) is expressed in a variety of hematological malignancies including AML, MDS, B-ALL, Hodgkin's lymphoma, hairy cell leukemia, systemic mastocytosis, plasmacytoid dendritic cell leukemia and CML. In AML, the majority of AML blasts express CD123 and this receptor is selectively over expressed on CD34+CD38− leukemic stem cells (LSC) compared to normal hematopoietic stem cells. This difference may provide a biological advantage to the leukemic cells given the survival and proliferation-promoting activities of IL-3, whilst at the same time providing an opportunity to target these malignant cells selectively. We have shown previously that 7G3, a mouse monoclonal antibody (mAb) which blocks IL-3 binding to CD123, is capable of eliminating human LSC in a mouse model of human AML by a combination of mechanisms, including engagement of the innate immune system via Fc-dependent mechanisms (Jin et al., 2009 Cell Stem Cell, 5:31). We have subsequently humanised and affinity-matured this antibody and, in addition, have engineered the Fc-domain to optimise potential cytotoxicity against AML cells. The resultant antibody, CSL362, retains the ability to neutralise IL-3 and has enhanced affinity for the FcγRIIIa (CD16) on NK cells. In vitro studies have demonstrated that the increased affinity for CD16 correlates with greater antibody-dependent cell-mediated cytotoxicity (ADCC) against CD123 expressing cell lines compared to CSL360, a non Fc-engineered anti-CD123 mAb. The improved activity was evident as both an increased maximal level of target cell lysis and as a shift in the EC50 of the antibody to lower concentrations. Importantly, both primary AML blasts and CD34+CD38−CD123+LSC were susceptible to CSL362-induced ADCC and this was seen even in samples that were resistant to ADCC by a non Fc-engineered anti-CD123 mAb. In an AML xenograft mouse model, where treatment with the antibody was initiated 4 weeks after engraftment of leukemia cells, CSL362 was more effective in reducing leukemic growth than the non Fc-engineered anti-CD123 mAb. The evaluation of neutrophils, monocytes, macrophages and NK cells in ADCC assays has revealed that the major effector cell responsible for CSL362-mediated cytotoxicity in human peripheral blood is the NK cell. In clinical samples we have been able to demonstrate autologous depletion ex vivo of target AML blasts (collected at diagnosis and cryopreserved) following incubation with CSL362 and peripheral blood mononuclear cells (taken from the same patient at first remission), indicating that NK cell number and function is sufficiently preserved in such patients for CSL362-directed killing of leukemic target cells. The pre-clinical data generated thus far strongly support the clinical development of CSL362 for the treatment of AML in patients with adequate NK cell function. A Phase 1 study of CSL362 in patients with CD123 positive AML in remission is underway (Clinical Trials.gov identifier: NCT01632852). Disclosures: Busfield: CSL Limited: Employment. Biondo:CSL Limited: Employment. Wong:CSL Limited: Employment. Ramshaw:CSL Limited: Research Funding. Lee:CSL Limited: Research Funding. Martin:CSL Limited: Employment. Ghosh:CSL Limited: Employment. Braley:CSL Limited: Employment. Tomasetig:CSL Limited: Employment. Panousis:CSL Limited: Employment. Vairo:CSL Limited: Employment. Roberts:CSL Limited: Research Funding. DeWitte:CSL Behring: Employment. Lock:CSL Limited: Consultancy, Research Funding. Lopez:CSL Limited: Consultancy, Research Funding. Nash:CSL Limited: Employment.

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NK Cell Mediated Cytotoxicity Against Malignant Promyelocytes Enhanced By Arsenic Trioxide: Potential Clinical Relevance

Blood ◽

10.1182/blood.v122.21.1455.1455 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1455-1455

Author(s):

Ansu Abu Alex ◽

Hamenth Kumar P ◽

Saravanan Ganesan ◽

Nithya Balasundaram ◽

Kavitha M Lakshmi ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Cell Lines ◽

Nk Cell ◽

Single Agent ◽

Adaptive Immune System ◽

Cytolytic Activity ◽

Leukemic Cells ◽

Ligand Expression ◽

The Impact

Abstract NK cells are primary effectors of the innate immune response against cells that have undergone malignant transformation. Several lines of evidence indicate that the expression level of NK ligands on leukemic cells affects the sensitivity of the leukemic cells to cytolytic activity by NK cells. Various agents have been evaluated for their ability to induce these ligands on leukemic cells to augment the NK cell mediated anti-leukemia effect. There is substantial evidence that has established the importance of the adaptive immune system in the treatment of acute promyelocytic leukemia (APL) (Rose Ann Padua et al. Nat Med 2003). While there is significant data which address the mechanisms of arsenic trioxide (ATO) on malignant promyelocytes, limited data is available of its effect on the innate and adaptive immune system. We undertook a series of experiments to address the impact of ATO on NK cell receptor and malignant promyelocyte ligand expression and its effect on NK cell mediated cytotoxicity. We also evaluated NK cell reconstitution in patients treated with ATO and the impact of KIR genotypes on relapse. We first evaluated the cytotoxic activity of NK92MI (NK cell line) against 5 different myeloid (K562, U937, HL60, UF1, NB4) and 2 lymphoid cell lines (Jurkat E6.1, SUP-B15) by CFSE/ 7AAD cytotoxicity assay. Target (T) cells (1x 105/100 µL/well) pre-treated with CFSE were co-cultured with effector NK cells (E) at a E:T ratio of 1:1, 2:1 and 5:1 for 5 hours at 37°C in 96 well plates. The percentage cytolytic activity of the NK cells was then calculated after adding 7AAD and acquired in FACS Calibur (Becton Dickinson, San Jose, CA, USA). Significant cytolytic activity was noted against K562 and NB4 cell lines. At the highest E:T ratio there was a median 22% cytolytic activity against NB4 (N=5). We observed that NB4 when treated overnight with 1µM ATO (>99% viability retained after this exposure) significantly increased the cytotoxic effect of NK92MI cell line at all the E:T ratios as shown in figure 1A (n=5; P=0.0023). No other cell line showed a similar increase in cytotoxic effect following exposure to ATO at these concentrations (data not shown). We next evaluated the effect of exposure of NB4 cells to ATO at 1µM for 6 hours on NK ligand expression by flowcytometry. As shown in figure 1B there was a significant increase in activating ligand MICA/B in NB4 cell lines (n=3; P=0.016) which was not seen in any of the other cell lines. Similar significant increased expression of Nectin-2 (DNAM-1 ligand) and HLA Class I was seen. Exposure of NK92MI to ATO for 6 hours at 1uM (non cytotoxic dose:IC50-3.8uM) resulted in increased expression of activating receptors NKG2D, NKP30 and KIR2DS4 (figure 1C) and inhibitory receptor NKG2A and decrease in inhibitory receptors KIR3DL1/DL2. There were no changes in the expression of NKP46, KIR2DL1, KIR2DL2 and DNAM1 receptors. We undertook a prospective study to evaluate the pattern of NK (CD56+CD3-) reconstitution in patients with newly diagnosed APL treated at our center with a single agent ATO regimen (Mathews et al. JCO 2011). The mean NK cell counts in patients were below the 2SD deviation level of the normal range even after completion of therapy (approximately a year)(figure 1D). All other subsets evaluated (CD4, CD8, CD3, CD19, CD56+CD3+, CD4CD45RO) had returned to levels within the normal range by the end of consolidation therapy (approximately 3 months from diagnosis). KIR genotyping was done on 55 patients with APL who received treatment with single agent ATO based regimen. The median follow up of this cohort was 20 months and 14 cases relapsed following initial therapy. The presence or absence of 17 KIR genes was done by PCR-SSP method (KIR Typing kit, Miltenyi Biotech Inc, CA). There was no association with any specific genotype or haplotype with risk of relapse. In summary we have noted that there is up regulation of receptors on NK cells and ligands on malignant promyelocytes following exposure to ATO that favors NK cell mediated cytotoxicity. In-vitro we have demonstrated a significant increase in NK cell mediated cytolytic activity against malignant promyelocytes exposed to ATO even at relatively low E:T ratios. This could be an important mechanism by which ATO induces durable remissions in patients with APL. The delayed NK cell recovery following treatment with ATO raises the possibility of using NK cell therapy to augment the effect of ATO in the treatment of patients.Figure 1Figure 1. Disclosures: No relevant conflicts of interest to declare.

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Exome Sequencing of Aggressive Natural Killer Cell Leukemia and Drug Profiling Highlight Candidate Driver Pathways in Malignant Natural Killer Cells

Blood ◽

10.1182/blood.v126.23.700.700 ◽

2015 ◽

Vol 126 (23) ◽

pp. 700-700

Author(s):

Olli Dufva ◽

Tiina Kelkka ◽

Shady Awad ◽

Nodoka Sekiguchi ◽

Heikki Kuusanmäki ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Exome Sequencing ◽

Cell Lines ◽

Hierarchical Clustering ◽

Research Funding ◽

Drug Sensitivity ◽

Somatic Mutations ◽

Nk Cell ◽

Negative Regulator

Abstract Background Natural killer (NK) cell malignancies are rare lymphoid neoplasms characterized by aggressive clinical behavior and poor treatment outcomes. Clinically they are classified as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK cell leukemia (ANKL). Both subtypes are almost invariably associated with Epstein-Barr virus (EBV). Recently, genomic studies in NKTCL have identified recurrent somatic mutations in JAK-STAT pathway molecules STAT3 and STAT5b as well as in the RNA helicase gene DDX3X in addition to previously detected chromosomal aberrations. Here, we identified somatic mutations in 4 cases of ANKL in order to understand whether these entities share common alterations at the molecular level. To further establish common patterns of deregulated oncogenic signaling pathways operating in malignant NK cells, we performed drug sensitivity profiling using NK cell lines representing ANKL, NKTCL and other malignant NK cell proliferations. We aimed to identify sensitivities to agents that selectively target components of pathways required for survival of malignant NK cells in an unbiased manner. Methods Exome sequencing was performed on peripheral blood or bone marrow of ANKL patients using the NK cell negative fraction or other healthy tissue as control. Profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs. The NK cell lines KAI3, KHYG-1, NKL, NK-YS, NK-92, SNK-6 and YT and IL-2-stimulated and resting NK cells from healthy donors were used as sample material. All drugs were tested on a 384-well format in 5 different concentrations over a 10,000-fold concentration range for 72 h and cell viability was measured. A Drug Sensitivity Score (DSS) was calculated for each drug using normalized dose response curve values. Results The ANKL patients displayed mutations in genes reported as recurrently mutated in NKTCL, such as FAS, TP53, NRAS, STAT3 and DDX3X. Additionally, novel alterations in genes previously implicated in the pathogenesis of NKTCL were detected. These included an inactivating mutation in INPP5D (SHIP), a negative regulator of the PI3K/mTOR pathway and a missense mutation in PTPRK, a negative regulator of STAT3 activation. Interestingly, the total number of nonsilent somatic mutations in 3 out of 4 ANKL patients (97, 82 and 45) was remarkably high compared to other hematological malignancies analyzed in our variant calling pipeline. Analysis of drug sensitivities in NK cell lines showed a close correlation between all cell lines and a markedly higher correlation with those of IL-2 stimulated than resting healthy NK cells, suggesting that malignant NK cells may share a common drug response pattern. Furthermore, in an unsupervised hierarchical clustering the NK cell lines formed a distinct group from other leukemia cell lines tested (Fig. A). Among pathway-selective compounds (namely, kinase inhibitors and rapalogs), the drugs most selective for malignant NK cells fell into two major categories: PI3K/mTOR inhibitors (e.g. temsirolimus, buparlisib) and inhibitors of aurora and polo-like kinases such as rigosertib and GSK-461364 (Fig. B). JAK inhibitors (e.g. ruxolitinib, gandotinib) and CDK inhibitors (e.g. dinaciclib) showed strong efficacy in both malignant NK cells and IL-2 activated healthy NK cells. Conclusions Our exome sequencing results suggest that candidate driver alterations affecting similar signaling pathways underlie the pathogenesis of ANKL as has been reported in NKTCL. Drug sensitivity profiling highlights the PI3K/mTOR pathway as a potential major driver of malignant NK cell proliferation, whereas JAK-STAT signaling appears to be essential in both healthy and malignant NK cells. Components of these pathways harbored mutations in our small cohort of ANKL patients and have been shown to be deregulated by mutations or other mechanisms in previous studies, underlining their importance as putative drivers. The systematic large-scale characterization of drug responses also identified these pathways as potential targets for novel therapy strategies in NK cell malignancies. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Disclosures Mustjoki: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

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A Novel HIV Envelope Bi-Specific Killer Engager Enhances Natural Killer Cell Mediated ADCC Responses Against HIV-Infected Cells

Blood ◽

10.1182/blood.v128.22.2517.2517 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2517-2517 ◽

Cited By ~ 3

Author(s):

Zachary B. Davis ◽

Todd Lenvik ◽

Louis Hansen ◽

Martin Felices ◽

Sarah Cooley ◽

...

Keyword(s):

T Cell ◽

Nk Cells ◽

Cell Lines ◽

Research Funding ◽

Nk Cell ◽

Mhc I ◽

Variable Heavy ◽

Infected Cells ◽

T Cell Lines ◽

Hiv Envelope

Abstract Natural Killer (NK) cells, a critical component of the immune response to viral infection, recognize and destroy cells with diminished expression of major histocompatibility class-I (MHC-I) molecules and expression of ligands for activating NK receptors such as NKG2D. Down-modulation of MHC-I is a hallmark of viral infection, as it allows infected cells to evade a CD8 T-cell response. Stalling of the cell cycle to enhance viral replication induces NK activation ligands such as the NKG2D ligands unique long binding proteins (ULBP)-1 and -2 which could trigger NK destruction of infected cells. Unfortunately, incomplete down-modulation of MHC-I by HIV leaves HLA-C on the cell surface, which inhibits the majority of NK cells from killing infected targets. CD16, the low affinity Fc receptor, is the most potent NK cell activating receptor. It mediates antibody dependent cell-mediated cytotoxicity (ADCC), and can override inhibition by MHC-I. We designed a series of bi-specific killer-engager (BiKE) constructs to direct NK cell ADCC against an HIV-infected target. We linked the Fab portions of broadly neutralizing (bn)Abs to a novel llama-derived nanobody EF91 that binds CD16 at high affinity and signals strong activation. We chose to use EF91 as its structure is unique compared to the use of a single chain variable fragment (scFv). Rather than being composed of a variable heavy (VH) and variable light (VL) chain, the nanobody is composed of a single variable heavy (VHH) domain. A distinct advantage to using a CD16 nanobody over a scFv is in the purity of the generated product. During protein folding it is not uncommon for the wrong VH to associate with the wrong VL; the result of which is a nonfunctional product. Since the nanobody is single VHH, and does not require association with another domain, there is less risk of a misfolded product. Nanobodies are also known to have similar, if not increased, affinity for their target molecules. In the case of EF91, this may result in more robust activation of NK cells than with a traditional scFv. We tested a BiKE constructed with the bnAb, VRC01, which recognizes the CD4 binding domain of HIV-Env. The specificity of our novel anti-CD16 nanobody was demonstrated by binding of our BiKE construct to CD16+ NK cells (Figure 1A). Function of our BiKE construct was tested by incubating it with chronically infected T-cell lines (HIV-IIIB and ACH-2) or with their respective uninfected counterparts (H9 and CEM). We only observed binding to infected cells (Figure 1B), demonstrating HIV-Env binding specificity to the HIV strains ACH-2 (LAI strain) and HIV-IIIB. The ability of the anti-Env BiKE construct to mediate ADCC and IFNγ production was tested against two uninfected CD4 T-cell lines or their infected counterparts. While NK cells degranulated when incubated with the infected cell lines (50% against HIV-IIIB and 20% against LAI), this response was markedly enhanced when co-incubated with the HIV-Env specific BiKE (80% against HIV-IIIB and 60% against LAI) (Figure 1C). Furthermore, the HIV-Env BiKE enhanced IFNγ production against HIV-infected T-cell lines compared to responses in the absence of BiKE (28% against HIV-IIIB compared to 36% with BiKE; 15% against ACH-2 compared to 37% with BiKE) (Figure 1D). Our data demonstrate that a BiKE construct containing the Fab of an HIV bnAb and an anti-CD16 component can eliminate HIV-infected targets that express the HIV-envelope on their surface. The reservoir of latently infected CD4 T cells lack expression of any recognizable virus protein on the cell surface, we plan to combine our BiKE strategy with cellular activation using IL-15. Alternatively, we can construct a tri-specific engager (TriKE) with an IL-15 segment that may activate CD4 T cells while enhancing NK cell killing. Disclosures Cooley: Fate Therapeutics: Research Funding. Vallera:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

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Engineered Interleukin-15 Autocrine Signaling Invigorates Anti-CD123 CAR-NK Cells

Blood ◽

10.1182/blood-2021-146609 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2806-2806

Author(s):

Ilias Christodoulou ◽

Michael Koldobskiy ◽

Won Jin Ho ◽

Andrew Marple ◽

Wesley J. Ravich ◽

...

Keyword(s):

Nk Cells ◽

Peripheral Blood ◽

Research Funding ◽

Nk Cell ◽

Xenograft Model ◽

Blood Analysis ◽

Target Cells ◽

Interleukin 15 ◽

Peripheral Blood Analysis

Abstract Introduction : Acute Myeloid Leukemia (AML) is an aggressive neoplastic disorder with poor outcomes in children and adults. NK cell adoptive transfer is an anti-cancer immunotherapy that has promise for AML treatment. We aimed to improve NK cell anti-tumor efficacy with expression of a Chimeric Antigen Receptor (CAR) on the cell surface. Our CAR consists of an extracellular single-chain variable fragment targeting the AML-associated antigen CD123 (IL3Rα) and intracellular domains derived from 2B4 and TCRζ. We sought to improve the persistence and long-term functionality of our CAR-NKs by introducing transgenic interleukin-15 (IL15). Methods: CD3-depleted PBMCs were first activated with lethally irradiated feeder cells, then transduced with transiently produced replication incompetent γ-retrovirus (αCD123.2B4.ζ, αCD123.2B4.ζ-IRES-sIL15, sIL15-IRES-mOrange) on day 4 of culture. CAR expression was measured on day 8 using FACS. Secretion of IL15 was verified with ELISA. Cytotoxicity was measured using ffLuc expressing target cells and bioluminescence (BL) measurement. In serial stimulation assays, target cells were repleted daily to maintain a 1:1 effector:target ratio. Immunophenotype and cell counts were assessed by FACS. Transcriptomic analysis (RNAseq) was performed on RNA derived from NK cells purified on D10. Xenograft modeling was performed using NSG mice engrafted with MV-4-11.ffLuc or MOLM-13.ffLuc AML cell lines. Mice were treated with NK cells on D4 or D4-7-10. Untreated mice served as controls. Tumor growth was serially tracked in vivo using BL imaging. NK cell persistence and expansion were measured in peripheral blood. Results: The 2B4.ζ CAR was well expressed on the surface of transduced NK cells (median transduction efficiency 95%, range 85-97%, n=3). 2B4.ζ CAR-NK treatment prolonged survival of AML engrafted mice when compared to treatment with unmodified NKs (median survival: 63 vs 55 days; n=8 mice; p=0.014). Serial peripheral blood analysis revealed a steady decline in circulating NK cells, which were undetectable in all cohorts within 21 days. NK cells were then engineered for constitutive secretion of IL15, with and without CAR expression. 2B4.ζ/sIL15 CAR-NKs had the most potent 24h-cytotoxicity against CD123+ targets (Fig. 1). After a 10-day chronic stimulation with MV-4-11, 2B4.ζ/sIL15- and sIL15-NKs expanded (x1.2 and x5.9 respectively), while NK cells without sIL15 decreased in number. In this assay, only 2B4.ζ/sIL15 CAR-NKs exhibited sustained tumor killing. Transcriptomic analysis after 10 days of serial stimulation showed sample clustering dependent on IL15 secretion. Differential gene expression analysis (DESeq2) identified upregulation of genes associated with cell cycle progression, apoptosis regulation, chemokine signaling, and NK cell mediated cytotoxicity in NK cells secreting IL15 compared to those without. In multiparameter flow cytometric analysis, 2B4.ζ/sIL15 CAR-NKs had a higher percentage of NK cells populating clusters defined by higher surface expression of NK cell activating receptors (NKp30, NKG2D, LFA-1) compared to 2B4.ζ and unmodified NK cells. In our MV-4-11 xenograft model, NKs armed with secreted IL15 expanded in vivo and had improved persistence. A single dose (D4) of 2B4.ζ/sIL15 CAR-NKs demonstrated an initial antitumor response, equivalent to that seen following 3 doses (D4-7-10) of 2B4.ζ CAR-NKs. However, mice treated with IL15-secreting NKs had short survival (Fig. 2). Compared to control mice, peripheral blood analysis showed increasing systemic hIL15 and higher levels of hTNFα. In our more aggressive MOLM-13 xenograft model, both single dose 2B4.ζ/sIL15 CAR-NK and multiple dose 2B4.ζ CAR-NK treatment prolonged survival compared to treatment with unmodified NKs. (27 and 26 vs 20 days; n=5 mice; p<0.01; Fig. 2). Conclusion: 2B4.ζ CAR-NKs have limited antitumor efficacy and short persistence in vivo. NK cells armored with secreted IL15 have enhanced anti-AML cytotoxicity and in vitro persistence. Introduction of IL15 secretion confers a distinctly activated phenotype that is maintained during chronic antigen stimulation. Constitutive local IL15 secretion improves in vivo NK cell persistence but may cause lethal toxicity when employed against AML. These results warrant further study and should impact the development of CAR-NK clinical products for patients with AML. Figure 1 Figure 1. Disclosures Ho: Rodeo Therapeutics/Amgen: Patents & Royalties; Exelixis: Consultancy; Sanofi: Research Funding. Bonifant: Kiadis Pharma: Research Funding; BMS: Research Funding; Merck, Sharpe, Dohme: Research Funding.

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Hypersialylation Protects Multiple Myeloma Cells from NK Cell-Mediated Immunosurveillance and This Can be Overcome By Targeted Desialylation Using a Sialyltransferase Inhibitor

Blood ◽

10.1182/blood-2019-126613 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 138-138

Author(s):

John Daly ◽

Subhashis Sarkar ◽

Alessandro Natoni ◽

Robert Henderson ◽

Dawn Swan ◽

...

Keyword(s):

Multiple Myeloma ◽

Sialic Acid ◽

Nk Cells ◽

Board Of Directors ◽

Cell Lines ◽

Immune Evasion ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees

Introduction: Evading Natural Killer (NK) cell-mediated immunosurveillance is key to the development of Multiple Myeloma (MM). Recent attention has focused on the role of hypersialylation in facilitating immune-evasion of NK cells. Abnormal cell surface sialylation is considered a hallmark of cancer and we have implicated hypersialylation in MM disease progression. Certain sialylated glycans can act as ligands for the sialic acid-binding immunoglobulin-like lectin (Siglec) receptors expressed by NK cells (Siglec-7 and Siglec-9). These ITIM motif-containing inhibitory receptors transmit an inhibitory signal upon sialic acid engagement. We hypothesized that desialylation of MM cells or targeted interruption of Siglec expression could lead to enhanced NK cell mediated cytotoxicity of MM cells. Methodology: MM cells were treated with the sialidase neuraminidase prior to co-culture with primary NK (PNK) cells. MM cells were treated with 300µM 3Fax-Neu5Ac (sialyltransferase inhibitor) for 3 days prior to co-cultures with PNK cells. PNK cells were expanded, IL-2 activated (500U/ml) overnight, or naïve (resting). Primary MM samples/MM cell lines were screened with Siglec-7/9 chimeras (10µg/ml). PNK (IL-2 activated) cells were stained with anti-Siglec-7 and anti-Siglec-9 antibodies. Siglec-7 was targeted for knockout (KO) using the CRISPR/Cas9 system, a pre-designed guideRNA and the MaxCyteGT transfection system. MM cells were treated with 10µg/ml of Daratumumab prior to co-culture with expanded PNK cells. Results: Using recombinant Siglec-7/9 chimeras a panel of MM cell lines (MM1S, RPMI-8226, H929, JJN3 and U266) were shown to express ligands for Siglec-7 and Siglec-9 (>85%, n=3). Primary MM cells isolated from BM of newly diagnosed (n=3) and relapsed patients (n=2) were also shown to express Siglec-7 ligands (72.5±17.5%, 36.5% respectively). PNK cells express Siglec-7 and Siglec-9 (94.3±3.3% and 61±8.8% respectively, n=6). Desialylation of the MM cell lines JJN3 and H929 using neuraminidase significantly enhanced killing of MM cells by healthy donor (HD) derived PNK cells (expanded, IL-2 activated and naïve, n=7) at multiple effector:target (E:T) cell ratios. Furthermore, de-sialylation of JJN3 and H929 using neuraminidase resulted in increased NK cell degranulation (CD107α expression), compared to a glycobuffer control (n=7). De-sialylation, using 300µM 3Fax-Neu5Ac, resulted in strongly enhanced killing of MM1S by expanded HD-derived PNK cells at multiple E:T ratios (n=5, p<0.01 at 0.5:1, p<0.001 at 1:1, p<0.01 at 2.5:1). Furthermore, CD38 expression on H929 MM cells significantly increased after treatment with 300µM 3Fax-Neu5Ac for 3 days (p<0.01, n=3). In a cytotoxicity assay, expanded PNK cell-mediated antibody dependent cellular cytotoxicity (ADCC) of H929 MM cells pre-treated with Daratumumab (anti-CD38 moAb) and 3Fax-Neu5Ac was significantly higher than H929 cells pre-treated with Dara (p<0.05 at 0.5:1, p<0.01 at 1:1) or 3Fax-Neu5Ac (p<0.01 at 0.5:1, p<0.01 at 1:1) alone (n=5). Using CRISPR/Cas9, over 50% complete KO of Siglec-7 was observed on expanded PNK cells, yet did not result in enhanced NK cell-mediated cytotoxicity against either H929 or JJN3 (n=7). Siglec-9 KO using CRISPR/Cas9 is ongoing. Discussion: Hypersialylation of MM cells facilitates immune evasion and targeted removal of sialic acid strongly enhances the cytotoxicity of NK cells against MM. However, to date the role of Siglecs remains inconclusive. Nevertheless, our data suggest that targeted desialylation is a novel therapeutic strategy worth exploring in MM. In particular, upregulation of CD38 provides a strong rationale for combinatory strategies employing targeted desialylation with CD38 moAbs such as Daratumumab, with the goal of maximizing ADCC. Disclosures Sarkar: Onkimmune: Research Funding. O'Dwyer:Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; GlycoMimetics Inc: Research Funding; AbbVie: Consultancy.

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Selinexor Enhances NK Cell Activation Against Lymphoma Cells Via Downregulation of HLA-E

Blood ◽

10.1182/blood-2021-144836 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2411-2411

Author(s):

Jack Fisher ◽

Christopher J. Walker ◽

Peter Johnson ◽

Mark S Cragg ◽

Francesco Forconi ◽

...

Keyword(s):

Nk Cells ◽

Peripheral Blood ◽

Research Funding ◽

Cell Activation ◽

Nk Cell ◽

Cell Activity ◽

Fold Increase ◽

Target Cells ◽

Lymphoma Cells ◽

Pre Treatment

Abstract Introduction: Natural killer (NK) cells are powerful immune effectors which induce direct cytotoxicity, promote adaptive immune responses and mediate antibody dependent cellular cytotoxicity (ADCC). Enhancement of NK cell activity against cancer is currently the focus of intense research efforts and strategies include CAR-NK, stimulatory antibodies, cytokines and checkpoint inhibitors. Upregulation of exportin-1 (XPO1) is common in human cancers and high expression is negatively associated with survival in various cancers including diffuse large B cell lymphoma (DLBCL). Targeted inhibition of XPO1 by the selective inhibitor selinexor leads to cancer cell death via accumulation of tumour suppressor proteins in the nucleus, dysregulation of growth regulatory proteins and blockade of oncogene protein translation. The therapeutic efficacy of XPO1 inhibition has led to FDA approval of the oral XPO1 inhibitor selinexor for the treatment of multiple myeloma and DLBCL. The effect of selinexor on NK cell activity has not previously been investigated and was therefore addressed in this study. Methods: The B lymphoma cell lines JeKo-1, SU-DHL-4 and Ramos were incubated with selinexor (50-2000nM) for 18 hours before analysis. Flow cytometry was used to assess cell surface expression of activating and inhibitory ligands for NK cells. For NK based assays, peripheral blood derived NK cells were isolated from healthy donors and incubated with IL-15 (1ng/ml) overnight prior to co-culture with target lymphoma cells for a further 4 hours. Cytotoxicity was assessed using propidium iodide staining of target cells and degranulation of NK cells was assessed by measurement of CD107a. Whole blood samples from colorectal cancer patients (n=11) at pre-treatment and 3 weeks post selinexor monotherapy were assessed by flow cytometry for CD45+CD3-CD19-CD56+ NK cells. Results: Selinexor pre-treatment of target lymphoma cells significantly increased NK cell mediated cytotoxicity against SU-DHL-4 (2.2 Fold increase, p<0.01), JeKo-1 (2 Fold increase, p<0.01) and Ramos (1.7 Fold increase, p<0.01) cells. In accordance with this, selinexor pre-treatment of target cells also increased the activation of NK cells against SU-DHL-4, JeKo-1 and Ramos cells as measured by CD107a expression in both CD56 bright and CD56 dim NK cell sub-groups. To identify the mechanism behind this, we measured expression of activating and inhibitory ligands for NK cells on SU-DHL-4 cells after incubation with selinexor. No significant changes in expression of activating ligands (MICA/B, ULBP-2/5/6, ULBP-1, Vimentin, B7H6, CD54) were evident. In contrast, selinexor significantly (p<0.001) reduced the surface expression of HLA-E on SU-DHL-4 cells by 50%. Selinexor mediated downregulation of HLA-E was also evident in Ramos (60% reduction, p<0.001) and JeKo-1 cells (20% reduction, p<0.01). HLA-E binds the ITIM containing receptor NKG2A, a key inhibitory receptor for NK cells and subsets of T cells. In accordance with this, selinexor pre-treatment of SU-DHL-4 cells selectively increased NKG2A+ NK cell activation (p<0.01) following co-culture. To examine the effect of selinexor on NK cells in patients, we assessed the proportion of NK cells in the peripheral blood of 11 colorectal cancer patients at pre-treatment and three weeks post selinexor monotherapy. % NK cells of CD45+ peripheral blood lymphocytes following treatment with selinexor was increased 2-fold (Median 5% pre-treatment vs 10% post selinexor). In addition, increased abundance of the less mature and less cytotoxic CD56 bright subset of NK cells was associated with poor response to therapy (Median 4% responders (n=3) vs 20% non-responders (n=8)). Larger patient datasets are required to confirm these effects and this analysis is currently ongoing. The effect of selinexor on NK cells in patients with lymphoma is also currently under investigation. Conclusions: The NKG2A:HLA-E axis is a novel immune checkpoint target and our data identifies that selinexor sensitises lymphoma cells to NK cell mediated killing via disruption of this interaction. In addition, we provide initial evidence that NK cells may be associated with clinical response to selinexor. This data indicates that NK cells may contribute to the therapeutic efficacy of selinexor and that selinexor may synergise with NK cell targeted therapies for the treatment of lymphoma. Disclosures Walker: Karyopharm Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Johnson: Morphosys: Honoraria; Kymera: Honoraria; Kite Pharma: Honoraria; Incyte: Honoraria; Genmab: Honoraria; Celgene: Honoraria; Bristol-Myers: Honoraria; Epizyme: Consultancy, Research Funding; Boehringer Ingelheim: Consultancy; Novartis: Honoraria; Takeda: Honoraria; Oncimmune: Consultancy; Janssen: Consultancy. Cragg: BioInvent International: Consultancy, Research Funding; GSK: Research Funding; UCB: Research Funding; iTeos: Research Funding; Roche: Research Funding. Forconi: Novartis: Honoraria; Roche: Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau; Gilead: Research Funding. Landesman: Karyopharm Therapeutics: Current Employment, Current equity holder in publicly-traded company. Blunt: Karyopharm Therapeutics: Research Funding.

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