ALBERT

Description: 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

Description: Background: The prognosis of children with relapsed/refractory B-cell (CD20+) non-Hodgkin lymphoma (B-NHL), including Burkitt lymphoma (BL), is dismal due to chemo-radiotherapy resistance (Cairo et al, JCO, 2012, Cairo et al, BJH, 2018). Rituximab has been widely used in frontline treatment of B-NHL, however, some patients retreated with rituximab will relapse which limits patient treatment options (Goldman/Cairo, Leukemia, 2013). Several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering of NK cells with chimeric antigen receptors (Chu/Cairo et al, Can Imm Res 2015), as well as second-generation engineered anti-CD20 antibodies (Tiwari/Cairo et al BJH 2015). To enhance NK based therapy, our group has successfully expanded the functionally activated peripheral blood NK cells (exPBNK) with irradiated feeder cells (Chu/Cairo, et al, Can Imm Res 2015). To enhance NK cell-based targeted therapy, we had successfully engineered expanded NK cells with chimeric antigen receptors (Chu/Cairo, et al, Can Imm Res 2015) and combined expanded NK with an anti-CD20 targeted IL-15 fusion protein N-820 (2B8T2M) targeting rituximab-sensitive and -resistant BL (Chu/Cairo, et al, ASH 2017). N-820 was generated by fusing ALT-803 to four single-chain antibody domains of rituximab (Liu/Wong, et al, JBC, 2016). ALT-803 is a superagonist of an IL-15 variant bound to an IL-15RαSu-Fc fusion with enhanced IL-15 biological activity (Zhu et al. 2009 J Immunol), longer half-life and increased potency (Han, et al. Cytokine. 2011). N-820 displayed tri-specific binding activity through its recognition of CD20 on tumor cells, activated NK cells to enhance antibody-dependent cellular cytotoxicity (ADCC), and induced apoptosis of B-lymphoma cells (Liu/Wong, et al, JBC, 2016). Objective: To investigate how N-820 modulates the crosstalk between BL tumor cells and expanded NK cells by monitoring cytokines, chemokines and growth factors released and the anti-tumor effects of N-820 on NK cells in BL xenografts. Method: PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells and isolated as we have previously described with K562-mbIL15-41BBL (Chu/Cairo, et al, Can Imm Res 2015). ALT-803 and N-820 were generously provided by Altor Bioscience. Equal doses of N-820, Rituximab (R), ALT-803, R+ALT-803, obinutuzumab (obinu, generously provided by Christian Klein, PhDfrom Roche) were used for comparison. IgG was used as control. Rituximab-sensitive Raji and -resistant BL cells Raji-2R and Raji-4RH were used as target cells. NK cells were sorted by Beckman Coulter MoFlo XDP high-speed sorter and NK purity was confirmed by flow cytometry. Cytokines, chemokines and growth factors mRNA levels in NK cells were monitored by real-time PCR. Secreted cytokines, chemokines and growth factors were examined by standard ELISAs. Raji-2R-Luc cells were injected into NSG mice. ExPBNK+N-820 and controls were injected to the xenografted NSG mice. The tumor burden was measured with the IVIS-200 system. Results: We found that ALT-803 and N-820 at equal doses significantly enhanced the expression of NK activating receptors such as NKG2D, NKp30, NKp44, and CD16 on exPBNK compared to IgG controls. Additionally, N-820 significantly enhanced exPBNK cytotoxicity against Raji, Raji-2R and Raji-4RH cells compared to the controls IgG, R, ALT-803, R+ALT-803, or obinu (p

Description: 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 and refractory MM, but the response is transient in most cases. Putative mechanisms of suboptimal efficacy of DARA include down-regulation of CD38 expression and over-expression 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 if 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. Additionally, transcriptomic and cellular metabolic analysis demonstrated that CD38KO NK cells have unique metabolic reprogramming with higher mitochondrial respiratory capacity. Lastly, we evaluate the impact of exposure to all-trans retinoic acid (ATRA) on wild type NK and CD38KO NK cells function and highlight potential benefit 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.

Description: Immunotherapy is a promising approach to improve treatment responses in hematological malignancies. Accumulating evidence suggests a role for natural killer (NK) cells in controlling hematological malignancies. However, mechanisms regulating sensitivity or resistance of hematologic cancer cells to the effector function of NK cells are incompletely understood. Here, we performed genome-scale CRISPR-Cas9 loss-of-function screens to systematically map genes that regulate sensitivity of hematologic malignancies to NK cells. To screen for genes involved in the interaction between NK and cancer cells, we infected human cancer cells expressing Cas9 with a genome-scale lentiviral guide RNA library (Figure). The resulting pool of knockout cells was exposed to NK cells expanded from peripheral blood of healthy donors. Enriched and depleted knockouts were detected by next-generation sequencing of the integrated sgRNA cassettes, enabling identification of genes conferring resistance or susceptibility to NK cell-mediated lysis. The screens were performed in cell lines from diverse hematological malignancies, including chronic myeloid leukemia (CML), B cell acute lymphoblastic leukemia, diffuse large B cell lymphoma (DLBCL), and multiple myeloma. We recovered several known mechanisms of NK cell/cancer cell interactions, demonstrating feasibility of the screening approach. Loss of genes encoding components of the MHC class I complex (B2M, HLA-A, HLA-C, HLA-E) sensitized multiple cancer cell lines to NK cell lysis, consistent with missing-self recognition. Furthermore, knockout of IFN-JAK-STAT signaling mediators led to increased tumor cell lysis, suggesting that MHC class I induction in response to NK cell-derived IFN gamma enables NK cell evasion by tumor cells. We also identified genes essential for effective NK cell-mediated lysis. NCR3LG1, encoding the B7-H6 ligand for the NKp30 activating NK cell receptor, was essential for NK cell lysis of CML cells. In contrast, knockout of apoptotic mediators and TRAIL pathway components conferred resistance to NK cell cytotoxicity in DLBCL cells, indicating heterogeneity in NK cell/cancer cell interactions between cancer types. Our data support a view that distinct mechanisms regulate sensitivity to NK cell cytotoxicity in different hematologic cancers. Importantly, our results indicate that loss-of-function mutations in the antigen-presenting machinery and the IFN-JAK-STAT pathway sensitize tumors to NK cell effector function. As alterations in these genes are associated with resistance to T cell immunotherapies such as PD-1 blockade, NK cell-based therapies could be employed to overcome resistance in these patients. In summary, we suggest that systematic identification of mechanisms governing tumor immune susceptibility has the potential to uncover novel immunotherapy targets. Figure. Figure. Disclosures Kankainen: Medix Biochemica: Consultancy. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mustjoki:Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria; Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.

Description: Background: Disease relapse following allogeneic stem-cell transplantation remains unacceptably high and there is an urgent need for new therapies that decrease relapse rates and improve survival post-transplant. Natural killer (NK) cells have potent antitumor effects, particularly those expended with mb-IL21 from peripheral blood. Preliminary data from a phase-1 dose-escalation study of up to 1x108 NK cells/Kg/dose and multiple dosing yielded promising results and a favorable safety profile (Ciurea SO.Blood.2017;130:18657). This report presents long-term follow-up from a phase-1/2 clinical trial in patients with high-risk myeloid malignancies (AML/MDS/CML) (clinicaltrials.gov NCT01904136) and a comparison with CIBMTR controls. Methods: Patients received conditioning with fludarabine 160 mg/m2, melphalan 140 mg/m2 and 2GyTBI, post-transplant cyclophosphamide-based GVHD prophylaxis and bone marrow graft from a haploidentical donor. Ex vivo expanded NK cells were generated from peripheral blood mononuclear cells of the same donor with a K562 feeder cells expressing mb-IL21 and 41BB and infused fresh on Day-2, and cryopreserved on Day+7 and +28 (up to Day+90). 1x108/Kg/dose was chosen for the phase 2 trial. An independent matched-pair analysis was done using controls from the CIBMTR database stratified by conditioning intensity. Results: 24/26 patients treated to date were evaluable (one short follow-up and one excluded as ineligible). 80% (19/24) of patients received all 3 doses of NK cells. The median age was 45 years (range 18-59), median follow-up was 43.6 months (range 15.1-60.9). Thirteen patients (54%) were females. 5 patients had donor-specific anti-HLA antibodies (DSA). The median HCT-CI was 2 (range 0-8), 12 patients (50%) had HCT-CI〉3. 17 patients (72%) had AML/MDS and 7 (28%) advanced CML. Of AML/MDS patients, 10 (59%) had high-risk cytogenetics, 7 (41%) had measurable residual disease, 9 (53%) had intermediate/adverse-risk ELN2017 and 5 (29.4%) had primary induction failure. No infusion reactions or significant adverse events were observed to date. All patients (100%) achieved engraftment after a median of 19 days (range 14-42). The cumulative incidence (CI) of grade 2-4 aGVHD was 29.2% at Day100 and 41.7% at 1-year post-transplant. Only one patient developed severe grade 3-4 aGVHD and one patient had extensive cGVHD. Only one patient relapsed (a patient with DSA who did not receive desensitization prior to transplantation), 1-year CI of relapse was 5.9%. The CI of TRM at 1-year for patients without DSA was 21%. The median overall survival and progression-free survival (PFS) were not reached. The 1-year and 3-year PFS for all patients and patients without DSA was 70.8% and 66.1%, and 79% and 72.9% for patients without DSA, respectively (Figure 1). One-year and 3-years GRFS for all patients and patients without DSA was 70.8% and 66.1%, and 79% and 72.9%, respectively. An independent matched-pair analysis (at least 1:1) was conducted by CIBMTR after the first 18 patients treated on study in 07/2018 with RIC (N=57) or MAC (N=61) controls. The relapse was 1/18 vs 25/57 for RIC (p=0.037) and 15/61 for MAC (p=0.07), while the 1-year PFS was 82% vs 49% for RIC and 64% for MAC (p=0.21) (Figure 1). Updated results of this analysis will be presented at the meeting. Conclusions: Results from this long-term follow-up analysis confirm very low relapse rate and excellent GRFS after haploSCT for patients treated with high-doses of NK cells expanded with mbIL21 stimulation. A prospective multi-center phase 2 BMTCTN study will evaluate the safetly and efficacy of high doses of NK cells for the prevention of relapse in patents with AML/MDS receiving haploSCT. Figure 1 Disclosures Ciurea: Miltenyi: Research Funding; Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees, Other: stock holder; MolMed: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees. Bashir:Kite: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; StemLine: Research Funding; Acrotech: Research Funding; Celgene: Research Funding; Imbrium: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Pasquini:Novartis: Research Funding; Kite Pharmaceuticals: Research Funding; BMS: Research Funding; Medigene: Consultancy; Amgen: Consultancy; Pfizer: Consultancy. Lee:Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Description: Introduction Immunotherapy represents a new weapon in the fight against multiple myeloma. Current clinical outcomes using CAR-T cell therapy against multiple myeloma show promise in the eradication of the disease. However, these CARs observe relapse as a common phenomenon after treatment due to the reemergence of neoantigens or negative cells. CARs can also be targeted using non-antibody approaches, including the use of receptors, as NKG2D with a wider range of ligands, and ligands to provide target specificity. Different cell types have been used to improve CAR cell therapy. CAR-T cells are the most commonly used. However, despite its effectiveness, there are still problems to face. The toxicity of the cytokine release syndrome is well known, that is why memory CD45RA- T cells are used to avoid collateral effects, although having lower efficacy. However, CAR-NK cells may have less toxicity and provide a method to redirect these cells specifically to refractory cancer. The objective of this work was to compare the anti-tumor activity of CAR-T, NKAEs and CAR-NK cells from multiple myeloma patients. Methods The activated and expanded NK cells (NKAE) were generated by coculture of peripheral blood mononuclear cells with the previously irradiated CSTX002 cell line. The CD45RA- T cells were obtained by depletion with CD45RA magnetic beads and subsequent culture. The NKAE and T were transduced with an NKG2D-CAR with signaling domains of 4-1BB and CD3z. The expansion of NKAE and the expression of NKG2D-CAR were evaluated by flow cytometry based on the percentage of NK cell population and transduction efficiency by the expression of NKG2D. Europium-TDA release assays (2-4 hours) were performed to evaluate in vitro cytotoxic activity. The antitumor activity of the NKAE (n=4) and CD45RA- (n=4) cells against MM U-266 cells was studied. Methylcellulose cultures were performed to assess the activity against the clonogenic tumor cell. In vivo studies were carried out in NSG mice receiving 5.106 of U266-luc MM cells i.v. injected at day 1. At day 4, mice received 15.106 i.v. injected of either CAR-NKAE or untransduced NKAE cells. Results In vitro. The killing activity of primary NKAE cells (n=4) was 86.6% (± 13.9%), considerably higher than that of CD45RA- lymphocytes (16.7% ± 13.6%) from the same patient (n=4). Even CD45RA- T cells from healthy donors (n=4) exhibit lower anti tumoral capacity (28.2% ± 9.7%) than NKAE cells. The transduction with an NKG2D CAR (MOI=5) improved the activity of autologous NKAE cells by 10% (96.4% ± 19%) leading to a nearly complete destruction of U-266 MM cells, and that of CD45RA- allogenic healthy cells in 19% (47.4% ± 12.6%). Nevertheless, CD45RA- autologous T cells transduced with NKG2D-CAR minimally improved their activity by 5.8% (22.5% ± 10.6%). Additionally, the CAR-NKAE cells were able to destroy the clonogenic tumor cell responsible for the progression of the MM from RPMI-8226 cell line. At an 8:1 ratio the CAR-NKAE cells were able to destroy 71.2% ± 2.5% of the clonogenic tumor cells, while the NKAE reached 56.5% ± 2.6% at a maximum ratio of 32: 1. The toxicity of the CAR-NKAE cells on healthy tissue from the same patient was assessed, and no activity against autologous PBMCs was observed, 1,8% at a maximun ratio of 32:1 (effector:target). In vivo. NKAE cells and CAR-NKAE cells were efficient in abrogating MM growth. However, CAR-NKAE cells treatment showed higher efficiency 14 days after tumor cells injection. Forty-two days after tumor cells injection, only animals receiving CAR-NKAE cells treatment remain free of disease (Figure 1). Conclusions It is feasible to modify primary NKAE cells and CD45RA- T cells from primary MM cells to safely express an NKG2D-CAR. Our data show that CD45RA- T cells from patients are not effective in vitro against MM even once transduced with our CAR. The resulting CAR-NKG2D NKAE cells are the most appropriate strategy for the destruction of MM in vitro and in vivo in our model. These results form the basis for the development of an NKG2D-CAR NK cell therapy in MM. Disclosures Rio: Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Lee:Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Martinez-Lopez:Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Vivia: Honoraria; Pfizer: Research Funding; BMS: Research Funding; Novartis: Research Funding.

Description: Genetic modification of NK cells to enhance cancer immunotherapy has potential application to treat a wide range of cancers. Recently, we developed a new strategy in which CRISPR/Cas9 elements are introduced into NK cells as ribonucleoproteins (RNPs) via electroporation, followed by expansion on feeder cells expressing 4-1BBL and membrane-bound IL-21 to generate large numbers of genetically modified NK cells. This method was used to genetically modify several genes in primary and expanded NK cells including suppressor of cytokine signaling 3 (SOCS3). SOCS3 negatively regulates cytokine signaling through the JAK/STAT pathway. We hypothesized that disruption of SOCS3 in primary NK cells using Cas9/RNPs could potentially maintain STAT3 signaling levels and subsequently increase their proliferation and cytotoxic function. We designed gRNAs to target exon 2 of the SOCS3 gene and electroporated them along with Cas9 protein as Cas9/RNPs into primary NK cells using the Lonza 4D electroporator. We tested 6 different conditions of gRNAs alone or in combination. The NK cells in the control group were electroporated with no Cas9/RNPs. After electroporation, the cells were rested in culture media supplemented with 100 IU of human IL-2 for 48 hours and then expanded using irradiated feeder cells. At day 7, an equal number of cells were restimulated with irradiated feeder cells to test the effect of SOCS3 KO on proliferation. Western blot was used to assay the knock out efficacy at the protein level. Calcein assay and IncuCyte Zoom (Essen) were performed to measure cytotoxicity against two cancer cell lines, K562 and Daoy. Our results showed a significant reduction in SOCS3 protein levels in 3 conditions (gRNA1, gRNA3 and gRNA1+ gRNA3) in comparison to the control group. The calcein assay and IncuCyte zoom showed the modified SOCS3 KO NK cells could kill the tumors more efficiently in comparison to the control. Proliferation data showed the SOCS3 KO cells can grow faster than the control group. In conclusion, our data demonstrate the role of SOCS3 and JAK/STAT pathway in NK cell function and suggests that SOCS3 may be a good target for genetic modification to improve cancer immunotherapy using NK cells. Disclosures Lee: CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: In patients with acute leukemia, relapse remains one of the most frequent causes of treatment failure after allogeneic transplantation (allo-HCT). Early after transplant, natural killer (NK) cells predominate and thus, they have been implicated in graft vs. leukemia (GVL) reactions after allo-HCT. Killer Ig Receptors (KIR) are a family of 15 distinct genes that both positively and negatively regulate NK cell function. Individuals vary in the number of genes that they possess within their genome and thus, may vary in their GVL capacity. Accordingly, numerous studies have investigated the association of donor KIR gene content (B/x vs. A/A), KIR B gene score (neutral, better, best), KIR composite score (taking into account CenB/x and TelA/A gene status) and KIR ligand mismatching in the setting of allo-HCT. These studies have largely focused on adult transplant recipients, yielding variable outcomes. However, pediatric patients may differ from adult transplant recipients in a variety of ways including differential sensitivity of leukemic blasts to NK cytotoxicity, distinct cytogenetic changes, the higher propensity to receive myeloablative conditioning and an earlier adoption of minimal residual disease-driven timing of transplantation. The purpose of this analysis was to investigate the impact of KIR gene content and KIR ligand mismatching on outcomes of children transplanted with acute lymphoblastic and acute myeloid leukemia (ALL and AML, respectively), reported to CIBMTR. The study cohort included pediatric patients (0-

Description: Introduction Therapy of marrow borne disease such as leukemias or myelomas require the eradication of tumor cells that are resident in the bone marrow. Cell based therapies are increasingly a promising modality toward potential complete cures of such marrow malignancies. In particular, natural killer (NK) cell therapeutics are an attractive therapeutic modality due to their inherent ability to broadly detect and kill tumors. However, for NK cells to kill the marrow resident blasts, they must traffic to the bone marrow. Key for marrow homing is the proper fucosylation of extracellular proteins (such as e.g. P-selectin glycoprotein ligand-1, PSGL) on NK cells which is necessary for E-selectin binding. PM21 particles are plasma membrane particles prepared from CSTX002 cell line (K562 cells modified to express membrane bound IL-21 [mbIL21] and 4-1bbl, K562.mbIL21.41BBL) and can be used to potently stimulate expansion of NK cells that have high anti-tumor potency, similarly as to the expansion with CSTX002 feeder cells that have mbIL21 stimulation. Shown herein is that NK cells stimulated with PM21 particles have increased fucosylation of E-selectin binding ligands and have increased homing to the bone marrow in mice. Methods NK cells were expanded from PBMCs obtained from healthy donors by stimulating with PM21 particles (200 µg/mL) in SCGM media supplemented with 10% FBS and IL-2 (100 U/mL). PM21 particles were prepared from CSTX002 cell line as previously described. The fucosylation status of E-selectin ligands on expanded NK cells were analyzed by flow cytometry with the HECA452 mAb that only binds the fucosylated forms. ChIP-seq analysis was performed on NK cells expanded from feeder cell co-cultures with CSTX002 and by probing with anti-STAT3. For physiological trafficking studies, whole PBMCs were pre-activated in vitro for two days with PM21 particles (200 µg/mL) and then injected ip into NSG mice and treated or not with PM21-particles (2x/weekly). Organs were harvested 16 days later and analyzed by flow cytometry for the presence of human NK cells. Results NK cells expanded using PM21 or other stimulation methods were comparatively analyzed for fucosylation status by staining with HECA452. The NK cells stimulated with mbIL21, either through PM21 or CSTX002 feeder cells, were found to have highest HECA452 staining compared NK cells stimulated with IL-2, soluble IL-21, soluble 4-bbl, unmodified K562, or K562 modified with only mbIL21 (no 4-1bbl). To determine transcriptional changes responsible for the apparent increase in fucosylation, anti-STAT3 ChIP-seq analysis was performed on naïve NK cells or those expanded with CSTX002. STAT3 is a transcription factor that is activated upon IL-21 stimulation. The anti-STAT3 ChIP-seq analysis elucidated that CSTX002 stimulated NK cells have enhanced STAT3 binding to the FUT7 gene and that FUT7 expression increased. To determine if stimulation with PM21 particles can enhance homing of NK cells to bone marrow, NSG mice were injected ip with NK cells stimulated in vitro for 2 days with PM21, and then the mice were treated with or without PM21 particles (ip, 800 µg, 3x per week). Analysis of the bone marrow from sacrificed animals showed that a greater amount of human NK cell were present in the marrow in animals treated with ip doses of PM21 particles. Conclusions NK cells expanded with PM21 particles had increased fucosylation of likely E-selecting ligands. This higher degree of fucosylation is likely due to the enhanced expression of fucosyl transferase 7, shown bye ChIP-seq analysis of CSTX002 stimulated NK cells. In vivo studies show that stimulation with PM21 does allow for increased trafficking of NK cells to the bone marrow. Disclosures Oyer: Kiadis Pharma: Equity Ownership, Patents & Royalties. Igarashi:Kiadis Pharma: Employment, Equity Ownership, Patents & Royalties. Altamore:Kiadis Pharma: Equity Ownership, Patents & Royalties. Lee:Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Copik:Kiadis Pharma: Equity Ownership, Patents & Royalties, Research Funding.

Description: Natural killer (NK) cells have been suggested to control progression and relapse in several hematological malignancies. Enhancing NK cell reactivity represents an attractive approach to improve treatment responses. However, mechanisms enabling evasion of hematologic cancer cells from NK cells are incompletely understood. To identify cancer cell-intrinsic factors enabling resistance to NK cell cytotoxicity, we conducted genome-wide CRISPR screens in a range of hematological malignancies. Cas9-expressing cancer cells from diverse hematological malignancies, including acute and chronic myeloid leukemia (AML and CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), and B cell acute lymphoblastic leukemia were infected with a genome-scale lentiviral sgRNA library and exposed to primary human NK cells. Genes essential for NK cell cytotoxicity were discovered from surviving cancer cells which were enriched with gene knockouts inducing reduced sensitivity to NK cell killing. Additional data from a gain-of-function screen using a genome-scale CRISPR activation system were generated using the MM.1S myeloma cell line. Results from the functional genomic screens were integrated with transcriptomic data from 〉 9,000 patients across hematological cancers, as well as multi-omics data from AML and DLBCL, and both public and in-house single-cell RNA-sequencing data from AML patients. Knockout of genes encoding components involved in antigen presentation (B2M, HLA-A, HLA-C, HLA-E, TAP1, TAP2) sensitized multiple blood cancer cell lines to NK cell cytotoxicity, consistent with the inhibitory effect of MHC I on NK cells. Furthermore, knockout of interferon and JAK-STAT signaling components sensitized cancer cells to NK cell lysis, suggesting that MHC class I induction in response to interferon promotes NK cell resistance across cancer types. Interestingly, genes and pathways whose disruption conferred resistance for NK cell-mediated lysis exhibited partial overlap but also substantial variability across blood cancer types. Knockout of NCR3LG1 (B7-H6, ligand for the NKp30 activating NK cell receptor), resulted in resistance of K562 CML cells to NK cell cytotoxicity. In contrast, disruption of genes encoding apoptotic mediators (FADD, PMAIP1, BID) and TRAIL receptors (TNFRSF10B) conferred resistance to NK cell cytotoxicity in SUDHL4 DLBCL cells. The same pathways were identified in the MM cell line MM.1S, in which knockout of FAS, CASP2, and CASP8 as well as the TRAIL receptor TNFRSF10A induced NK cell resistance. Furthermore, loss of CD48, a ligand of the non-MHC binding receptor CD244 on NK cell surface, resulted in resistance and a genome-scale CRISPR gain-of-function screen in the same cell line showed sensitization upon CD48 overexpression. A CRISPR screen in the AML cell line MOLM14 identified disruption of TNFRSF1B encoding TNFR2 as strongly conferring NK cell resistance. Interestingly, TNFRSF1B overexpression sensitized the MM cell line MM.1S to NK cell treatment in the gain-of-function screen. Integration with genomic data from patients with hematological malignancies revealed selective expression of TNFRSF1B in AML. Within AML, TNFRSF1B expression was enriched in myelomonocytic and monocytic subtypes as well as in MLL-rearranged cases represented by the MOLM14 cell line. Further dissection at the single-cell level revealed increased expression of TNFRSF1B with differentiation of AML cells along the monocytic lineage. Consistently, the less differentiated MOLM13 cell line established from the same patient as MOLM14 was resistant to NK cell killing, suggesting that a less differentiated phenotype of AML cells confers resistance to NK cell cytotoxicity through lack of TNFRSF1B expression. Our data suggest that different lineages of hematological malignancies may be susceptible to NK cells through distinct mechanisms. In some cases, this heterogeneity is governed by lineage-specific expression of susceptibility genes, such as TNFRSF1B in monocytic AML. Particularly, our data identify a mechanism of NK cell evasion in less differentiated AML cells, suggesting potential for enhancing immune clearance of AML cells through differentiating therapies. figure Disclosures Lee: Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Mitsiades:Takeda: Other: employment of a relative ; Ionis Pharmaceuticals: Honoraria; Fate Therapeutics: Honoraria; Arch Oncology: Research Funding; Sanofi: Research Funding; Karyopharm: Research Funding; Abbvie: Research Funding; TEVA: Research Funding; EMD Serono: Research Funding; Janssen/Johnson & Johnson: Research Funding. Mustjoki:BMS: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Research Funding.