Education and Cytokine Receptor Balance Play a Critical Role in Long Term Survival of the Most Differentiated Natural Killer (NK) Cells.

Abstract Abstract 2148 NK cells can effectively treat advanced acute myelogenous leukemia if they survive and maintain functional competency after adoptive transfer. In the peripheral blood of healthy human donors the balance between Killer-cell immunoglobulin-like receptor positive (KIR+) NK cells, which are more differentiated and functional in terms of tumor killing and cytokine production, and KIR− NK cells is maintained by proliferation, differentiation, and survival. The mechanism that governs this process is largely unknown. Given that we and others have shown that KIR− NK cells proliferate better than KIR+ NK cells with IL-15 in vitro, we chose to study whether the balance between these two subsets could be maintained by enhanced survival of KIR+ NK cells. To explore this further, peripheral blood mononuclear cells (PBMCs) from healthy human donors were put in serum free conditions overnight and NK cell survival was assessed. A significantly larger proportion of the KIR− NK cells died (33±2.41%) compared to well characterized KIR+ NK cells: KIR2DL1/DS1 (8.9±1.22%), KIR2DL2/DL3 (6.7±0.51%), and KIR3DL1 (6.1±0.95%) (all P < 0.0001). A similar effect was seen when looking at Annexin V+ cells that still had membrane integrity. In order to further understand why KIR+ NK cells exhibited superior survival, we quantified expression of anti-apoptotic markers and found that KIR-expressing NK cells express more Bcl-xL, by Q-PCR and Western blotting. They also express more Bcl-2, by Q-PCR and flow median fluorescence intensity (MFI), particularly when the cells are treated with IL-15 overnight (P ≤ 0.0005). We next investigated expression of cell death family receptors Fas and Fas-Ligand (FasL). Under every condition tested Fas was expressed at significantly higher levels in the KIR− NK cells (P ≤ 0.03). FasL on the other hand was only expressed at higher levels in KIR− NK cells post 60 hrs in culture alone or with IL-2 but not IL-15. Although NK cells can signal through IL-7 or IL-2, only IL-15 mouse knockouts demonstrate a complete deficit of NK cells. Both IL-15 and IL-2 signal through 2 common shared components, IL-2Rb and the common g chain, and an individual cytokine specific component, IL-2Ra for IL-2 and IL-15Ra for IL-15. Interestingly, although all the components for IL-2 signaling are present on the NK cell, IL-15Ra bound to IL-15 is presented in trans from other cells or in complexes in order to achieve IL-15 signaling in NK cells, thereby discarding the necessity for IL-15Ra expression on the NK cell itself. Based on this information, the expression of all of these components was evaluated on KIR+ and KIR− NK cells. We found that IL-2Ra is expressed, by MFI, at statistically significant lower levels (P ≤ 0.0023) on KIR+ NK cells when compared to KIR− NK cells. In contrast, the common g chain is expressed, by MFI, at statistically higher levels (P < 0.0001) on KIR+ NK cells. No statistical differences were seen in IL-15Ra and IL-2Rb expression. We hypothesized that competition for common components, IL-2Rb and the common g chain, between IL-2Ra and IL-15Ra (in trans) plays a role in regulating survival through IL-15. In agreement with this premise, IL-2Ralo NK cells died significantly less than IL-2Rahi NK cells with transpresentation of IL-15 (3.5±1.5% vs. 30.8±2.6%, P = 0.0025). Transient overexpression of IL-2Ra ablates this enhanced survival. Finally we wanted to see if the educational status of the NK cell has a role in its survival. Of the KIR+ NK cells the educated NK cells, which have KIRs corresponding to self-ligands, represent the most differentiated and functional subset. We found that educated NK cells consistently had more Bcl-2 than uneducated NK cells (P ≤ 0.0021). This was associated with enhanced common g chain expression in the educated NK cell population (P = 0.0001). Previous studies have shown that CD57+ NK cells are long lived. We show here that educated NK cells had a statistically larger proportion and density of CD57 expression on a per cell basis (P < 0.0001). These data indicate that educated NK cells could survive better, through enhanced Bcl-2 upregulation, and persist longer, as determined by CD57 expression, than their uneducated counterparts. In summary we find that the mechanism for maintenance of more differentiated NK cells involves enhanced cell survival and differential responses to cytokines limited by the alpha chains of IL-2 and IL-15, which may be manipulated for therapeutic purposes. Disclosures: Miller: Celgene: Membership on an entity's Board of Directors or advisory committees; Coronado Bioscience: Membership on an entity's Board of Directors or advisory committees.

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CD38 Down-Regulation on Ex Vivo Activated and Expanded NK Cells for Cell Therapy Persists after Infusion

Blood ◽

10.1182/blood-2021-152522 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4796-4796

Author(s):

Hareth Nahi ◽

Michael Chrobook ◽

Stephan Meinke ◽

Charlotte Gran ◽

Nicole Marquardt ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Peripheral Blood ◽

Nk Cell ◽

Ex Vivo ◽

Multiparameter Flow Cytometry ◽

Advisory Committees ◽

Natural Form ◽

Cellular Therapies ◽

Privately Held

Abstract Introduction: Immunotherapies are gaining more and more importance in the treatment of multiple myeloma (MM). Antibodies directed against MM antigens like CD38, SLAMF7 or BCMA are used either in their natural form, conjugated to drugs, or in the form of bispecific T-cell engagers. Cellular therapies make use of cytotoxic lymphocytes, i.e. T cells or NK cells that can also be modified to express chimeric antigen receptors to target MM cells. Combinations of antibody and cellular therapies could further improve the outcome as, for example, NK cells can mediate antibody dependent cellular cytotoxicity (ADCC). However, NK cells also express CD38 and SLAMF7 and would be targeted by the therapeutic antibodies against these antigens. We have recently reported our clinical study infusing multiple doses of ex vivo activated and expanded autologous NK cells in six patients with MM post autologous stem-cell transplantation (EudraCT 2010-022330-83). Here, we report results of a phenotypic analysis of the ex vivo expanded NK cells and peripheral blood NK cells before and after infusion with implications for possible combination therapies. Methods: Ex vivo activated and expanded NK cells and NK cells in peripheral blood of the patients were analyzed by multiparameter flow cytometry. Peripheral blood cells were taken from the non-NK cell infusion arm before and at three different timepoints after infusion. NK-cell sub-populations within these samples were analyzed using t-SNE clustering. Results: Upon ex vivo activation and expansion, we observed that the NK cells gained a unique activated phenotype including populations of CD56 brightCD16 +Ki67 +HLA-DR + NK cells. Interestingly, these NK cells showed a reduced expression of CD38 compared to peripheral blood NK cells. Clustering analyses of data from peripheral blood samples revealed the gradual appearance of a new NK cell population with a similar phenotype in a dose-dependent fashion over four hours following infusion of the NK cell product. Infused NK cells could be detected in circulation up to four weeks after the last infusion. Like the NK cell infusion product, these cells expressed little to none CD38, high levels of NKG2D, 2B4, TIM-3, and TIGIT and similar levels of SLAMF7 compared to peripheral blood NK cells. Conclusions: The persistent high expression of CD16 and the low expression of CD38 in infused NK cells offers the choice to combine ex vivo activated and expanded NK cells with anti-CD38 antibody therapy without concern for antibody-mediated NK-cell death. Based on these findings, we have started a clinical trial testing this combined therapy (NCT04558931). Disclosures Nahi: XNK Therapeutics AB: Consultancy. Chrobook: XNK Therapeutics AB: Consultancy. Meinke: XNK Therapeutics AB: Consultancy, Current holder of stock options in a privately-held company. Gilljam: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company. Stellan: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company. Walther-Jallow: XNK Therapeutics: Other: Shareholder in the company. Liwing: XNK Therapeutics AB: Current Employment. Gahrton: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company; Fujimoto Pharmaceutical Corporation Japan: Membership on an entity's Board of Directors or advisory committees. Ljungman: Takeda: Consultancy, Other: Endpoint committee, speaker; OctaPharma: Other: DSMB; Enanta: Other: DSMB; Merck: Other: Investigator, speaker; AiCuris: Consultancy; Janssen: Other: Investigator. Ljunggren: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Alici: XNK Therapeutics AB: Current holder of individual stocks in a privately-held company.

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Sequential Immune Cell Modulation in Nordic Follicular Lymphoma Patients in the SAKK 35/10 Randomized Trial with Rituximab and Lenalidomide

Blood ◽

10.1182/blood.v126.23.1535.1535 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1535-1535 ◽

Cited By ~ 2

Author(s):

Sandra Lockmer ◽

Björn E Wahlin ◽

Bjorn Ostenstad ◽

Åsa Jeppson-Ahlberg ◽

Birgitta Sander ◽

...

Keyword(s):

Follicular Lymphoma ◽

Nk Cells ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Immune Cell ◽

Nk Cell ◽

Disease Stage ◽

Advisory Committees ◽

Immune Cell Subsets

Abstract Background Follicular lymphoma (FL) is the second most common lymphoma in adults. Although responsive to therapies it is still considered incurable. The introduction of the CD20 antibody rituximab is well known to have improved outcome. Rituximab acts through complement-mediated cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC) and direct induction of apoptosis. To enhance the efficacy of rituximab, different combination regimens have been used, mostly with chemotherapy but also with cytokines. Lenalidomide, an immunomodulatory agent commonly used in the treatment of multiple myeloma, has been shown to induce durable responses with manageable toxicity in indolent lymphomas and mantle cell lymphoma, especially in combination with rituximab. It acts on both the malignant cells and their microenvironment. The drug modulates signaling pathways, enhances the capacity of T cells and increases ADCC by natural killer (NK) cells, as well as suppresses angiogenesis. When combined with rituximab, the clinical effects seem to be synergistic (Fowler 2014). We aimed to investigate the dynamics of immune cell subsets in peripheral blood in patients given rituximab with or without lenalidomide. Patients and Methods FL patients included in a multicenter randomized phase II trial performed by the Swiss Group for Clinical Cancer Research (SAKK) in collaboration with the Nordic Lymphoma Group (NLG) were randomized 1:1 to treatment either with rituximab alone or rituximab and lenalidomide. Inclusion criteria were histologically confirmed CD20+ FL grade 1, 2 or 3A in disease stage Ann Arbor III-IV (or II not suitable for radiotherapy). Patients had to be in need of systemic therapy because of clinical symptoms, cytopenia, bulky disease or significant disease progression. In both treatment arms rituximab was administered as 4 single infusions of 375 mg/m2 weeks 1, 2, 3 and 4; in patients who showed at least a minor response 4 additional infusions were administered at weeks 12, 13, 14 and 15. In the combination arm, lenalidomide, 15 mg p.o. daily, was started 14 days before the first infusion and given continuously until 14 days after the last. Peripheral blood cells were sequentially sampled: at baseline, after 2 weeks' use of lenalidomide, 24 hours after first rituximab infusion and at follow-up at weeks 10 and 23. Analyses of CD3+, CD4+, CD8+ and CD56+CD3- (NK) cells were performed with flow cytometry. Results Immune cell activity was assessed on blood samples of 28 Norwegian and Swedish patients until July 2015. In all patients, irrespective of treatment arm, NK cell numbers markedly decreased at 24 hours after the first rituximab infusion compared to baseline counts (P=0.046), but returned to baseline levels by week 10 in most. However, patients in the combination arm exhibited a heterogeneous response with a diverse NK cell depletion/proliferation pattern, some showing a transient rise already after 14 days of lenalidomide use (Figure 1). CD3 levels were not affected at 24 hours after rituximab but increased over time in 15 of 18 patients (without differences between treatment arms). The increase at week 23 was statistically significant (P=0.004) with a median of 1.4 x 109 /L CD3+ cells compared to a baseline median of 0.88 x 109/L. In all patients, independent of treatment arm, the CD4/CD8 ratio increased compared to baseline already 24 hours after rituximab (P=0.011) and persisted throughout the study (week 10, P=0,005; week 23, P=0.019). The increased ratio was due to a large rise in CD4 counts (week 10, P=0.014; week 23, P=0.003), and a less pronounced rise in CD8 counts (week 10, P=0.094; week 23, P=0.007; Figure 2). Conclusion We found changes in the composition of immune cell subsets in peripheral blood in rituximab treated FL patients, with a larger interindividual variation when combined with lenalidomide. Ongoing analyses will reveal whether these patterns of immune cell response correlate with clinical outcome and long-term treatment effects. Figure 1. NK cell absolute counts (x 109/L) in (a) patients treated with rituximab and in (b) patients treated with rituximab plus lenalidomide. 1=baseline, 2=after 14 days of lenalidomide (b only), 3=24h after rituximab, 4=week 10, 5=week 23. Figure 1. NK cell absolute counts (x 109/L) in (a) patients treated with rituximab and in (b) patients treated with rituximab plus lenalidomide. 1=baseline, 2=after 14 days of lenalidomide (b only), 3=24h after rituximab, 4=week 10, 5=week 23. Figure 2. CD4/CD8 ratios in all 28 patients. The y scale is logarithmic. 1=baseline, 2=after 14 days of lenalidomide (14 patients only), 3=24h after rituximab, 4=week 10, 5=week 23. Figure 2. CD4/CD8 ratios in all 28 patients. The y scale is logarithmic. 1=baseline, 2=after 14 days of lenalidomide (14 patients only), 3=24h after rituximab, 4=week 10, 5=week 23. Figure 3. Figure 3. Disclosures Off Label Use: Lenalidomide was used together with rituximab in a randomized clinical trial.. Kimby:Gilead: Membership on an entity's Board of Directors or advisory committees; Jansen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding.

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PD-1 Is Expressed at Low Levels on All Peripheral Blood Natural Killer Cells but Is a Significant Suppressor of NK Function Against PD-1 Ligand Expressing Tumor Targets

Blood ◽

10.1182/blood-2019-127261 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 621-621

Author(s):

Zachary Davis ◽

Martin Felices ◽

Todd R Lenvik ◽

Sujan Badal ◽

Peter Hinderlie ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Cytokine Production ◽

Nk Cell ◽

Fold Increase ◽

Advisory Committees ◽

Fund Research

Checkpoint blockade has become a promising immunotherapy for the treatment of a variety of malignancies. In particular, the receptor programmed death-1 (PD-1) has become a focus of intense study due to its expression on and negative regulation of T-cell function. The ligand for PD-1, PD-L1, is upregulated on many tumors and, as a result, can suppress antigen-specific T-cells thereby limiting their anti-tumor response. Pharmacological PD-1/PD-L1 axis disruption can occur with either Pembrolizumab and Nivolumab (PD-1 antagonists) and Avelumab and Atezolizumab (PD-L1 antagonists). These antibodies (mAbs) are being used to treat melanoma, non-small cell lung cancer, kidney, bladder and head and neck cancer with varying degrees of success. Like T-cells, natural killer cells (NK) also have potent antitumor cytolytic properties. The expression and functional effects of PD-1 on NK cells remain unclear due to difficulties in receptor detection and efficacy of receptor blockade by available commercial reagents. While some studies have been unable to detect PD-1 on resting NK cells, others have identified PD-1 expression only on specific NK populations under certain conditions (e.g. Cytokine stimulation or virus infection). Here, we identify PD-1 expression on peripheral blood NK cells. Using commercial reagents (Figure 1A) and a FITC-labeled clinical mAb (Pembrolizumab, Pembro), we detect low yet consistent PD-1 expression on all circulating, resting NK cells. Since FITC-Pembro mean fluorescent intensity was low and a high proportion of FITC labeled NK cells overlapped with the isotype control (Figure 1B), we designed a short-chain variable fragment (scFv) of the mAb to determine whether the smaller scFv molecule has better binding and functional activity than the intact mAb. The Pembro scFv bound to resting NK cells with a distinct fluorescent peak compared to the native Prembro from which the scFv was derived (Figure 1B). Compared to intact Prembro, use of the Pembro scFv as a PD-1 antagonist resulted in a 2-fold increase of NK cell cytolytic activity and a 3-4 fold increase in cytokine production against the PD-L1 expressing CML target, K562 (Figure 1C-D) and the AML target, THP-1 (Figure 1E-F). While PD-1 blockade enhanced NK cell degranulation and target cell killing, a greater functional enhancement was seen for interferon-γ production. PD-1 signaling inhibits PI3K induced pAkt and NK function. PD-1/PD-1 ligand blockade by the Pembro scFv resulted in increased NK cell pAKT in the presence of PD-L1 and NK activating NKG2D-ligand-expressing THP-1 cells. In addition to natural cytotoxicity, NK-mediated ADCC was also enhanced with PD-1 blockade. CD33 mAb immunoconjugates have been used to treat AML. Combined anti-CD33 mAb and PD-1 blockade against THP-1 cells resulted in a small but significant increase in NK cell degranulation and a 4-fold increase in cytokine production compared to anti-CD33 mAb without PD-1 blockade (Figure 1G-H). Since stimulation with IL-15, a cytokine that effectively lowers the NK activation threshold, abrogated the benefits of Pembro scFv in diminishing PD-1 inhibitory effects on NK cells, PD-1 control of NK function appears limited to be mostly relevant to resting NK cells. To understand the physiologic expression of PD-1 in vivo, we studied samples taken from AML patients receiving matched sibling donor transplantation at the University of Minnesota. Increased PD-1 on reconstituting NK cells in BMT recipients up to day 100 post-transplant was shown by both flow-cytometric (Figure 2A) and mass-cytometric (CyTOF) analyses (Figure 2B). Blockade of PD-1 on these cells significantly enhanced both NK degranulation (Figure 2C) and cytokine production (Figure 2D) against K562 targets. A similar increase in NK function was observed with PD-1 blockade in AML patients receiving umbilical cord transplants (not shown). These data indicate that PD-1 is present on human NK cells and PD-1 ligation negatively regulates NK function against PD-L1 expressing tumor targets. The observation that functional PD-1 is expressed on NK cells under resting conditions strongly suggests that the use of a PD-1 antagonist, in combination with NK cell therapy, should be clinically effective for treatment of cancer. Disclosures Felices: GT Biopharma.: Other: consulting funds, Research Funding. Blazar:Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Leukemia and Lymphoma Society: Research Funding; Abbvie Inc: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees. Vallera:GT Biopharma, Inc.: Consultancy, Research Funding. Miller:Fate Therapeutics, Inc: Consultancy, Research Funding; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CytoSen: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Keytruda. PD-1 blockade on NK cells for tumor immunotherapy

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Tim-3, a Novel Immune Receptor, Is Constitutively Expressed on Human Natural Killer Cells and Functions as An Activating Coreceptor

Blood ◽

10.1182/blood.v116.21.106.106 ◽

2010 ◽

Vol 116 (21) ◽

pp. 106-106

Author(s):

Michelle Gleason ◽

Todd Lenvik ◽

Valarie McCullar ◽

Sarah Cooley ◽

Michael Verneris ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Cell Activation ◽

Low Dose ◽

Cell Function ◽

Nk Cell ◽

Effector Function ◽

Advisory Committees ◽

Immune Receptor ◽

Ifn Γ

Abstract Abstract 106 NK cells are an attractive option for immunotherapy as they do not require pre-sensitization for anti-tumor activity and do not induce graft versus host disease (GvHD) in an allogeneic transplant setting. The potential of NK cells in controlling human hematological malignancies has been increasingly recognized in recent years, as the adoptive transfer of alloreactive NK cells in hematopoietic cell transplantation (HCT) clinical trials have demonstrated therapeutic anti-leukemia effects. NK cell function is regulated by the integration of antagonist signals received from cell surface activating and inhibitory receptors. Tim-3 is a novel immune receptor that is a member of the T cell immunoglobulin and mucin-containing domain (TIM) family of glycoproteins. While its role in T cells and antigen presenting cells has been described, little is known about its function in human NK cells. While Tim-3 is present on a variety of immune cells, resting NK cells constitutively express Tim-3 compared to other lymphocyte populations (NK: 73±3%; NKT: 6±1%; T: 1±1%; n=14) and we hypothesized that Tim-3 may be important in mediating NK cell function. The unique subset of cytokine producing CD56Bright NK cells exhibited significantly lower resting Tim-3 expression compared to CD56Dim NK cells (53±3% vs. 75±3%; p<0.001, n=14). Distinct Tim-3 expression patterns were found on resting CD56Dim NK cells and activation with low dose IL-12 (1ng/mL) and IL-18 (10ng/mL), intended to more closely mimic physiologic conditions, resulted in further differentiation of this unique expression pattern dividing NK cells into 4 distinct populations: Tim-3 was homogeneously up-regulated on all CD56Bright NK cells after activation while CD56Dim NK cells were further stratified into 3 defined populations with Tim-3hi, Tim-3lo and Tim-3neg expression. The only identified ligand of Tim-3 is galectin-9 (Gal-9), a β-galactoside binding lectin, which is expressed on a wide range of healthy and malignant cells. To investigate the potential function of Tim-3, an expression vector containing human Gal-9 was transduced into K562 and Raji cells, both without endogenous Gal-9 expression. Resting NK cytotoxicity (51Cr release) was found to be increased in the presence of Gal-9 compared to the non-Gal-9 expressing targets [E:T=0.7:1, K562 vs. K562-Gal-9: 25±3% vs. 33±3% (n=8, p<0.05); E:T=20:1, Raji vs. Raji-Gal-9: 8±1% vs. 17±2% (n=4, p<0.05)]. Analysis of CD107a degranulation showed that resting Tim-3+ CD56Bright cells were more functional against Gal-9 expressing targets than Tim-3− CD56Bright cells, suggesting that Tim-3 might also play a role in IFN-γ production. To further investigate this, resting NK cells were activated with low-dose IL-12/IL-18 overnight and IFN-γ levels were measured in response to soluble rhGal-9 (0, 2.5, 5, 10 and 20nM). Exposure to soluble rhGal-9 alone without IL-12/IL-18 did not induce IFN-γ production. For both the CD56Bright and CD56Dim IL-12/IL-18 activated NK populations, only Tim-3+ NK cells displayed a dose dependent increase in IFN-γ production upon exposure to soluble rhGal-9 compared to Tim-3− NK cells. To understand the relevance of the distinct Tim-3 populations circulating in resting blood, CD56Bright, CD56Dim/Tim-3hi, CD56Dim/Tim-3lo and CD56Dim/Tim-3neg populations were sorted, cultured overnight in IL-12/IL-18 and exposed to soluble rhGal-9. Results showed the Tim-3 expressing populations contain the predominant IFN-γ producing cells that were responsive to rhGal-9 (results for the sorted CD56Dim/Tim-3lo population shown in the figure below). This increase in IFN-γ production within the Tim-3 expressing NK cell populations was abrogated by the addition of β-lactose, a β-galactoside that binds and blocks Gal-9 activity. Lastly, Western blot and immunohistochemistry analysis of human primary acute leukemia blasts revealed high Gal-9 expression. As the presence of ligands for NK cell activating receptors on tumors provide an important prerequisite for NK cell activation and effector function, we show a novel functional role for the receptor Tim-3 in human NK cell biology in the presence of its ligand Gal-9. We, therefore, propose a model where constitutively expressed Tim-3 is up-regulated by NK cell activation and effector function is enhanced by Tim-3/Gal-9 interaction, which may potentiate the elimination of Gal-9 positive tumors by NK cells. Disclosures: Niki: GalPharma: Membership on an entity's Board of Directors or advisory committees. Hirashima:GalPharma: Membership on an entity's Board of Directors or advisory committees.

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The Influence Of KIR Haplotype In ITP Incidence, Treatment Response and Bleeding Symptoms

Blood ◽

10.1182/blood.v122.21.2316.2316 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2316-2316

Author(s):

Bethan Psaila ◽

Nayla Boulad ◽

Emily Leven ◽

Naznin Haq ◽

Christina Soo Lee ◽

...

Keyword(s):

Platelet Count ◽

B Cells ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees ◽

Kir Genes ◽

Number Of Patients

Abstract The pathogenesis of immune thrombocytopenia (ITP) is multifactorial, with both cellular and humoural immune dysfunction. The role of NK cells has not been well defined in ITP but in other diseases NK cells have a role in rejecting “foreign” eg transplanted organ or tumor, and also acting against self as occurs in autoimmunity. NK cell activity is orchestrated by the balance of activating vs. inhibitory signalling, in particular via the killer cell immunoglobulin-like receptor (KIR) family of receptors. Significant variation exists in KIR allelic subtype and copy number for the KIR between individuals, and associations have been made with certain haplotypes and a number of autoimmune disorders including rheumatoid arthritis, scleroderma and diabetes. Previous reports have demonstrated a reduction in natural killer (NK) cell number and function in ITP and expression of inhibitory KIR genes is increased in patients in remission vs. active ITP. Methods To explore whether a particular KIR haplotype might predispose to ITP, and also affect response to ITP treatment, we performed KIR genotyping using the Invitrogen SSP kit on 92 patients attending a haematology centre in New York and compared the results to data from 213 controls taken from the USA Eastern Database. Genomic DNA was typed for the inhibitory KIR genes KIR2DL1, KIR2DL2, KIR2DL5A (alleles 001 and 002), KIR2DL5B (alleles 002-004, 06, and 007), KIR3DL1, KIR3DL3; the activating KIR genes KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1; the framework genes KIR2DL3, KIR2DL4, KIR3DL2, KIR3DP1; and the pseudogene KIR2DP1. The patients with ITP had been or were receiving treatment with IVIG (n=64), corticosteroids (72) and rituximab (37). Bleeding symptoms were recorded. Response to treatment was defined as complete - platelet count increase to > 100 x 109/mL; partial - platelet count increase to > 50 x 109/mL; or no response. For the purpose of analysis, PRs and CRs were combined. A comprehensive database allowed a logistic regression, assessing both responses to treatments, platelet counts, neutrophil counts, CRP, lymphocyte subsets and bleeding symptoms. Results The expression of two inhibitory KIR genes, 2DL1 and 3DL1, was significantly lower in the patients with ITP as compared to controls (87% 2DL1 and 87% 3DL1 compared to 99% in controls - P < 0.02). Response to rituximab was strongly related to KIR haplotype expression. 2DL1 expression was higher among nonresponders to Rituximab (100% of non responders compared to 82% of responders), whereas 2DL3 expression was significantly lower (79% compared to 90%) (P < 0.05, Figure 1B). Separately, patients with the 2DS3 allele, an activatory KIR, were 5.5 times more likely to have experienced significant bleeding. Conclusions Although these findings are preliminary and require further investigation, these data suggest that increased cytotoxic autoimmunity due to reduced KIR inhibition may be associated with the development of ITP and possibly contribute importantly to the pathogenesis. Anti-CD20 targeting therapy directed at B cells was strongly influenced by 2 different KIRs (1 upregulated and one down-regulated) emphasizing the potential role of NK cells in elimination of tissue-based (nodal) B cells. Finally a more pronounced clinical phenotype with a markedly higher incidence of severe bleeding associated with an increased activatory KIR expression demonstrates the role of NK cells in bleeding presumably via their effects on either endothelial cells or platelet function. These exciting findings will be pursued for confirmation in a larger number of patients. Disclosures: Bussel: Amgen: Family owns stock Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Genzyme: Research Funding; GlaxoSmithKline: Family owns stock, Family owns stock Other, Membership on an entity’s Board of Directors or advisory committees, Research Funding; IgG of America: Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Shionogi: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Sysmex: Research Funding; Symphogen: Membership on an entity’s Board of Directors or advisory committees.

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Functional NK Cell Repertoires Are Determined By Survival Mechanisms Through IL-2Ra and FasL

Blood ◽

10.1182/blood.v122.21.786.786 ◽

2013 ◽

Vol 122 (21) ◽

pp. 786-786

Author(s):

Martin Felices ◽

Todd Lenvik ◽

Dave Ankarlo ◽

Bree Foley ◽

Julie Curtsinger ◽

...

Keyword(s):

Cell Death ◽

Cell Survival ◽

Nk Cells ◽

Nk Cell ◽

Fold Increase ◽

Myelogenous Leukemia ◽

Cell Numbers ◽

Transplant Patients ◽

Cmv Reactivation

Abstract NK cell based immunotherapy can be used to treat advanced acute myelogenous leukemia and data shows that success depends on the function of NK cells and how long the cells are maintained in the recipient. An important determinant of NK cell function is expression of Killer-cell immunoglobulin-like receptors (KIRs), which are involved in the maintenance of self tolerance and acquisition of NK cell functional competence, a process termed education. Little is known about how NK cell education influences NK cell survival. Utilizing a serum starvation assay we found that KIR+ and educated NK cells survived better when compared to those that did not have a KIR or KIR matching cognate HLA-ligand respectively (Figure 1A and 1B). Under basal conditions both KIR+ and educated NK cells had more anti-apoptotic proteins (Bcl-2 and Bcl-xL) and expressed less Fas. When NK cells were incubated with serum and IL-15 (10 ng/ml) followed by IL-15 withdrawal, a substantial increase of pro-apoptotic Bim was found on uneducated NK cells (1.4 fold increase; P = 0.01). Under this same condition educated NK cells expressed more FasL (1.5 fold increase; P = 0.0003), indicating that they could be driving cell death on neighboring NK cells when cytokine is limiting. Since both NK cell survival and homeostasis are mediated by cytokine signaling, we studied expression of IL-15 and IL-2 signaling components. The most significant change was IL-2Ra, which was expressed at higher levels on uneducated NK cells (3 fold increase; P = 0.0004) when the cells were stimulated with serum and IL-15 (1 ng/ml) for 72 hrs. Higher IL-2Ra expression correlated well with cell death and Bim expression so we decided to test if modulating its expression would alter NK cell survival in an IL-15 withdrawal setting. Compared to the control, transient overexpression of IL-2Ra lead to a 1.55 fold decrease in NK cell numbers (P = 0.01) while siRNA knockdown of IL-2Ra lead to a 1.5 fold increase in NK cell numbers (P = 0.07). Importantly, at the time of harvest there was a 1.4 fold decrease in cell death in the IL-2Ra knockdown condition when compared to the control (P = 0.0007). Given that no IL-2 or crosslinking signals were present, the mechanism must differ from the well-described role of IL-2 on activation induced cell death. Since FasL is upregulated on educated NK cells when IL-15 is limiting and IL-2Ra renders NK cells more sensitive to cell death, we tested if educated cells could drive cell death of IL-2Rahi NK cells when survival signals are scarce using a co-culturing assay and FasL blocking antibodies. IL-2Rahi NK cells were more sensitive to apoptosis when co-cultured with KIR+ NK cells (presumably enriched for educated subsets) than with KIR- NK cells from the same donor that were subjected to IL-15 stimulation and withdrawal (34±4.8% vs. 20.6±4.7%; P = 0.002). The effect was reduced when FasL was blocked (P = 0.003), and no differences in killing were seen on the IL-2Ralo NK cells regardless of the treatment. Taken together these findings indicate that educated NK cells outlive their counterparts through two mechanisms: decreased expression of proteins involved in cell death and by killing competing NK cells when IL-15 is limiting. Finally, since we have previously reported on expansion of educated NK cells on transplant patients post CMV reactivation and since CMV reactivation can be associated with decreased relapse, we wanted to investigate if CMV reactivation could alter NK cell survival. There was increased survival on the NK cells from adult donor HCT (2 fold increase at 6 months; P = 0.03) and umbilical cord blood (3 fold increase at 100 days; P = 0.01) allogeneic transplant patients that had undergone CMV reactivation supporting the physiologic role of NK cell survival in vivo from a pathologic challenge. Taken together, these findings show that NK cell functional repertoires are determined by class I interactions, infection, and NK-NK interactions through IL-2Ra, Bim, and FasL to mediate clonal dominance that might be exploited in order to enhance NK cell survival and function after adoptive transfer of allogeneic NK cells for therapeutic use in cancer. Disclosures: No relevant conflicts of interest to declare.

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Off-the-Shelf, Multiplexed-Engineered iPSC-Derived NK Cells Mediate Potent Multi-Antigen Targeting of B-Cell Malignancies with Reduced Cytotoxicity Against Healthy B Cells

Blood ◽

10.1182/blood-2021-148654 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 407-407

Author(s):

Frank Cichocki ◽

Jode P Goodridge ◽

Ryan Bjordahl ◽

Svetlana Gaidarova ◽

Sajid Mahmood ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees ◽

Car T Cells ◽

Car T

Abstract Treatments for B-cell malignancies have improved over the past several decades with clinical application of the CD20-specific antibody rituximab and chimeric antigen receptor (CAR) T cells targeting CD19. Despite the success of these therapies, loss of CD20 after rituximab treatment has been reported in leukemia and lymphoma patients. Additionally, up to 50% of all patients receiving anti-CD19 CAR T-cell therapy relapse within the first year with many of those patients exhibiting CD19 loss. Thus, new therapeutic approaches are needed to address tumor antigen escape. Accordingly, we generated triple gene-modified iPSC-derived NK (iNK) cells, termed "iDuo" NK cells, tailored to facilitate multi-antigen targeting. The iPSC line was clonally engineered to express high-affinity, non-cleavable CD16a (hnCD16), an anti-CD19 CAR optimized for NK cell signaling, and a membrane-bound IL-15/IL-15R fusion (IL-15RF) molecule to enhance NK cell persistence (Fig. 1A). To model antigen escape, we generated CD19 knockout AHR77 lymphoma cells alongside wild type AHR77 cells (both CD20 +) as targets in cytotoxicity assays. Activated peripheral blood NK (PBNK) cells, non-transduced iNK cells, and iDuo NK cells were tested as effectors. Unlike PBNK cells or non-transduced iNK cells, iDuo NK cells efficiently eliminated wild type AHR77 cells with or without the addition of rituximab at all tested E:T ratios. Similarly, iDuo NK cells in combination with rituximab were uniquely able to efficiently eliminate CD19 KO AHR77 cells due to enhanced antibody-dependent cellular cytotoxicity (ADCC) driven by hnCD16 (Fig. 1B-E). Cytotoxicity mediated by iDuo NK cells was also evaluated using primary chronic lymphocytic leukemia (CLL) cells. Compared to expanded PBNK cells and non-transduced iNK cells, only iDuo NK cells (in the absence of rituximab) were able to kill primary CLL cells (Fig. 1F). Expression of IL-15RF by iDuo NK cells uniquely supports in vitro expansion without the need for cytokine supplementation. To determine whether IL-15RF supports in vivo persistence of iDuo NK cells, CD19 CAR iNK cells (lacking IL-15RF) and iDuo NK cells were injected into NSG mice without the addition of cytokines or CD19 antigen availability. iDuo NK cell numbers peaked within a week after injection and persisted at measurable levels for ~5 weeks, in marked contrast to CD19 CAR iNK cell numbers that were undetectable throughout (Fig. 1G). To evaluate the in vivo function of iDuo NK cells, NALM6 leukemia cells were engrafted into NSG mice. Groups of mice received tumor alone or were treated with 3 doses of thawed iDuo NK cells. iDuo NK cells alone were highly effective in this model as evidenced by complete survival of mice in the treatment group (Fig. 1H). To assess iDuo NK cells in a more aggressive model, Raji lymphoma cells were engrafted, and groups of mice received rituximab alone, iDuo NK cells alone, or iDuo NK cells plus rituximab. Mice given the combination of iDuo NK cells and rituximab provided extended survival compared to all other arms in the aggressive disseminated Raji lymphoma xenograft model (Fig. 1I). One disadvantage of anti-CD19 CAR T cells is their inability to discriminate between healthy and malignant B cells. Because NK cells express inhibitory receptors that enable "self" versus "non-self" discrimination, we reasoned that iDuo NK cells could have higher cytotoxicity against tumor cells relative to healthy B cells. To address this, we labeled Raji cells, CD19 + B cells from healthy donor peripheral blood mononuclear cells (PBMCs) and CD19 - PBMCs. Labeled populations of cells were co-cultured with iDuo NK cells, and specific killing was analyzed. As expected, iDuo NK cells did not target CD19 - PBMCs. Intriguingly, iDuo NK cells had much higher cytotoxic activity against Raji cells compared to primary CD19 + B cells, suggesting a preferential targeting of malignant B cells compared to healthy B cells. Together, these results demonstrate the potent multi-antigen targeting capability and in vivo antitumor function of iDuo NK cells. Further, these data suggest that iDuo NK cells may have an additional advantage over anti-CD19 CAR T cells by discriminating between healthy and malignant B cells. The first iDuo NK cell, FT596, is currently being tested in a Phase I clinical trial (NCT04245722) for the treatment of B-cell lymphoma. Figure 1 Figure 1. Disclosures Cichocki: Gamida Cell: Research Funding; Fate Therapeutics, Inc: Patents & Royalties, Research Funding. Bjordahl: Fate Therapeutics: Current Employment. Gaidarova: Fate Therapeutics, Inc: Current Employment. Abujarour: Fate Therapeutics, Inc.: Current Employment. Rogers: Fate Therapeutics, Inc: Current Employment. Huffman: Fate Therapeutics, Inc: Current Employment. Lee: Fate Therapeutics, Inc: Current Employment. Szabo: Fate Therapeutics, Inc: Current Employment. Wong: BMS: Current equity holder in publicly-traded company; Fate Therapeutics, Inc: Current Employment. Cooley: Fate Therapeutics, Inc: Current Employment. Valamehr: Fate Therapeutics, Inc.: Current Employment. Miller: Magenta: Membership on an entity's Board of Directors or advisory committees; ONK Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vycellix: Consultancy; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees; Wugen: Membership on an entity's Board of Directors or advisory committees.

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Gene Set Enrichment Analysis Suggests That Increased Rituximab-Mediated NK Cell Cytotoxicity after Vitamin D Substitution Is Driven By Upregulation of Interferon Alpha (IFN-a) Isoforms

Blood ◽

10.1182/blood-2018-99-111817 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3696-3696

Author(s):

Konstantinos Christofyllakis ◽

Frank Neumann ◽

Stephan Stilgenbauer ◽

Dominic Kaddu-Mulindwa ◽

Evi Regitz ◽

...

Keyword(s):

Vitamin D ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Cytokine Production ◽

Nk Cell ◽

Cell Cytotoxicity ◽

Advisory Committees ◽

Vitamin D Levels ◽

Nk Cell Cytotoxicity

Abstract Introduction: We recently showed that vitamin D deficiency leads to decreased overall survival of DLBCL-patients treated with rituximab-chemotherapy (Bittenbring et al, JCO, 2014). We hypothesized that rituximab-mediated NK cell-cytotoxicity is more effective at higher vitamin D levels. This was confirmed by vitamin D substitution of healthy volunteers, which increased their rituximab-mediated cytotoxicity in vitro against the Daudi lymphoma cell line. To unveil the molecular mechanisms behind this finding, resting NK cells before and after vitamin D supplementation were isolated from those volunteers and a whole transcriptome analysis was performed. Methods: We collected PBMCs from eight healthy volunteers with vitamin D deficiency before and after vitamin D substitution to > 30 ng/ml 25-OH vitamin D3. NK cells were isolated from PBMCs by magnetic depletion of all non-NK cells. Purity of the CD16+ cells was confirmed by flow cytometry. After isolating total RNA, we performed a microarray analysis using an Affymetrix Gene-Chip 2.0 ™. The signals were normalized using the LMA algorithm. For pathway analysis, gene set enrichment analysis (GSEA) was used. A two-step approach was chosen. Firstly, we separated 7.705 genes due to their involvement in the NK cell-mediated immune response according to the Gene Ontology database, irrespective of their differential expression. This dataset was used separately for specific analysis of the NK cell-cytotoxicity pathway to increase sensitivity. Secondly, the complete data set of 48.145 genes was used in an exploratory analysis in an attempt to screen for other dysregulated pathways involved in the immune response and vitamin D homeostasis. We used gene sets provided from the Molecular Signature Database. A significance level of < 0.05 for p and False Discovery Rate (FDR) was chosen. Real-time quantitative PCR was performed to confirm the results. Results: The NK cell-associated cytotoxicity pathway was found to be significantly upregulated after restoration of normal vitamin D levels in the specific analysis. The most significantly overexpressed genes in the gene set were five IFN-α subtypes (IFN-α2, IFN-α4, IFN-α6, IFN-α7, and IFN-α10) as well as IFN-κ. The exploratory analysis showed an upregulation of the response to type I interferon pathway and regulation of type I interferon mediated signaling pathway. The most upregulated genes in those pathways were again the IFN-α subtypes mentioned above. Other pathways involved in the immune response were found to be downregulated after vitamin D substitution, like interferon gamma response; cytokine production and chemotaxis. The common denominator of these pathways was the downregulation of three toll-like receptor genes (TLR-8, TLR-7, TLR-2). Conclusion: The increased expression of specific IFN-α subtypes could explain the increased rituximab-mediated NK cell-cytotoxicity after vitamin D substitution in deficient individuals. To the best of our knowledge, this is the first study to suggest a role for vitamin D in IFN-α regulation. TLRs are known to stimulate cytokine production in NK cells including IFN-α. It can be assumed, that the observed upregulation of IFN-α genes after vitamin D substitution leads to a negative feedback on positive regulators of cytokine production like TLR, causing their downregulation once vitamin D levels are restored. This implies a comprehensive role of vitamin D in IFN-α biosynthesis in human NK cells. Disclosures Stilgenbauer: AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Results of a Phase 1 Trial of Gda-201, Nicotinamide-Expanded Allogeneic Natural Killer (NK) Cells in Patients with Refractory Non-Hodgkin Lymphoma (NHL) and Multiple Myeloma

Blood ◽

10.1182/blood-2020-142419 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 6-6 ◽

Cited By ~ 1

Author(s):

Veronika Bachanova ◽

Joseph Maakaron ◽

David H. McKenna ◽

Qing Cao ◽

Todd E. DeFor ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Phase 1 ◽

Advisory Committees ◽

Non Hodgkin Lymphoma ◽

Cell Manufacturing ◽

Cell Current

Background: The innate capacity of natural killer (NK) cells to kill tumor targets has been translated into cancer immunotherapy. GDA-201 is a novel allogeneic NK cell product derived from NK cells from healthy donors, expanded ex-vivo with nicotinamide (NAM) and IL-15. We previously reported improved killing function, in vivo proliferation, organ trafficking, and augmented resistance against exhaustion in pre-clinical models. We conducted a phase 1 study of GDA-201 in combination with monoclonal antibodies to enhance NK cell targeting through antibody-dependent cellular cytotoxicity (ADCC). We now report safety data in patients (pts) with relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) and multiple myeloma (MM), and report efficacy outcomes in pts with NHL. Methods: Following donor apheresis, CD3-depleted mononuclear cells were cultured for 14-16 days with NAM (5mM) and IL-15 (20ng/ml), resulting in a 40-fold increase in NK cells and increased expression of CD62L from 2.9% to 21%. GDA-201 contained ~98% NK cells, and CD3 content was maintained at <0.5% (<5x105/kg/dose). Pts with R/R B-cell NHL or MM received lymphodepleting (LD) therapy with cyclophosphamide (400mg/m2 IV x 3d) and fludarabine (30 mg/m2 /d IV x 3d), followed by GDA-201 (days 0 and 2) and low-dose IL-2 (6 million units sc x 3 doses). Pts with NHL or MM received rituximab (375 mg/m2) or elotuzumab (10 mg/kg), respectively, x 3 weekly infusions. Results: 30 pts were enrolled:15 with NHL and 15 with MM, in 3 cohorts of escalating GDA-201 dose; 15 pts received the maximum target dose (median dose 12.4 [range 2.0-26.0] x 107 cells/kg). There were no dose limiting toxicities. The most common grade 3/4 adverse events were thrombocytopenia (n=9), hypertension (n=5), neutropenia (n=4), febrile neutropenia (n=4), and anemia (n=3). There were no neurotoxic events, confirmed cytokine release syndrome, graft versus host disease, or marrow aplasia. One patient died of E-coli sepsis. In pts with NHL, histologies included diffuse large B cell lymphoma (DLBCL) (de novo n=5, transformed n=3), follicular lymphoma (FL) (n=6), and mantle cell lymphoma (n=1). Median age was 64 (range 48-83 years). Pts had a median of 3 lines of prior therapy (range 1-8); most were multiply relapsed or refractory (n=2), and 87% had advanced stage. Median follow-up was 10.8 months (range 4.3-27.5 months). Ten pts had complete response (CR): 6/6 pts with FL and 4/8 with DLBCL; 1 pt had partial response (PR), and overall response rate in pts with NHL was 73.3%. Median duration of response was 8.7 months (range 4.3-25 months). Flow cytometry confirmed the persistence of GDA-201 in peripheral blood for 7-10 days (range 2-92% donor NK cells on day 7), as well as enhanced in vivo proliferation (median Ki 67 99%). Flow cytometry of biopsied tissues at day 4 demonstrated trafficking to bone marrow and lymph nodes. Four pts underwent re-treatment with GDA-201 without LD chemotherapy; GDA-201 cells were detectable in blood after the re-treatment and likely contributed to deepening of response in 2 patients. Post-GDA-201 therapy included allogeneic (n=2) and autologous (n=1) hematopoietic stem cell transplantation. One-year estimates of progression-free survival and overall survival were 66% (95% CI 36-84%) and 82% (95% CI 42-95%), respectively. Conclusions: Cellular therapy using GDA-201 with monoclonal antibodies to enhance ADCC was well-tolerated, and demonstrated significant clinical activity in heavily pretreated pts with advanced NHL. Data support the future testing of multiple infusions to potentially enhance anti-tumor effect. The omission of lymphodepleting chemotherapy is feasible and contributes to safety of this approach. Phase II studies in aggressive and indolent NHL cohorts are planned. Disclosures Bachanova: Incyte: Research Funding; FATE: Research Funding; Kite: Membership on an entity's Board of Directors or advisory committees; Karyopharma: Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Gamida Cell: Membership on an entity's Board of Directors or advisory committees, Research Funding. McKenna:Gamida: Other: Cell Manufacturing; Fate Therapeutics: Other: Cell Manufacturing; Intima: Other: Cell Manufacturing; Magenta: Other: Cell Manufacturing. Janakiram:Takeda, Fate, Nektar: Research Funding. Simantov:Gamida Cell: Current Employment. Lodie:Gamida Cell: Current Employment. Miller:Vycellix: Consultancy; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics, Inc: Consultancy, 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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