Low-Dose Interleukin-2 Therapy Enhances Cytotoxicity of CD56bright NK Cells in Patients with Chronic Gvhd

Abstract Introduction: Natural killer (NK) cells play an important role in defense against infections and cancer. Two major subsets of mature NK cells have been described. CD56bright CD16- NK cells, which normally represent only 5-10% of circulating NK cells, are thought to exhibit less cytolytic activity and greater immune regulatory functions than CD56dim CD16+ NK cells. However, recent studies have reported that cytolytic activity of CD56bright NK cells increases when these cells are stimulated with IL-15or the combination of IL-12, IL-15 and IL-18. Previous studies from our center have shown that daily administration of low-dose IL-2 in patients with chronic graft-versus-host disease (cGVHD) induces selective expansion of CD4+FoxP3+Helios+ regulatory T cells and CD56bright NK cells and improves clinical manifestations of cGVHD. The function of CD56bright NK cells expanded by low-dose IL-2 has not previously been studied. Methods: Single cell mass cytometry (CyTOF) with a panel of 35 metal tagged antibodies was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from 10 adult patients with active cGVHD receiving daily low-dose IL-2 therapy. Patients in this clinical trial received extracorporeal photopheresis (ECP) for 8 weeks prior to starting daily low dose IL-2. ECP therapy (twice weekly) was continued when patients began low-dose IL-2 (1x106 IU/M2/day x 8 weeks). The analytic panel included 26 cell surface markers to identify distinct lymphocyte subsets and 9 intracellular markers to measure functional status and activation of specific signaling pathways. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data. NK cytolytic activity was measured in flow cytometry-based cytotoxicity assays. CD56bright and CD56dim NK cells from 8 adult patients were purified from cryopreserved PBMC by cell sorting and incubated with labeled K562 cells for 4 hours followed by staining with 7-AAD and Annexin-V. E:T ratio of 1:1 was used for incubation with K562 targets. Results: No changes in extracellular or intracellular NK cell markers or quantitative changes in NK cells were observed during the initial 8 week ECP treatment period. Selective expansion of CD56bright NK cells was noted after 1 week of IL-2 therapy (9W) and continued during 8 weeks of daily IL-2 therapy. Increased expression of NKp30, Nkp46, NKG2D, HLA-DR and Ki67 occurred in expanded CD56bright NK cells with peak expression at 1 week after starting IL-2 (9W). At later time points during IL-2 therapy, expression of NKG2D, HLA-DR and Ki67 returned to baseline (Figure 1A). Expression of CD56, CD122 and NKG2A continued to increase during IL-2 treatment. In contrast, expression of CD25 by expanded CD56bright NK cells decreased during IL-2 treatment. Expression of phosphorylated signaling proteins did not change in any NK cell subset during IL-2 treatment.　Cytolytic activity was measured in CD56bright and CD56dim NK cell subsets at different times during ECP and IL-2 therapy. After thawing, flow-sorted NK cell subsets were cultured for 16-20 hours with IL-2 (100 IU/ml). Cells were then washed and incubated with tumor targets (K562) for 4 hours and % killing was assessed by flow cytometry. Compared to pre IL-2 treatment, cytolytic activity of CD56bright NK cells increased during IL-2 treatment while cytotoxicity of CD56dim NK cells did not change. Notably, cytotoxicity of CD56bright NK cells became significantly higher than CD56dim NK cells during IL-2 therapy (Figure 1B). Conclusion: Single cell mass cytometry revealed that daily low dose IL-2 therapy induces selective expansion, activation and increased expression of activating NK receptors in CD56bright NK cells. CD56dim NK cells were not affected by IL-2 therapy. In vitro assays revealed that cytolytic activity of CD56bright NK cells increased during IL-2 treatment and exceeded the cytotoxicity of CD56dim NK cells. CD56bright NK cells, traditionally considered to be minimally tumor-responsive, are effectively stimulated by daily low dose IL-2 exposure to enable potent cytotoxicity in response to tumor targets. In patients receiving low-dose IL-2 after allogeneic HSCT, expanded CD56bright NK cells may contribute to graft versus leukemia (GVL) and help prevent relapse after transplant. Disclosures Nikiforow: Kite Pharma: Consultancy. Ho:Jazz Pharmaceuticals: Consultancy. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Soiffer:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

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Low-Dose Interleukin-2 Therapy Activates Circulating T Follicular Regulatory Cells and Suppresses Circulating T Follicular Helper Cells in Patients with Chronic Gvhd

Blood ◽

10.1182/blood.v130.suppl_1.74.74 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 74-74

Author(s):

Yusuke Kamihara ◽

Edouard Forcade ◽

John Koreth ◽

Hongye Liu ◽

Tomohiro Kubo ◽

...

Keyword(s):

B Cell ◽

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Low Dose ◽

Cell Mass ◽

Mass Cytometry ◽

Advisory Committees ◽

Healthy Donors

Abstract Introduction: T follicular helper (TFH) and T follicular regulatory (TFR) cells play important roles in the regulation of B-cell immunity. While TFH promote B cell functions in the germinal center (GC), TFR function as negative regulators of the GC response. Previous studies in murine models established that TFH and GC B cells are required for the development of chronic graft-versus-host disease (cGVHD). We previously reported that circulating TFH (cTFH) were more functionally activated in patients with active cGVHD compared with patients with no cGVHD. Low-dose IL-2 therapy has been shown to selectively expand CD4Treg and improve cGVHD symptoms. In the current study, we examined the effects of IL-2 therapy on cTFH and circulating TFR (cTFR) in patients with steroid resistant cGVHD. Methods: Single cell mass cytomtery (CyTOF) was performed on cryopreserved peripheral blood mononuclear cells (PBMC) from healthy donors and 17 adult patients with active cGVHD receiving daily low-dose IL-2 therapy (Koreth et al. Blood 2016). A panel of 35 metal-tagged monoclonal antibodies was used to simultaneously examine the phenotypic and functional effects of low-dose IL-2 on lymphocyte populations in vitro and in vivo. The analytic panel included 22 cell surface markers to identify distinct lymphocyte subsets and 13 intracellular markers to measure functional status and activation of specific signaling pathways. Before staining for surface and intracellular antigens, serial samples from individual patients were barcoded to ensure uniformity of analysis. viSNE was used to visualize of high-dimensional data on a two-dimensional map and quantify single cell mass cytometry data. Results: In PBMC from healthy donors, expression of CD25 (IL-2Rα), CD95, CTLA-4, BLIMP-1 and GITR was higher in cTFR compared with cTFH. To examine the response to IL-2 in vitro, PBMC from healthy donors were stimulated with IL-2 for 15 minutes (Figure 1A). At low IL-2 concentrations (1 to 10 IU/mL), phospho-STAT5 (p-STAT5) was selectively activated in cTFR compared with cTFH. At high IL-2 concentrations (100 to 1,000 IU/mL), p-STAT5 was activated in both cTFR and cTFH. To examine the response to IL-2 in vivo, we used mass cytometry to examine serial PBMC samples from cGVHD patients receiving daily low dose IL-2 therapy (1x106 IU/M2/day). Selective expansion of cTFR was noted after 1 week of treatment and cTFR expansion remained stable for the 12 week duration of therapy. Expanded cTFR increased expression of p-STAT5, FoxP3, BCL6, HLA-DR (Figure 1B) and CD25, CD95, CTLA-4, ICOS, Ki67 and Helios 1 week after starting IL-2. cTFR:cTFH ratio increased rapidly after starting low dose IL-2 and paralleled the increased Treg:Tcon ratio (Figure 1C). Activated TFH and TFR can be identified by expression of ICOS and PD-1. The expansion of ICOS+PD-1+ cTFR was evident after 1 week of IL-2 and remained elevated at the end of therapy. In contrast, ICOS+PD-1+ cTFH increased 1 week after starting IL-2 therapy but subsequently decreased and fell below baseline 6 and 12 weeks after starting IL-2 (Figure 1D). Activated ICOS+PD-1+ cTFR expressed higher levels of p-STAT5, BCL-6, FoxP3, HLA-DR and CD25 during low dose IL-2 therapy. In contrast, these functional markers were not increased in ICOS+PD-1+ cTFH during IL-2 therapy (Figure 1B). Conclusion: Single cell mass cytometry analysis revealed that daily low dose IL-2 therapy induces selective activation and increased expression of functional proteins in ICOS+PD-1+ cTFR. In contrast, activated ICOS+PD-1+ cTFH were suppressed during IL-2 therapy. The selective activation of cTFR and suppression of cTFH provide a mechanism whereby low dose IL-2 therapy can promote B cell tolerance as well as T cell tolerance in patients with cGVHD. Disclosures Forcade: Neovii: Other: Travel grant. Koreth: Amgen Inc.: Consultancy; Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Nikiforow: Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand: Infinity: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Otsuka: Research Funding; Tensha: Research Funding; Sequenta/Adaptive: Research Funding; Genmab: Consultancy; Affimed: Research Funding; Sigma Tau: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Roche: Research Funding. Cutler: Bristol-Myers Squibb: Consultancy; Pfizer: Consultancy; Kite: Consultancy; Pharmacyclics: Consultancy; Incyte: Consultancy; Astellas: Consultancy.

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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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Identification of Novel Immune Cell Populations in Lenalidomide Refractory Relapsed Multiple Myeloma Patients Treated with Pomalidomide and Low Dose Dexamethasone

Blood ◽

10.1182/blood-2019-130154 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3186-3186

Author(s):

Anna Kalff ◽

Tiffany Khong ◽

Malarmathy Ramachandran ◽

Sam Norton ◽

Andrew Mitchell ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Statistical Significance ◽

Advisory Committees ◽

Travel Costs ◽

Trial Investigator ◽

Hla Dr ◽

Advisory Role

The ALLG MM14 trial evaluated the impact of low dose dexamethasone (LoDEX) withdrawal in lenalidomide (LEN) refractory and relapsed (RR) multiple myeloma (MM) patients achieving initial disease control with pomalidomide (POM) and LoDEX re-induction. As previously reported, patients continuing with POM LoDEX had superior progression free survival (PFS) compared to maintenance with POM alone, however, this early PFS benefit was lost and by 18m was reversed to favour POM only. In patients who received post-progression therapy, more durable responses (second PFS: 12.7m vs 4.6m, p=0.034) and superior survival (OS: 19.4m vs 12.5m, p=0.092) were seen in those previously treated with POM alone. Here we present findings from the preliminary correlative immune studies of this trial. Aims To undertake mass cytometry (CyTOF) based immune profiling in patients with advanced MM receiving treatment with POM LoDEX. Methods MM14 was a multicentre, open-label, randomised phase 2 study of LEN refractory RRMM patients who had received ≥ 2 prior lines of therapy. Patients were treated with POM 4mg d1-21 (28d cycle) and LoDEX (40mg weekly). After 4 cycles (induction), patients with stable disease or better (≥SD) were randomised to receive maintenance with ongoing POM-LoDEX or POM alone. Therapy continued until toxicity/progression. PBMCs were collected at baseline and sequentially while on treatment. Cells were barcoded using the Cell-ID 20-Plex Pd barcoding kit (Fluidigm) followed by staining with sub-set/function defining antibodies (targeting myeloid, B, T and NK cells: CD16, CD24, CD11c, CD45RO, CD314, CD38, CD336, HLA-DR, CD14, CD56, CD158a, CD27, CD28, CD159a, CD8, CD19, CD45RA, CD11b, CD4, IgD, CD335, FOXP3, CD25, CD66b, CD3, CD337, CD20, CD158b, CD127 CD57, CD197, CD194, CD304 and CD279). Samples were acquired on the Helios instrument. Data were clustered in the VORTEX package. Significant differences in cluster frequency were assessed by Mann-Whitney test for statistical significance. Cluster phenotypes were determined and validated via multiple visualisation approaches. CD3-CD19-CD56+ NK cells were pre-gated from patient datasets. We then performed Boolean gating using seven NK cell activation/inhibitory markers - CD158a, CD158b, CD159a, CD314, CD335, CD336 and CD337. Boolean populations that comprised 3% or greater of the total NK cell population (median) were then compared. A Mann-Whitney test was used to determine statistical significance. Results 154 patients from 11 Australian sites were enrolled. The median number of prior treatment lines was 4.5, 82.5% were double refractory. 78 patients who achieved ≥SD were randomised to maintenance: POM n = 40, Pom LoDEX n = 38. CD336+CD20+ cells ("NK-B-cells") were identified in the pre-induction samples of all patients and were significantly more frequent in responders (median 2% of total cells) than in non-responders (0.8% of total cells, p<0.0001). These cells also variably expressed CD19, IgD, HLA-DR, CD158b, CD38 and CD45RA. Preliminary validation of this observation has also been successfully undertaken in an independent cohort of MM patients utilising multi-parameter flow cytometry. In the patients who achieved ≥SD, 5 out of the 8 large clusters (each at least 3% [median] of total nucleated cells evaluated) that were significantly enriched (p<0.0001) following POM LoDEX induction were neutrophil populations. These populations all expressed CD66b but with variable expression of CD24, CD16, CD11c, CD11b and CD45RO. Inhibited NK cells (CD3-CD19-CD56+) based on CD159a, CD314 and CD158a expression were enriched pre-induction and significantly decreased following POM LoDEX (p<0.0001), while activated NK cells expressing CD337 and CD336 and no inhibitory receptors were significantly increased following POM LoDEX (p<0.0001). Conclusion Utilising CyToF, we have identified a novel "NK B cell" population in RRMM patients, with a higher baseline frequency of these cells being associated with a greater likelihood of response to POM LoDEX. Importantly, we have also confirmed the presence of these cells in an independent MM cohort. Moreover, subsequent to POM LoDEX exposure we have demonstrated the enrichment of heterogeneous neutrophil populations as well as an increase in activated NK cells and commensurate decrease in inhibited NK cells. These novel observations may provide new insights into the mechanisms of action of pomalidomide in MM. Disclosures Kalff: Amgen: Honoraria; Celgene: Honoraria; pfizer: Honoraria. Khong:Novartis Oncology: Research Funding. Reynolds:Alfred Health: Employment, Other: Biostatistician for trials funded by the Australian government and Abbvie, Amgen, Celgene, GSK, Janssen-Cilag, Merck, Novartis, Takeda, but sponsored by Alfred Health.; AUSTRALASIAN LEUKAEMIA & LYMPHOMA GROUP (ALLG): Consultancy; Novartis AG: Equity Ownership; Novartis Australia: Honoraria. Quach:GSK: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees. Ho:Novartis: Other: Trial Investigator meeting travel costs; La Jolla: Other: Trial Investigator meeting travel costs; Celgene: Other: Trial Investigator meeting travel costs; Janssen: Other: Trial Investigator meeting travel costs. Mollee:Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Spencer:Takeda: Other: Consulting/advisory role, Research Funding; Janssen Oncology: Other: Consulting/advisory role, Research Funding, Speakers Bureau; Amgen: Other: Consulting/advisory role, Research Funding; AbbVie: Other: Consulting/advisory role, Research Funding; Servier: Other: Consulting/advisory role; Secura Bio: Other: Consulting/advisory role; Haemalogix: Other: Consulting/advisory role; Celgene: Other: Consulting/advisory role, Research Funding, Speakers Bureau; Sanofi: Other: Consulting/advisory role; Specialised Therapeutics Australia: Consultancy, Honoraria.

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Combination of Melphalan Flufenamide and Anti-PD-L1 or Anti-CD38 Antibodies Enhances Anti-Myeloma Immunity

Blood ◽

10.1182/blood-2021-153705 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4357-4357

Author(s):

Arghya Ray ◽

Ting DU ◽

Nina N. Nupponen ◽

Fredrik Lehmann ◽

Jakob Lindberg ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Board Of Directors ◽

Tumor Cells ◽

Research Funding ◽

Mononuclear Cells ◽

Nk Cell ◽

Cytolytic Activity ◽

Publicly Traded ◽

Advisory Committees

Abstract Introduction Melphalan flufenamide (Melflufen; Oncopeptides AB) is a novel enzyme-activated analogue of melphalan that enables a more rapid and higher intracellular accumulation of melphalan in tumor cells than is achievable by direct exposure to equimolar doses of melphalan. Our preclinical study showed that melflufen is a more potent anti-myeloma (MM) agent than melphalan, overcomes drug-resistance, and induces synergistic anti-MM activity in combination with bortezomib, lenalidomide, or dexamethasone (Chauhan et al, Clinical Cancer Res 2013;19:3019). However, the effect of melflufen on the immunosuppressive and tumor-promoting MM-host bone marrow (BM) accessory cells such as immunologically dysfunctional plasmacytoid dendritic cells (pDCs; CD123/IL-3Rα) remains unclear. Here, we utilized our coculture models of pDCs, T-, and NK cells with autologous patient MM cells to examine whether a combination of melflufen and immune checkpoint inhibitor anti-PD-L1 Ab, or daratumumab (anti-CD38 Ab), restores anti-MM immunity. Methods MM patient BM and PB samples (N=10; obtained after informed consent), and cell lines were used for the study. Minimally cytotoxic concentration of melflufen (0.1 µM) was used to assess immune functions. CTL/NK activity assays MM CD8 + T- or NK-cells were cultured with autologous pDCs (1:10 pDC:T/NK ratio) with melflufen (0.1 μM) alone, and with anti-PD-L1 (5 μg/ml) or anti-CD38 (0.5 μg/ml) Abs for 3-5 days; cells were washed to remove the drugs, and then cultured for another 24h with pre-stained target MM cells (10:1 E/T ratio; T/NK:MM), followed by quantification of viable MM cells by flow. Results 1) Both MM tumor cells and pDCs showed higher PD-L1 and CD38 levels vs normal plasma cells; 2) Treatment of MM patient total BM mononuclear cells or purified MM cells with melflufen (0.1 µM) increased PD-L1 expression on MM cells (1.84-fold, treated vs untreated; p<0.05). Importantly, treatment of MM cells with melflufen and anti-PD-L1 Abs enhanced anti-MM cytotoxicity; 3) Combination of melflufen and anti-PD-L1 Ab triggers activation of CD3 + T cells, evidenced by an increase in CD69 expression on CD3 + T cells (1.15-fold, treated vs untreated, p<0.05); 4) Combination of melflufen and anti-PD-L1 Ab induced a more robust autologous MM-specific CD8 + cytotoxic T lymphocyte (CTL) activity than melflufen alone (% MM lysis: melflufen: 20%; melflufen plus anti-PD-L1 Ab: 60%; n=5; p=0.013); 5) Meflufen and anti-PD-L1 also triggered pDC-induced NK cell-mediated MM-specific cytolytic activity (p<0.05); and finally, 6) Low doses of melflufen and anti-CD38 Abs enhanced pDC-induced NK cell-mediated MM-specific cytolytic activity (%Viability: melflufen: 75%; melflufen + anti-CD38 Ab: 12.5%; n=4; p=0.001). Conclusions The combination of melflufen and anti-PD-L1 increases pDC-induced T- and NK cell-mediated cytolytic activities against MM. Moreover, combined melflufen and anti-CD38 Abs modestly enhance pDC-induced NK cell-mediated MM-specific cytolytic activity. Our preclinical data suggest targeting PD-L1 in combination with melflufen as well as support an ongoing clinical trial of melflufen with anti-CD38 Abs to enhance anti-MM immunity. Disclosures Nupponen: Oncopeptides AB: Consultancy. Lehmann: Oncopeptides AB: Current Employment. Lindberg: Oncopeptides: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Other: Travel, Accommodations, Expenses; Camurus: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Affibody: Membership on an entity's Board of Directors or advisory committees. Gullbo: Oncopeptides AB: Consultancy. Richardson: Takeda: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Janssen: Consultancy; Sanofi: Consultancy; Protocol Intelligence: Consultancy; Karyopharm: Consultancy, Research Funding; GlaxoSmithKline: Consultancy; Regeneron: Consultancy; AstraZeneca: Consultancy; Secura Bio: Consultancy; AbbVie: Consultancy; Oncopeptides: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Oncopeptides: Consultancy; Stemline Therapeutics: Consultancy. Anderson: Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

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Phenotypic Characterization by Single-Cell Mass Cytometry of Human Intrahepatic and Peripheral NK Cells in Patients with Hepatocellular Carcinoma

Cells ◽

10.3390/cells10061495 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1495

Author(s):

Yuichi Yoshida ◽

Sachiyo Yoshio ◽

Taiji Yamazoe ◽

Taizo Mori ◽

Yuriko Tsustui ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Nk Cells ◽

Single Cell ◽

Nk Cell ◽

Checkpoint Inhibitors ◽

Cell Mass ◽

Phenotypic Characterization ◽

Advanced Hepatocellular Carcinoma ◽

Mass Cytometry ◽

Cell Phenotypes

Overall response rates of systemic therapies against advanced hepatocellular carcinoma (HCC) remain unsatisfactory. Thus, searching for new immunotherapy targets is indispensable. NK cells are crucial effectors and regulators in the tumor microenvironment and a determinant of responsiveness to checkpoint inhibitors. We revealed the landscape of NK cell phenotypes in HCC patients to find potential immunotherapy targets. Using single cell mass cytometry, we analyzed 32 surface markers on CD56dim and CD56bright NK cells, which included Sialic acid-binding immunoglobulin-type lectins (Siglecs). We compared peripheral NK cells between HCC patients and healthy volunteers. We also compared NK cells, in terms of their localizations, on an individual patient bases between peripheral and intrahepatic NK cells from cancerous and noncancerous liver tissues. In the HCC patient periphery, CD160+CD56dim NK cells that expressed Siglec-7, NKp46, and NKp30 were reduced, while CD49a+CD56dim NK cells that expressed Siglec-10 were increased. CD160 and CD49a on CD56dim NK cells were significantly correlated to other NK-related markers in HCC patients, which suggested that CD160 and CD49a were signature molecules. CD49a+ CX3CR1+ Siglec-10+ NK cells had accumulated in HCC tissues. Considering further functional analyses, CD160, CD49a, CX3CR1, and Siglec-10 on CD56dim NK cells may be targets for immunotherapies of HCC patients.

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Characterization of Peripheral Blood Mononuclear Cells Addback Following CD34 Enrichment, Engraftment and T and NK Cells Immune Reconstitution in Patients with High Risk Sickle Cell Disease (SCD) (IND 14359)

Blood ◽

10.1182/blood-2020-140425 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 20-21

Author(s):

Yaya Chu ◽

Julie-An Talano ◽

Lee Ann Baxter-Lowe ◽

Carolyn A. Keever-Taylor ◽

Erin Morris ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Immune Reconstitution ◽

Mononuclear Cells ◽

Nk Cell ◽

Advisory Committees ◽

Hla Dr

Background: CD3/CD19 cell depletion (Barfiled RC, et al, Cytotherapy, 2004), αβ T-cell/CD19 cell depletion (Locatelli F, et al, Blood, 2017), CD34+ positive selection (Aversa F, et al, NEJM, 1998) are designed to deplete T cells and reduce AGVHD following allogeneic stem cell transplantation (AlloSCT). These approaches achieved low rates of AGVHD, but the grafts had few T and B cells. To improve immune reconstitution we undertook an alternative approach to addback small numbers and percentages of immune cells in the final HSCT product. We previously reported a very low incidence of AGVHD in pediatric recipients receiving CD34 enriched HPC products with peripheral blood mononuclear cells (PBMNC) addback containing a fixed dose of 2 x 105 CD3/kg from MUD donors (Geyer/Cairo et al, BJH, 2012). Recently we demonstrated that despite a 5 log depletion of T cells, PBMNC addback (fixed at 2 x 105 CD3/kg) facilitated rapid hematopoietic engraftment, high levels of donor chimerism and immune reconstitution with a low probability of Grade II-IV AGVHD. Patients had a 1 yr OS of 90% following familial haploidentical (FHI) CD34 Enriched Stem Cell Transplantation in patients with SCD (Cairo, JAMA Pediatr, 2020). Objective: To determine the final immune cell concentration following CD34 enrichment and PBMNC (2 x 105 CD3/kg) addback and determine the effect on engraftment and T and NK cell immune reconstitution. Methods: Patients and/or their guardians signed written informed consents and/or assents (NCT NCT02675959). CD34+ enrichment was performed using a CD34+ reagent system (CliniMACS; Miltenyi Biotec). Mononuclear cells (2 × 105 CD3 cells/kg of recipient body weight) were removed from the leukapheresis collection prior to CD34+ enrichment and were cryopreserved as a source of MNC addback (T cells). The addback products were analyzed for CD3+CD56- T cells, CD3-CD56+ NK cells, CD3+CD56+ NKT cells, Lin-CD123+ HLA-DR+ DC cells and Lin-CD11c+ HLA-DR+ DC cells by multicolor flow cytometry analysis. Th1/Th2 cytokines were measured by multiplex assays. T cell activity was measured by viral T cells IFN-g and plasma cytokines. NK function was measured by NK receptor expression by flow cytometry analysis and in vitro cytotoxicity. Results: We identified in the PBMNC addback, mean+SEM white blood cell (WBC) percentage of: CD3+ CD56- T cells = 56.4±5%; CD3- CD56+ NK cells = 4.6±1%; CD3+ CD56+ NKT cells = 5.1±0.6%; CD19+ B cells = 29.9±3.5%. Lin- WBC consisted of: CD123+ HLA-DR+ DC cells = 18.4±8.2%; CD11c+ HLA-DR+ DC cells = 6.0±3.0%. There were 20.0+9.1e6 T cells, 1.1+0.3e6 NK cells, 1.6+0.7 e6 NKT cells, 8.6+2.5e6 B cells, 1.2+0.6e6 CD123+DC and 0.8+0.5e6 CD11c DC in the final infused products (Fig.1). We found that percentages of IFN-g+ in CD4 cells in response to CMV (pp65), ADV (hexon) and EBV (BZLF1), ranged from 0.2%+0.1% to 0.5%+0.1%, while percentages of IFN-g+ in CD8 cells in response to the antigens ranged from 0.7%+0.3% to 3.7%+1.8% when examined at days 180, 270 and 365. NK (CD3- CD56+) reconstitution was extremely rapid and occurred as early as day 30 (35.5±8.6%, 2710+1624.4 cells/ul total cells; p<0.01 vs pre-t). There were no significant differences pre-HSCT vs day 365 in plasma cytokines (Th1 and Th2) and growth factors released including IFN-g, TNF-a, IL-18, IL-4, IL-5, IL-6, IL-10, G-CSF, MCP-1 and MIP1a. There was also robust expression of NK receptor expression including NK cytotoxicity receptors, NK KIR receptors, and C-type lectin-like receptors at day 30 as compared to pre-HSCT. NK cytotoxicity, as measured using PBMC cells from recipients at different time points against K562 (E:T=10:1), was also significantly increased at day 30 (26.2±2.8%) and day 180 (28.3±3%) vs pre-HSCT (16.1±2.1%) (p<0.01). As a NK cell activation marker, CD107a expression and granzyme B levels in gated NK cells peaked at day 30. Conclusion: PBMNC addback to CD34 enriched HPC products, with a final dose of 2 × 105 CD3 cells/kg, led to stem cell products with a diverse mixture of T, NK, NKT, DC1, and DC2 cells. Immune reconstitution following PBMNC addback to CD34 enriched cells resulted in excellent CD4 and CD8 responses to CMV, ADV and EBV, and rapid functional NK cell reconstitution (Supported by FDA R01FD004090 (MSC)). Disclosures Baxter-Lowe: CHLA: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Patents related to HLA typing, Research Funding. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Research Funding. Cairo:Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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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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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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