scholarly journals A Phase I Study of FT538, a First-of-Kind, Off-the-Shelf, Multiplexed Engineered, iPSC-Derived NK Cell Therapy As Monotherapy in Relapsed/Refractory Acute Myelogenous Leukemia and in Combination with Daratumumab or Elotuzumab in Relapsed/Refractory Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-3
Author(s):  
Murali Janakiram ◽  
Ravi Vij ◽  
David S. Siegel ◽  
Ted Shih ◽  
Sara Weymer ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Myelogenous Leukemia ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Cell Therapies ◽  
Acute Myelogenous

Background: Allogeneic natural killer (NK) cell therapies have been well-tolerated with documented anti-tumor activity in patients with relapsed/refractory (r/r) hematologic malignancies including acute myelogenous leukemia (AML) and multiple myeloma (MM) (Lupo et al. 2019). Allogeneic NK cell therapies may offer an improved safety profile characterized by the absence of cytokine release syndrome and neurologic toxicity compared with T-cell therapies (Liu et al. 2020). However, in comparison to T cells, NK cells have limited in vivo expansion and a short half-life, and the potential to generate deeper and more durable anti-tumor responses through multi-dose administration is limited by the inability to consistently manufacture and administer more than one dose of allogeneic NK cells. The monoclonal antibodies (mAbs) daratumumab and elotuzumab have demonstrated clinical benefit and are approved for the treatment of MM (Darzalex® USPI, Empliciti® USPI). However, durable responses and disease-free survival remain limited. Engagement of the Fc portion of the mAb with CD16 on NK cells, which promotes antibody-dependent cellular cytotoxicity (ADCC), is a major contributor to the efficacy of daratumumab and elotuzumab. It is hypothesized that more clinically meaningful outcomes may be achieved by combining therapeutic mAbs with allogeneic NK cells engineered to enhance ADCC. FT538 is an investigational, first-of-kind, multiplexed engineered NK cell therapy generated from a clonal master engineered induced pluripotent stem cell (iPSC) line, which can be used as a renewable source for the mass production of off-the-shelf NK cells for broad patient access. FT538 is engineered with three modalities for enhanced innate immunity: (1) high-affinity 158V, non-cleavable CD16 Fc receptor for augmented ADCC; (2) interleukin (IL)-15/IL-15 receptor fusion that promotes cytokine-autonomous persistence; and (3) CD38 knockout to mitigate NK cell fratricide by CD38-directed mAbs. In preclinical studies, FT538 combined with daratumumab against MM targets demonstrated avoidance of daratumumab-mediated fratricide and significantly enhanced ADCC in vitro in a serial stimulation cytotoxicity assay compared with peripheral blood NK cells, and the combination of FT538 with daratumumab led to highly effective tumor control compared with daratumumab alone in an in vivo MM xenograft model (Bjordahl et al. 2019). Study Design and Methods: This study is a multicenter, multi-dose, Phase I clinical trial of FT538 in patients with r/r AML or r/r MM. The primary objectives are to determine the recommended Phase II dose of FT538 as monotherapy in r/r AML and in combination with daratumumab or elotuzumab in r/r MM. Key secondary objectives include evaluation of FT538 safety and tolerability, anti-tumor activity, and pharmacokinetics (PK) as monotherapy in r/r AML and combined with mAbs in r/r MM. Exploratory objectives include characterization of FT538 pharmacodynamics as assessed by peripheral blood biomarkers, assessment of minimal residual disease, and characterization of the tumor microenvironment in pre- and post-treatment tumor biopsies. The dose-escalation part of the trial utilizes a 3+3 design to identify the maximum tolerated dose of up to three doses of FT538 on Days 1, 8, and 15 as a monotherapy in r/r AML (Regimen A) and in combination with daratumumab (Regimen B) or elotuzumab (Regimen C) in r/r MM. The dose-expansion part of the trial will further characterize the safety, efficacy, and PK of FT538 in all regimens. The trial will test up to five FT538 dose levels ranging from 50 million to 1.5 billion cells. Up to 105 patients will be enrolled. The mAbs in Regimens B and C will be administered based on dosing schedules per their respective prescribing information. Lympho-conditioning consisting of three consecutive days of fludarabine and cyclophosphamide will be administered prior to the first dose of FT538. Key inclusion criteria include r/r disease after standard approved therapies for r/r AML or r/r MM, as applicable, measurable disease for r/r MM, and adequate organ function. Key exclusion criteria include active central nervous system disease, need for systemic immunosuppressive therapy, and prior allograft organ transplant. This trial is expected to begin patient enrollment in 2020. Disclosures Janakiram: Takeda, Fate, Nektar: Research Funding. Siegel:Karyopharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; Merck: Consultancy, Honoraria, Speakers Bureau; Celulatiry: Consultancy; BMS: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau. Shih:Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Weymer:Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company. Chu:Roche Holding AG: Current equity holder in publicly-traded company; Fate Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Miller:Fate Therapeutics, Inc: Consultancy, Patents & Royalties, Research Funding; GT Biopharma: Consultancy, Patents & Royalties, Research Funding; Vycellix: Consultancy; Onkimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Cyclophosphamide and fludarabine will be used as lympho-conditioning therapy prior to FT538 administration.

scholarly journals 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 ◽  
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.

scholarly journals 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 ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-6 ◽  
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.

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

Blood ◽  
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.

The JAK Inhibitor Ruxolitinib Substantially Affects NK Cells in MPN Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3169-3169
Author(s):  
Kathrin Schönberg ◽  
Janna Rudolph ◽  
Maria Vonnahme ◽  
Isabelle Cornez ◽  
Sowmya Parampalli Yajnanarayana ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Receptor Expression ◽  
Target Cells ◽  
Jak Inhibitor ◽  
Advisory Committees ◽  
Nk Cell Differentiation

Abstract Introduction: Ruxolitinib (INCB018424) is the first JAK inhibitor approved for treatment of myelofibrosis (MF). Ruxolitinib-induced reduction of splenomegaly and symptoms control is linked to a substantial suppression of MF-associated circulating pro-inflammatory and pro-angiogenic cytokines. However, an increased rate of infections in ruxolitinib-exposed patients with MF was recently described. Natural killer (NK) cells are innate immune effector cells eliminating malignant or virus-infected cells. Thus, the aim of this project was to define in more detail the impact of JAK inhibition on NK cell biology both in vitro and in vivo. Methods: 28 patients with myeloproliferative neoplasms (MPN) with or without ruxolitinib therapy and 12 healthy donors were analyzed for NK cell frequency, NK receptor expression and function. Phenotypic and functional NK cell markers (e.g. CD11b, CD27, KIR, NKG2A, NKG2D, NKp46, CD16, granzyme B, and perforin) were analyzed by FACS. NK cell function was evaluated by classical killing assays upon stimulation with MHC class I-deficient target cells K562. Finally, a set of additional in vitro experiments (e.g. analysis of lytic synapse formation by FACS and confocal microscopy) were performed to define in more detail the characteristics and potential mechanisms of ruxolitinib-induced NK cell dysfunction. Results: In addition to our recent finding that ruxolitinib induces NK cell dysfunction in vitro (e.g. reduced killing, degranulation and IFN-γ production), we here demonstrate that NK cell proliferation and cytokine-induced receptor expression as well as cytokine signalling are drastically impaired by ruxolitinib. Interestingly, reduced killing is at least in part due to a reduced capacity to form a mature lytic synapse with target cells. The significance of the in vitrofindings is underscored by a dramatically reduced proportion and absolute number of NK cells in ruxolitinib-treated MPN patients when compared to treatment-naïve patients or to healthy controls (mean percentage of NK cell frequency: ruxolitinib-naïve MPN patients 12.63% ±1.81; healthy donors 13.51% ±1.44; ruxolitinib-treated patients 5.47% ±1.27). A systematic analysis of NK cell receptor expression revealed that the reduction of NK cells in ruxolitinib-exposed individuals is most likely due to an impaired NK cell differentiation and maturation process, as reflected by a significantly increased ratio of immature to mature NK cells. Finally, the endogenous functional NK cell defect in MPN is further aggravated by intake of the JAK inhibitor ruxolitinib. Conclusion: We here provide compelling in vitro and in vivo evidence that inhibition of the JAK/STAT-pathway by ruxolitinib exerts substantial effects on the NK cell compartment in MPN patients due to the inhibition of NK cell differentiation and NK cell key functions. Our data may help to better understand the increased rate of severe infections and complement recent reports on ruxolitinib-induced immune dysfunction. Disclosures Koschmieder: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel, Accomodation, Expenses Other. Brümmendorf:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Wolf:Novartis: Consultancy, Honoraria, Research Funding, Travel and Accommodation Other.

scholarly journals Enhanced In Vivo Persistence and Proliferation of NK Cells Expanded in Culture with the Small Molecule Nicotinamide: Development of a Clinical-Applicable Method for NK Expansion

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 657-657 ◽  
Author(s):  
Tony Peled ◽  
Guy Brachya ◽  
Nurit Persi ◽  
Chana Lador ◽  
Esti Olesinski ◽  
...  
Keyword(s):  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Culture Method ◽  
Employment Equity ◽  
Advisory Committees ◽  
Nsg Mice ◽  
Equity Ownership

Abstract Adoptive transfer of cytolitic Natural Killer (NK) cells is a promising immunotherapeutic modality for hematologic and other malignancies. However, limited NK cell in vivo persistence and proliferation have been challenging clinical success of this therapeutic modality. Here we present a reliable, scalable and GMP-compliant culture method for the expansion of highly functional donor NK cells for clinical use. Nicotinamide (NAM), a form of vitamin B-3, serves as a precursor of nicotinamide adenine dinucleotide (NAD) and is a potent inhibitor of enzymes that require NAD including ADP ribosyltransferases and cyclic ADP ribose/NADase. As such, NAM is implicated in the regulation of cell adhesion, polarity, migration, proliferation, and differentiation. We have previously reported that NAM augments tumor cytotoxicity and cytokine (TNFα and IFN-γ) secretion of NK cells expanded in feeder-free culture conditions stimulated with IL-2 or IL-15. Immunophenotype studies demonstrated NK cells expanded with NAM underwent typical changes observed with cytokine only-induced NK cell activation with no significant differences in the expression of activating and inhibitory receptors. CD200R and PD-1 receptors were expressed at low levels in resting NK cells, but their expression was up-regulated following activation in typical cytokine expansion cultures. Interestingly, the increase in CD200R and PD-1 was reduced by NAM, suggesting these NK cells to be less susceptible to cancer immunoevasion mechanisms (Fig 1). In vivo retention and proliferation is a pre-requisite for the success of NK therapy. We have reported that NK expanded with NAM displayed substantially better retention in the bone marrow, spleen and peripheral blood of irradiated NSG mice. Using a carboxyfluorescein succinimidyl ester (CFSE) dilution assay, we demonstrated increased in vivo proliferation of NAM-cultured NK cells compared with cells cultured without NAM. These results were recently confirmed using a BrdU incorporation assay in irradiated NSG mice (Fig.2). These findings were mechanistically supported by a substantial increase in CD62L (L-selectin) expression in cultures treated with NAM. CD62L is pivotal for NK cell trafficking and homeostatic proliferation and its expression is down regulated in IL-2 or IL-15 stimulated cultures (Fig. 3). These data provided the foundation for the development of a feeder cell-free scalable culture method for clinical therapy using apheresis units obtained from healthy volunteers. CD3+ cells were depleted using a CliniMACS T cell depletion set. Following depletion, the CD3- fraction was analyzed for phenotypic markers and cultured in closed-system flasks (G-Rex100 MCS, Wilson Wolf) supplemented with 20ng/ml IL-15 or 50ng/ml IL-2 GMP, 10% human serum, minimum essential medium-α and NAM USP for two weeks. While at seeding, NK cells comprised 5-20% of total culture seeded cells, at harvest, NK cells comprised more than 97% of the culture. Although overall contamination of the NK cultures was low with either IL-15 or IL-2, a lower fraction of CD3+ and CD19+ cells was observed with IL-15 vs IL-2 (0.2±0.1% vs. 0.4±0.2% and 1.3±0.4% vs. 2.4±0.6%, respectively). Consequently, we decided to use IL-15 for clinical manufacturing. Optimization of NAM concentration studies showed similar expansion with 2.5 and 5 mM and a decrease in expansion with 7.5 mM NAM. Since NAM at 5 mM had a stronger impact on CD62L expression and on the release of IFNγ and TNFα than NAM at 2.5 mM, we selected 5mM NAM for clinical manufacturing. Overall median NK expansion after two weeks in closed G-Rex flasks supplemented with IL-15 and 5mM NAM was 50-fold (range 37-87). An additional and significant increase in expansion was obtained after doubling the culture medium one week post seeding. While there was a marked advantage for single culture feeding, more feedings had less impact on NK expansion and had a negative effect on the in vivo retention potential. Our optimized expansion protocol therefore involved one feeding during the two weeks expansion duration resulting in 162±30.7-fold expansion of NK cells relative to their input number in culture. Based on these data, we have initiated a clinical trial at University of Minnesota, to test the safety and efficacy of escalating doses (2 x 107/kg - 2 x 108/kg) of our novel NAM NK cell product in patients with refractory non-Hodgkins lymphoma and multiple myeloma (NCT03019666). Disclosures Peled: Gamida Cell: Employment, Equity Ownership. Brachya: Gamida Cell: Employment. Persi: Gamida Cell: Employment. Lador: gamida Cell: Employment, Equity Ownership. Olesinski: gamida cell: Employment. Landau: gamida cell: Employment, Equity Ownership. Galamidi: gamida cell: Employment. Peled: Biokine: Consultancy; Biosight: Consultancy. Miller: Celegene: Consultancy; Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Bachanova: Oxis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Zymogen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle-Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Haploidentical Mbil-21 Ex Vivo Expanded NK Cells (FC21-NK) for Patients with Multiple Relapsed and Refractory Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-12
Author(s):  
Stefan O. Ciurea ◽  
Jolie Schafer ◽  
Piyanuch Kongtim ◽  
Julianne Chen ◽  
Doris Soebbing ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Median Number ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Nk Cell Therapy

Background: Allogeneic stem-cell transplantation (alloSCT) remains the only curative treatment for patients with advanced AML. However, only a minority of these patients achieve disease control prior to transplantation. Natural Killer (NK) cells have potent anti-leukemic activity but are functionally deficient in AML. Adoptive NK-cell therapy using high-doses of functionally active NK-cells could overcome these limitations. We previously developed an ex vivo NK-cell expansion method based on K562 feeder cells modified to express membrane bound IL-21 (mbIL-21) and 4-1BB ligand, (FC21), which resulted in high numbers of hyperfunctional FC21-NK cells with enhanced cytotoxicity and cytokine production. Here we report outcomes of a phase I clinical trial designed to assess the safety, feasibility and maximum tolerated dose (MTD) of haploidentical FC21-NK cells for patients with relapse/refractory (R/R) AML at MD Anderson Cancer Center. Methods: Eligible patients were ≥18 years, KPS ≥70 with good organ function. Patients with relapsed AML after alloSCT were eligible if they had no active GVHD and did not require immunosuppression. Haploidentical donors were selected based on KIR characteristics, when multiple donors were available. Donor NK cells were expanded over 3 weeks and cryopreserved. Three dose levels between 106-108 cells/kg were planned. Patients received cytoreductive chemotherapy with fludarabine 30 mg/m2/day and cytarabine 2 g/m2/day for 5 days (4 days for age >60) and G-CSF (subsequently eliminated). 3-7 days after chemotherapy, patients received FC21-NK cell infusions 3 times per week, up to 6 infusions. Results: As of 4/14/2020, 15 patients were screened, 12 of whom were eligible and received the FC21-NK cells. Median age was 60 years (range 25-70); 6 (50%) had adverse cytogenetics, 8 (66.7%) had adverse ELN genetic risk, 6 (50%) had primary induction failure, 2 (16.7%) had CNS disease and 4 (33.3%) had secondary AML. Median number of prior treatment regimens was 5 (range 2-8), median blast count at enrollment was 47% (range 7-88). Median time from diagnosis to enrollment and to first NK-cell infusion was 16.6 (range 2.5-98.1) and 17.2 (range 3.1-98.6) months, respectively. Donor-recipient NK-cell alloreactivity was seen in 5 patients (41.7%). Median number of NK-cell infusion was 6 (range 3-6); 8 (66.7%) and 4 (33.3%) patients received NK-cell dose of 1 X106 and 1 X107 cells/kg, respectively. MTD was not reached. Seven patients had ANC recovery post-NK cell infusion with cumulative incidence (CI) of ANC recovery to 500/mm3 at 60 days of 58.3%. Eight patients (66.7%) achieved complete remission (CR) (N=4, 33.3%) or CR with incomplete hematologic recovery (CRi) (N=4, 33.3%) at 30 days post-NK cell infusion. One patient with CR had negative minimal residual disease (MRD). Five patients (41.7%) proceeded to haploidentical alloSCT from the same donor and were transplanted in CR/CRi, all but one with persistent MRD. With a median follow-up of 13 months (range 4.1-42.7), median OS and DFS were 17.6 and 3.3 months, and 28 and 20 months for patients receiving alloSCT, respectively. Other outcomes including 2-year OS, DFS, relapse and TRM are shown in Figure 1 and Table 1. No infusion related toxicity or cytokine release syndrome was observed. Two patients were evaluable for FC21-NK cell persistence with haplotype-specific anti-HLA antibodies. FC21-NK cells were detected 5 and 6 weeks after the last FC21-NK cell infusion, respectively. A progressive decrease of the blast population with progressive expansion of the FC21-NK cell population after repeated NK-cell infusions was noted in samples collected from one pt (Figure 2). Persistence is also being evaluated by STR chimerism. Conclusions: Multiple infusions of FC21-NK cells yielded unprecedented outcomes with 66.7% of patients responding and approximately half proceeding to alloSCT in a heavily pre-treated, ultra-refractory, high-risk patient population. Responses were observed irrespective of dose. FC21-NK cell therapy was very well tolerated with no attributable AEs and were shown to persist for at least 5 weeks after infusion. These encouraging results warrant further clinical evaluation of FC21-NK cells in R/R AML patients. Disclosures Ciurea: Kiadis Pharma: Current equity holder in publicly-traded company, Research Funding. Schafer:Kiadis Pharma: Current Employment. Shpall:Zelluna: Membership on an entity's Board of Directors or advisory committees; Adaptimmune: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Magenta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Licensing Agreement. Konopleva:Calithera: Research Funding; Eli Lilly: Research Funding; Kisoji: Consultancy; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Forty-Seven: Consultancy, Research Funding; Sanofi: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding; Ablynx: Research Funding; AbbVie: Consultancy, Research Funding; Ascentage: Research Funding; Rafael Pharmaceutical: Research Funding; Cellectis: Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding. Lee:Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Champlin:Actinium: Consultancy; Johnson and Johnson: Consultancy; Omeros: Consultancy; DKMS America: Membership on an entity's Board of Directors or advisory committees; Cytonus: Consultancy; Genzyme: Speakers Bureau; Takeda: Patents & Royalties.

scholarly journals A Phase I Clinical Trial Testing the Safety of IL-21-Expanded, Universally Alloreactive Donor-Derived Natural Killer Cells for Relapsed/Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1732-1732
Author(s):  
Sumithira Vasu ◽  
Nidhi Sharma ◽  
Alison R. Walker ◽  
Sarah A Wall ◽  
James S. Blachly ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Nk Cells ◽  
Board Of Directors ◽  
Dose Level ◽  
Research Funding ◽  
Nk Cell ◽  
Third Party ◽  
Advisory Committees ◽  
Refractory Aml

Abstract Background: Natural Killer (NK) cells are cytotoxic lymphocytes that are able to exert an anti-tumor effect in an MHC-I independent manner, but are dysfunctional and reduced in number in patients with leukemia. Several studies have shown therapeutic potential for related donor NK cells expanded and/or activated ex vivo when administered to cancer patients, including patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). However, personalized, donor-derived cell therapies require time for donor identification, manufacturing and product release, resulting in patient attrition. As NK alloreactivity and NKG2C expression play critical roles in mediating anti-tumor effects, we identified 'ideal' NK cell donors (in partnership with "Be the Match Biotherapies") for collection. Human leukocyte antigen (HLA) and Killer immunoglobulin receptor (KIR) genotyping were done to screen and select donors. Following this, a sample was collected from donors to expand NK cells under small-scale conditions. If donor NK cells showed robust expansion, they proceeded with apheresis. To ensure these therapeutic cells would be readily-available, we established a third-party NK cell bank through scalable, affordable mass-production with membrane-bound IL-21 feeder cells (FC21), and cryopreserved large numbers of these NK cells for immediate 'off-the-shelf' administration to recipients. Here, we report our initial experience in a Phase I trial of these off-the-shelf (OTS) alloreactive NK cells as immunotherapy for patients with relapsed/refractory AML and MDS. Methods: Patients were treated at Dose level 1, to allow infusion at a dose of 1 x 10e7 NK cells/ kg. Each dose level had two cohorts, stratified by age. Patients were enrolled into Cohort 1 (patients <60 yrs. received Fludarabine (Flu) 30 mg/m2/day & Cytarabine 2 g/m2/day on days -6 to -2) and Cohort 2 (patients ≥ 60 yrs. received Flu 30 mg/m2/day (days -5 to -2) & Decitabine 20 mg/m2/day (days -6 to -2). Patients were evaluated to see if they had any donor-directed antibodies. After chemotherapy, NK cells were infused thrice weekly for 6 doses, to be given over a period of 2 weeks (Clinicaltrials.gov: NCT04220684). Persistence of donor NK cells was determined by flow cytometry using haplotype-discriminating anti-HLA antibodies (Figure 1, 2). NK cells were completely HLA-mismatched between donor and recipients. Results: 6 patients (3/cohort) were treated at the first dose level of 1x10 7 NK cells/kg/dose. All 6 patients had relapsed/refractory AML and had received at least two prior lines of treatment. One patient (005) withdrew from the study prior to infusion of NK cells. 6 patients received the first NK cell dose as an inpatient and were discharged to receive the remaining 5 doses as an outpatient. 5 patients tolerated infusion of all 6 doses of NK cells administered over 2 weeks. One patient (006) developed a Cytokine response (CRS) - like syndrome in the context of streptococcus bacteremia and received only one dose of NK cells. Symptoms suggestive of CRS responded fully with steroids and patient was able to successfully wean off steroids and discharged to the outpatient setting. One patient proceeded to allogeneic hematopoietic cell transplant. No infusion-related reactions, neurotoxicity or graft versus host disease was observed. In vivo persistence/expansion NK cells were superior with FLU/CY lymphodepletion (cohort 1). Conclusions: Cryopreserved, third-party donor-derived NK cells are safe and feasible in relapsed/refractory AML patients, and even at this first dose level, completely HLA-mismatched NK cells can persist and expand to high levels in vivo. Figure 1 Figure 1. Disclosures Vasu: Kiadis, Inc.: Research Funding; Seattle Genetics: Other: travel support; Boehringer Ingelheim: Other: Travel support; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Walker: Newave: Other: clinical trial support; Geron: Other: clinical trial support; Novartis: Other: clinical trial support. Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. de Lima: Incyte: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec: Research Funding. Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company.

scholarly journals Engineered Memory-like NK Cars Targeting a Neoepitope Derived from Intracellular NPM1c Exhibit Potent Activity and Specificity Against Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-4
Author(s):  
Han Dong ◽  
Guozhu Xie ◽  
Yong Liang ◽  
James Dongjoo Ham ◽  
Juliana Vergara ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Nk Cell ◽  
Leukemia Cells ◽  
Tumor Resistance ◽  
Cell Therapies ◽  
Car T

Introduction: Acute myeloid leukemia (AML) continues to be a major therapeutic challenge. There is an emerging need to develop less toxic and more effective targeted therapies. Natural Killer (NK) cells possess many of the key attributes critical for effective cancer therapies- "born to kill" but without apparent risk of graft versus host disease, cytokine release syndrome, or neurotoxicity. Furthermore, their intrinsic propensity to target myeloid blasts makes them particularly attractive for AML. Despite promising clinical results in blood cancer, the development of NK cell-based therapy remains challenging mostly due to NK cells' short lifespan, inadequate proliferation and lack of specific tumor targeting. Here, we utilized a new approach to arm NK cells for adoptive immunotherapy based on innate cell memory. Chimeric antigen receptors (CARs) significantly enhance anti-tumor specificity and activity of immune effector cells. Our innovative CAR-NK cells target a tumor-specific neoepitope in AML and harness potent function pathways in their design to enhance efficacy and minimize toxicity. Methods: 1. Mutated NPM1c as a CAR Target in AML. Most CAR-T cell therapies target tumor-associated antigens (TAAs), which could lead to on-target/off-tumor toxicity as well as tumor resistance. One way to overcome these drawbacks is to target tumor-specific oncogenic driver mutations. The four-nucleotide duplication in nucleophosmin, referred to as NPM1c, is a driver oncogene mutation in about 35% of AML. The mutation creates a neoepitope that is presented by the most common HLA-A2 allele. Using yeast surface display, we have isolated a human single-chain variable fragment (scFv) that specifically binds to the NPM1c epitope-HLA-A2 complex, but not HLA-A2 alone or HLA-A2 loaded with control peptides. 2. Cytokine-Induced Memory-Like (CIML) NK Cells as a CAR Platform. CIML NK cells can provide a unique platform for development of NK cell CARs based on the favorable safety profile, increased proliferation, prolonged persistence and enhanced anti-leukemia function that we have observed in pre-clinical models (Romee et al, Blood 2012) and in patients (Romee et al, Science Trans Med 2016) treated with un-modified CIML NK cells. 3. Efficient Gene Editing in Primary NK Cells. We have overcome the transduction block in primary human and mouse NK cells by utilizing an unconventional pseudotyped lentivirus based on a unique protein with high expression on CIML NK cells. Results: 1. Engineered CAR-T cells with the isolated scFv exhibit potent cytotoxicity both in vitro and in vivo against NPM1c+HLA-A2+ leukemia cells (OCI-AML3) and primary AML blasts, but not NPM1c-HLA-A2+ leukemia cells (OCI-AML2) or HLA-A2- tumor cells (PC-3). 2. The in vivo anti-leukemia efficacy of anti-NPM1c CAR-T cells was however transient (overall survival extended from 28 to 42 days, median survival extended from 21 to 37 days, compared with the control mice adoptively transferred with untraduced T cells), with unneglectable toxicity. 3. Utilizing an unconventional pseudotyped lentivirus to transduce CIML NK cells from healthy donor blood (n = 5 donors), we have successfully generated anti-NPM1c CAR-NK cells with high transduction efficiency (using MOI = 10: transduction rate mean 48%, range 32% to 65%; compared with 2%, range 0.8% to 4.5% for the conventional approach with VSVG pseudotyped lentivirus). 4. Harnessing key cytokine pathways in the CAR design substantially promoted CAR-NK cell survival (indicated by the enhanced cell viability from 29.7% to 75.2%) and proliferation (marked by the increased levels of ki-67 from 60.2% to 94.5%). 5. Anti-NPM1c CAR significantly promoted anti-tumor function (represented by CD107a, IFN-gamma) and tumor-specific killing (measured by annexin V and 7-AAD) of CIML NK cells against AML with NPM1c oncogene (OCI-AML3). 6. Dual-armed CIML NK cells with CAR and cytokine signaling exhibited optimal specificity and sustainability against AML targets. Conclusion: These results demonstrate that the innovative CAR-CIML NK cells could be developed as an efficient cellular immunotherapy for treating NPM1c+HLA-A2+ AML with potentially reduced on-target/off-tumor toxicity and tumor resistance. Our study should drive novel conception and design of CAR-NK cell therapies against myeloid malignancies in the clinic. Figure Disclosures Ritz: Rheos Medicines: Consultancy; LifeVault Bio: Consultancy; Infinity Pharmaceuticals: Consultancy; Falcon Therapeutics: Consultancy; Avrobio: Consultancy; Kite Pharma: Research Funding; Equillium: Research Funding; Amgen: Research Funding; Talaris Therapeutics: Consultancy; TScan Therapeutics: Consultancy.

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

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 777-777
Author(s):  
Veronika Bachanova ◽  
David H. McKenna ◽  
Xianghua Luo ◽  
Todd E. DeFor ◽  
Fiona He ◽  
...  
Keyword(s):  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Phase 1 ◽  
Large Cell ◽  
Clinical Activity ◽  
Advisory Committees

Background: NK cells have the capacity to kill tumor targets, representing a novel immunotherapeutic approach to cancer. We have shown promising clinical activity in AML with a previous NK cell preparation. Limitations of NK therapies have included specificity, persistence after infusion, and potential for maximal activity of NK cells in vivo. GDA-201 is a cellular product composed of natural killer (NK) cells from healthy donors expanded ex vivo with nicotinamide (NAM) and IL-15; this is a unique ex vivo activation strategy to induce persistence of potent anti-tumor activity. Prior in vitro studies and pre-clinical models demonstrated that NAM-exposed NK cells exhibited augmented resistance against exhaustion and improved killing function, proliferation, and organ retention. We now report safety and efficacy from a phase 1 clinical trial of GDA-201 in patients (pts) with relapsed or refractory (R/R) NHL or MM. 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 CD 20-positive NHL or refractory MM received cyclophosphamide (400mg/m2 IV x 3d) and fludarabine (30 mg/m2 /d IV x 3d), followed by two doses of GDA-201 (Days 0 and 2) and low-dose IL-2 (6 million units sc). Pts with NHL or MM received rituximab (375 mg/m2 x 4 weekly or elotuzumab (10 mg/kg x 3 weekly), respectively, to enhance NK cell targeting through antibody-dependent cellular cytotoxicity (ADCC). Results: 20 pts were enrolled: 7 with NHL (4 follicular, 2 transformed, 1 diffuse large cell lymphoma) and 13 with MM, in 3 cohorts of escalating GDA-201 dose; 11 pts received the maximum target dose (median 1.7 x 108 cells/kg, range 1.6-2.0 x 108 cells/kg). There were no dose limiting toxicities. The most common grade 3/4 adverse events were neutropenia and thrombocytopenia, febrile neutropenia (n=2), increased creatinine, hyponatremia, pulmonary edema; all events were transient. One pt had grade 2 cytokine release syndrome at day 18, presenting with fever, hypoxemia and hypotension, responding to tocilizumab; pt later died of E Coli sepsis. There were no neurotoxic events, GVHD or marrow aplasia. Among 7 NHL pts, there were 3 CR and 2 PR with overall response rate of 71%. Median duration of response was 12 months (CR patients) and 5 months (PR patients). Figure 1A illustrates a 57-year-old man with history of CLL and Richter's transformation (large cell lymphoma), pre- GDA-201 and 6 months post therapy; the pt had continued response with 80% tumor shrinkage at 6 months. In MM patients, 1 patient with extramedullary disease had CR and 4 had SD with median duration 2.5 months. In our previous study using overnight-activated NK cells, persistence 7 days after adoptive transfer was limited. Using GDA-201, flow cytometry confirmed persistence of donor NAM-NK in peripheral blood up to day 7-10 (day 7 range 2-55% donor NK cells; Figure 1B), as well as enhanced in vivo proliferation (median Ki67 99%). Conclusions: Cellular therapy using GDA-201 with monoclonal antibodies was safe, and demonstrated early evidence of clinical activity in heavily pre-treated pts with advanced NHL and MM. The recommended dose of GDA-201 for phase 2 is 2.0 x 108 cells/kg. The clinical responses showed that NK cell targeting through ADCC can be efficacious and increase response. Laboratory studies showed that GDA-201 had better persistence than observed in our previous studies using overnight activated cytokine alone stimulated NK cells. This study demonstrated that GDA-201 has an efficacy signal, and larger phase II studies are warranted. Disclosures Bachanova: Incyte: Research Funding; Gamida Cell: Research Funding; Novartis: Research Funding; GT Biopharma: Research Funding; Celgene: Research Funding; Kite: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. McKenna:Fate Therapeutics: Research Funding; Magenta Therapeutics: Research Funding; CIBMTR BMT CTN (NIH): Other: Medical Monitor; Icahn School of Medicine, New York, New York: Consultancy; National Eye Institute (NIH): Other: DSMB (2); Gamida: Research Funding; NMDP: Other: Donor and Patient Safety Monitoring Advisory Group; Intima: Patents & Royalties: Royalities, Research Funding. Brachya:Gamida Cell: Employment, Equity Ownership. Peled:Gamida Cell: Employment, Equity Ownership. Miller: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; 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; Fate Therapeutics, Inc: Consultancy, Research Funding; OnKImmune: Membership on an entity's Board of Directors or advisory committees.

Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 580-580
Author(s):  
Mark Wunderlich ◽  
Mahesh Shrestha ◽  
Lin Kang ◽  
Eric Law ◽  
Vladimir Jankovic ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Employment Equity ◽  
Cell Product ◽  
Equity Ownership ◽  
Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

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