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.

Development of a Human Therapeutic L-Cyst(e)Ine-Degrading Enzyme for the Treatment of Hematological Malignancies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1587-1587
Author(s):  
Giulia Agnello ◽  
Susan Alters ◽  
Joseph Tyler ◽  
Jinyun Liu ◽  
Peng Huang ◽  
...  
Keyword(s):  
Research Funding ◽  
Hematological Malignancies ◽  
Ex Vivo ◽  
Redox Balance ◽  
Lymphocytic Leukemia ◽  
Employment Equity ◽  
Equity Ownership ◽  
Antioxidant Mechanisms

Abstract Cancer cells experience higher intrinsic oxidative stress than their normal counterparts and acquire adaptive antioxidant mechanisms to maintain redox balance. This increased antioxidant capacity has been correlated to malignant transformation, metastasis and resistance to standard anticancer drugs. This enhanced antioxidant state also correlates with cancer cells being more vulnerable to additional oxidative insults, therefore disruption of adaptive antioxidant mechanisms may have significant therapeutic implications. Hematological malignancies including Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM) are critically dependent on the cellular antioxidant glutathione (GSH), consistent with the higher intrinsic oxidative stress. L-cysteine is the rate-limiting substrate for GSH biosynthesis and adequate levels of cysteine are critical to maintain the intracellular homeostasis of GSH. CLL and a subset of ALL cells have been reported to rely on the stromal supply of cysteine to increase the synthesis of GSH in order to maintain redox balance, which in turn promotes cell survival and fosters drug resistance. One approach to target this cancer specific dependency is by therapeutic depletion of amino acids via enzyme administration; a clinically validated strategy for the treatment of ALL. Aeglea BioTherapeutics Inc. has developed a bioengineered cysteine and cystine degrading enzyme (Cyst(e)inase, AEB3103) and evaluated its therapeutic efficacy against hematological malignancies in in vitro, ex vivo and in vivo pre-clinical studies. The TCL1-TG:p53 -/- mouse model exhibits a drug resistant phenotype resembling human CLL with unfavorable cytogenetic alterations and highly aggressive disease progression. AEB3103 greatly decreased the viability of TCL1-TG:p53 -/- cells cultured in vitro, whereas the CLL therapeutic, fludarabine, showed minimal cytotoxic effects. In vivo treatment of TCL1-TG:p53 -/- mice with AEB3103 resulted in an increase in median survival time (7 months, p<0.0001) compared to the untreated control group (3.5 months, p<0.001) and a fludarabine treated group (5.3 months, p<0.001). These results indicate a superior therapeutic effect of AEB3103 compared to fludarabine. Additionally, evaluation of AEB3103 in in vitro 2D cultures of patient-derived CLL and MM cells, and in ex vivo 3D cultures of cells derived from ALL and AML PDx models resulted in significant cell growth inhibition with therapeutically relevant IC50 values. Collectively these results demonstrate the sensitivity of hematological malignancies to modulation of GSH levels via AEB3103-mediated cyst(e)ine depletion. Disclosures Agnello: Aeglea BioTherapeutics: Employment. Alters:Aeglea BioTherapeutics: Employment, Equity Ownership. Tyler:Aeglea BioTherapeutics: Employment, Equity Ownership. Huang:Aeglea BioTherapeutics: Research Funding. Stone:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Research Funding; University of Texas at Austin: Employment, Patents & Royalties: I am an inventor of technology related to this abstract. Georgiou:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Lowe:Aeglea BioTherapeutics: Employment, Equity Ownership. Rowlinson:Aeglea BioTherapeutics: Employment, Equity Ownership.

scholarly journals Cord Blood Derived Natural Killer Cells Loaded with a Tetravalent Bispecific Antibody Construct (AFM13) As Off-the-Shelf Cell Therapy for CD30+ Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 341-341
Author(s):  
Lucila Kerbauy ◽  
Mecit Kaplan ◽  
Pinaki P Banerjee ◽  
Francesca Lorraine Wei Inng Lim ◽  
Ana Karen Nunes Cortes ◽  
...  
Keyword(s):  
T Cell ◽  
Cord Blood ◽  
Nk Cells ◽  
Natural Killer ◽  
Research Funding ◽  
Bispecific Antibody ◽  
Nk Cell ◽  
Ex Vivo

Abstract Chimeric antigen receptors to redirect T cell specificity against tumor antigens have shown remarkable clinical responses against CD19+ malignancies. However, the manufacture of an engineered autologous T cell product is expensive and cumbersome. Natural killer (NK) cells provide an alternative source of immune effectors for the treatment of cancer. NK cell cytolytic function can be directed towards specific targets by exploiting their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) through the NK cell Fc receptor, CD16 (FcγRIIIa). AFM13 is a tetravalent bispecific antibody construct based on Affimed's ROCK™ platform. AFM13 is bispecific for CD30 and CD16A, designed for the treatment of CD30 expressing malignancies. It binds CD16A on the surface of NK cells, thus activating and recruiting them to CD30 expressing tumor cells and mediating subsequent tumor cell killing. Since autologous NK effector function is impaired in many patients with malignancies, we propose to overcome this by the use of allogeneic NK cells in combination with AFM13. Cord blood (CB) is a readily available ("off-the-shelf") source of allogeneic NK cells that can be expanded to large, highly functional therapeutic doses. The feasibility and safety of therapy with allogeneic ex vivo expanded CB-derived NK cells have been shown by our group and others. In this study, we hypothesized that we can redirect the specificity of NK cells against CD30+ malignancies by preloading ex vivo activated and expanded CB-derived NK cells with AFM13 prior to adoptive infusion. Briefly, mononuclear cells were isolated from fresh or frozen CB units by ficoll density gradient centrifugation. CD56+ NK cells were cultured with rhIL-12, rhIL-18 and rhIL-15 for 16 hrs, followed by ex vivo expansion with rhIL-2 and irradiated (100 Gy) K562-based feeder cells expressing membrane-bound IL-21 and CD137-ligand (2:1 feeder cell:NK ratio). After 14 days, NK cells were loaded with serial dilutions of AFM13 (0.1, 1, 10 and 100 mg/ml). After washing twice with PBS, we tested the effector function of AFM13-loaded NK-cells (AFM13-NK) compared to expanded CB-NK cells without AFM13 against Karpas-299 (CD30 positive) and Daudi (CD30 negative) lymphoma cell lines by 51Cr release and intracellular cytokine production assays. AFM13-NK cells killed Karpas-299 cells more effectively at all effector:target ratios tested than unloaded NK cells (Figure 1) and produced statistically more INFγ and CD107a (P=0.0034; P=0.0031 respectively, n=4). In contrast, AFM13-NK cells and unloaded NK cells exerted similar cytotoxicity against Daudi cells. Next, we established the optimal concentration of AFM13 for loading (determined to be 100 μg/ml) and the optimal incubation time to obtain maximal activity (1 h) in a series of in vitro experiments. We also confirmed that the activity of AFM13-NK cells against Karpas-299 cells remains stable for at least 72h post-wash (Figure 2). Additionally, we characterized the phenotype of AFM13-NK vs. unloaded NK cells by flow cytometry using monoclonal antibodies against 22 markers, including markers of activation, inhibitory receptors, exhaustion markers and transcription factors. Compared to unloaded NK cells, AFM13-NK cells expressed higher levels of CD25, CD69, TRAIL, NKp44, granzyme B and CD57, consistent with an activated phenotype. We next tested the in vivo anti-tumor efficacy of AFM13-NK cells in an immunodeficient mouse model of FFluc-Karpas-299. Briefly, six groups of NOD/SCID/IL2Rγc null mice (n=5 per group) were transplanted by tail-vein injection with 1 x 10e5 FFluc-transduced Karpas cells. Group 1 and 6 received tumor alone or tumor + AFM13 and served as a control. Groups 2-4 receive Karpas FFLuc with either expanded NK cells or AFM13-NK cells (NK cells loaded with AFM13) or expanded NK cells and AFM13 injected separately. Group 5 received AFM13-NK cells without tumor. Initial studies confirm the antitumor activity of AFM13-NK cells. In summary, we have developed a novel premixed product, comprised of expanded CB-NK cells loaded with AFM13 to 'redirect' their specificity against CD30+ malignancies. The encouraging in vitro and in vivo data observed in this study, provide a strong rationale for a clinical trial to test the strategy of an off-the-shelf adoptive immunotherapy with AFM13-loaded CB-NK cells in patients with relapsed/refractory CD30+ malignancies. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

scholarly journals Eomes and T-Bet Expression Are Required By Mature Primary Human NK Cells for Anti-Leukemia Responses In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 194-194
Author(s):  
Pamela Wong ◽  
Carly C. Neal ◽  
Lily Chang ◽  
Julia A Wagner ◽  
Melissa M. Berrien-Elliott ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
T Box ◽  
Nsg Mice ◽  
Healthy Donors ◽  
And Function

Abstract Natural Killer (NK) cells are innate lymphoid cells that respond to hematologic cancers via cytotoxicity (perforin/granzyme and death receptors) and cytokine/chemokine production, yet the molecular determinants underlying their proliferation, function, and persistence are poorly understood. There are promising reports of pre-clinical and clinical NK cell responses to leukemia and lymphoma, which represent a nascent cellular therapy for these blood cancers. The T-box transcription factors (TFs) Eomes and T-bet are expressed by NK cells throughout their lifespan, and are required for development as evidenced by NK cell loss in Eomes and T-bet deficient mice. However, the roles of these TFs in mature human NK cell molecular programs and functions remain unclear. We hypothesized Eomes and T-bet, which are the only T-box TFs expressed in NK cells, are critical regulators of NK cell homeostasis and functionality, and are necessary for proper mature NK cell responses. To address this, we utilized the CRISPR-Cas9 system to genetically delete both Eomes and T-bet in primary human NK cells isolated from healthy donors, and investigated their role beyond guiding NK cell development, specifically in the anti-leukemia response. Gene-editing of primary human NK cells has been technically challenging, thus most reports that modified NK cells were performed with cell lines, in vitro-differentiated, or highly expanded NK cells that likely do not reflect primary human NK cell biology. Here, we introduced Cas9 mRNA and sgRNA targeting T-bet and Eomes by electroporation into unexpanded primary human NK cells isolated from healthy donors using the MaxCyte GT system. We observed highly efficient reductions of Eomes and T-bet protein expression, quantified by flow cytometry (p &lt; 0.0001, Fig A-B) without viability differences between control (sgRNA targeting TRAC, an unexpressed locus in NK cells), and Eomes/T-bet double CRISPR-edited (DKO) cells after one week in vitro. To study Eomes and T-bet in NK cell anti-leukemia response, control or DKO primary human NK cells were engrafted into NSG mice, supported with human IL-15, and challenged with K562 leukemia cells. Utilizing bioluminescent imaging to visualize leukemia burden, we observed that NK cells lacking both TFs were unable to suppress leukemia growth in vivo. To understand the mechanism responsible for impaired leukemia control, we investigated in vivo persistence and proliferation, cytotoxic effector molecule expression, as well as ex vivo degranulation and cytokine production of DKO NK cells compared to control NK cells. DKO or control human NK cells were transferred into NSG mice and supported with human IL-15. After 2-3 weeks, significantly fewer (&lt;30%) DKO NK cells persisted compared to control NK cells: spleen (5-fold decrease, control 240e3±65e3 vs DKO 47e3±15e3 NK cells, p&lt;0.01, Figure C), blood (6-fold decrease, p&lt;0.01), and liver (4-fold decrease, p&lt;0.05). Using intracellular flow cytometry, double T-bet/Eomes CRISPR-edited NK cells that lacked both Eomes and T-bet protein after in vivo transfer were identified. A proliferative defect was evident in flow-gated DKO (62±6% undivided), compared to unedited (WT) NK cells (4±2% undivided) assessed by CellTrace Violet dilution (Figure D). In addition, there were marked reductions in granzyme B and perforin protein (p&lt;0.001) in flow-gated DKO NK cells compared to controls. To assess DKO NK cell functional capacity, we performed an ex vivo functional assay on NK cells from spleens of the NSG mice as effectors, and K562 targets or IL-12/15/18 stimulation for 6 hours. Degranulation to K562 targets was impaired (p&lt;0.05), and IFN-γ production was reduced (p&lt;0.0001) after cytokine stimulation in flow-gated DKO NK cells (Figure E). Thus, CRISPR-editing of unexpanded, primary human NK cells revealed that Eomes and T-bet are required by mature human NK cells for their function and homeostasis, distinct from their role in development. This is translationally relevant, as defects in proliferation and function of human DKO NK cells manifested markedly reduced response against human leukemia cells in vivo in xenografts. These findings expand our understanding of key molecular regulators of mature NK cell homeostasis and function, with the potential to provide new avenues to enhance NK cell therapy. Figure 1 Figure 1. Disclosures Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Foltz-Stringfellow: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Fehniger: HCW Biologics: Research Funding; Compass Therapeutics: Research Funding; Affimed: Research Funding; ImmunityBio: Research Funding; Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; Kiadis: Other; OrcaBio: Other; Indapta: Other.

Evaluating the Antitumor Activity of MLN9708 in a Disseminated Mouse Model of Double Transgenic iMyc Ca/Bcl-XL Plasma Cell Malignancy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3835-3835 ◽  
Author(s):  
Michael Fitzgerald ◽  
Yueying Cao ◽  
Bret Bannerman ◽  
Zhi Li ◽  
Olga Tayber ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Mouse Model ◽  
Lymph Nodes ◽  
Plasma Cell ◽  
Research Funding ◽  
Ex Vivo ◽  
Employment Equity ◽  
Equity Ownership

Abstract Abstract 3835 Poster Board III-771 Introduction The first generation proteasome inhibitor VELCADE® (bortezomib) is indicated for the treatment of patients with multiple myeloma (MM), a form of plasma cell malignancy (PCM). MLN9708 is our novel proteasome inhibitor that selectively and reversibly binds to, and potently inhibits the b5 site of the 20s proteasome in preclinical studies. We have recently demonstrated that MLN9708 significantly prolongs tumor-free survival of double transgenic iMycCa/Bcl-XL mice, a genetically-engineered mouse model of de novo PCM. Here we describe the in vivo evaluation of cell lines derived from double transgenic iMycCa/Bcl-XL mice and the antitumor activity of MLN9708 in a disseminated mouse model of iMycCa/Bcl-XL PCM. Materials MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. Double transgenic iMycCa/Bcl-XL mice develop de novo PCM, in which neoplastic plasma cell development is driven by the targeted expression of the oncoprotein Myc and anti-apoptotic Bcl-XL (J. Clin. Invest. 113:1763-1773, 2004). DP54 and DP42 are plasma cell tumor cell lines isolated from the bone marrow and lymph nodes, respectively, of syngeneic mice previously inoculated with iMycCa/Bcl-XL tumors (Cancer Res. 67:4069-4078, 2007). In vitro, DP54 and DP42 cells express both the Myc and Bcl-XL transgenes, various plasma cell and B-cell markers including CD38, CD138 and B220, and have gene expression profiles very similar to human MM. Methods Cell viability studies were performed to determine the antiproliferative effects of MLN2238 in DP54 and DP42 cells in vitro. To evaluate DP54 and DP42 cells in vivo, these cells were aseptically inoculated into the tail vein of NOD-SCID mice. Progressions of the resultant PCM were monitored and tumor burdens were evaluated by magnetic resonance imaging (MRI), ex vivo mCT imaging, and histopathology. Mouse plasma samples were collected at the end of the studies and levels of immunoglobulin were assessed. To establish a preclinical disseminated mouse model of iMycCa/Bcl-XL PCM, freshly dissociated DP54-Luc cells (constitutively expressing firefly luciferase under a mouse Ig-k promoter) were aseptically inoculated into the tail vein of NOD-SCID mice. Once tumor growth has been established, mice were randomized into treatment groups and then treated with vehicle, bortezomib (at 0.7mg/kg intravenously [IV] twice weekly [BIW]) or MLN2238 (at 11 mg/kg IV BIW) for 3 consecutive weeks. Tumor burden was measured by bioluminescent imaging. Results In vitro, both DP54 and DP42 cells were sensitive to MLN2238 treatment (LD50 values of 14 and 25 nM, respectively). In vivo, NOD-SCID mice rapidly succumbed to PCM after being inoculated with DP54 and DP42 cells (25 and 14 days post-inoculation, respectively), where the disease was accompanied by marked elevation of plasma immunoglobulins. MRI scans revealed the presence of multiple lesions and several abnormalities were found including: cranial deformation, bowel distortion, splenomegaly and renal edema. Tumor infiltrates, ranging from minor to extensive, were identified in multiple organ compartments (brain<kidney<liver<lymph nodes<spleen<bone marrow) by histopathological analysis. Ex vivo mCT imaging has also revealed signs of bone erosion in the cranial sagittal sutures. Dissemination of DP54-Luc cells after tail vein inoculations was detected by in vivo bioluminescent and confirmed by ex vivo imaging where luminescent tumor nodules were identified in the spleen, kidneys, liver, intestine, lymph nodes, spinal bone and cranium. To assess the antitumor activity of MLN2238, an efficacy study was performed using the DP54-Luc disseminated model. Tumor burden (bioluminescence), skeletal malformation (mCT) and overall survival after treatment with bortezomib and MLN2238 will be presented. Conclusion The DP54-Luc disseminated mouse model of double transgenic iMycCa/Bcl-XL PCM recapitulated several key features of human MM and provided real-time assessment of novel MM therapy preclinically. MLN9708 is currently in human clinical development for both hematological and solid tumor indications. Disclosures: Cao: Milllennium: Employment, Equity Ownership. Bannerman:Milllennium: Employment. Li:Milllennium: Employment. Bradley:Milllennium: Employment, Equity Ownership, Research Funding. Silverman:Milllennium: Employment. Janz:Milllennium: Research Funding. Van Ness:Milllennium: Research Funding. Kupperman:Milllennium: Employment. Manfredi:Milllennium: Employment. Lee:Milllennium: Employment, Equity Ownership.

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.

Nicotinamide, a Form of Vitamin B3, Promotes Expansion of Natural Killer Cells That Display Increased In Vivo Survival and Cytotoxic Activity,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4035-4035 ◽  
Author(s):  
Gabi M Frei ◽  
Nurit Persi ◽  
Chana Lador ◽  
Amnon Peled ◽  
Yael C Cohen ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Vitamin B3 ◽  
Employment Equity ◽  
Post Infusion ◽  
Equity Ownership ◽  
Cells Cultured

Abstract Abstract 4035 NK cells are cytotoxic lymphocytes that have drawn considerable attention in recent years as a promising tool for immunotherapy in patients with various refractory hematological malignancies and metastatic solid tumors. Clinical results of experimental protocols have shown only a partial response attributed mainly to the relatively low number of NK cells infused and their short in vivo persistence. An important challenge, therefore, in advancing the clinical applicability of NK cells is to expand ex vivo NK cells that display increased functionality upon in vivo infusion. In efforts to induce NK cell expansion, different combinations of cytokines have been studied. However, most reports show a modest expansion and demonstrate a need for additional stimuli. Nicotinamide (NAM) is a form of Vitamin B3 and a potent inhibitor of enzymes that use NAD for their activity. Hence, NAM is directly involved in the control of redox sensitive enzymes, mitochondrial functions, cell metabolism, the production of energy, and cell motility. Here we show that NAM (2.5–5 mM) enhances expansion (60-80 fold) of functional NK cells in feeder-free cultures stimulated with IL-2 and IL-15 for two weeks. This effect was observed in cultures initiated with purified CD56+ (CD56 enriched/CD3 depleted) or with CD3 depleted, peripheral blood and cord blood cells. Immunophenotyping of the cultured NK cells has so far revealed that NAM substantially modulates three cell surface receptors. CD200R and programmed death receptor-1 (PD-1) expressed on NK cells interact with their ligands on tumor cells which leads to a suppression in NK cell anti-tumor activity and tumor immunoevasion. These two receptors are down-regulated by NAM. CD62L (L-selectin) defines an NK subset with increased self-renewal capacity and its expression was reported to be pivotal for NK cells trafficking to lymphoid organs and their homeostatic proliferation. Following expansion in culture with IL-2, CD62L is down-regulated, whilst NAM increased its expression with a dose-dependent effect. Using a CFSE-based cytotoxicity assay we have demonstrated that NK cells cultured with NAM display higher cytotoxic activity against K562, BL2, NK-resistant COLO 205 cell lines and primary leukemia cells. In a Transwell migration assay, NK cells cultured with NAM demonstrated increased migration towards the CXCR4 ligand SDF-1. To test in vivo homing and retention, irradiated (350 RAD) NOD/SCID mice were transplanted with a similar number of cells (15–20×106 /mouse) derived from two week cultures treated with or without NAM. Mice were infused with 50μg/mouse IL-2 and 5μg/mouse IL-15 every other day. To test homing, mice were sacrificed 24 hour post infusion. Number of human NK cells (CD45+CD56+) detected in the spleen and BM were significantly (p< 0.05) higher in the cohort of mice infused with NK cells cultured with NAM (7.9 and 1.39 respectively) compared to mice infused with NK cells cultured without NAM (4.13 and 0, respectively). In a different set of experiments, persistence of human NK cells was analyzed 4 and 12 days post infusion. Four days post infusion, the percentage of human NK cells in the spleen, BM, lung and liver were substantially higher in mice infused with NK cells cultured with NAM compared to mice infused with NK cells cultured without NAM (Fig 1). Even though 12 days post infusion, a decrease in the number of human NK cells was observed in comparison to day 4, still cell retention in the spleen, liver and lung was significantly greater in the cohort infused with NK cells cultured with NAM (13.45, 0.6, 9.21% Vs. 1.26, 0.12, 2.85%, (p<0.05), respectively). The calculated decrease in the number of human NK cells from day 4 to 12 was 50% less in the NAM cohort, suggesting enhanced in vivo survival of NK cells cultured with NAM.Table 1:In vivo persistence of ex vivo expanded NK cellsTable 1:. In vivo persistence of ex vivo expanded NK cells In conclusion, expansion of NK cells with NAM was found to increase in vivo homing and survival and to augment tumor cytotoxic effect of NK cells. This suggests a potential for enhancing the clinical efficacy of adoptively transferred NK cells. Based on these intriguing findings we are developing a cell product for adoptive cell-mediated immune therapy. Disclosures: Frei: Gamida Cell: Employment. Persi:Gamida Cell: Employment. Lador:Gamida Cell: Employment, Equity Ownership. Peled:Gamida Cell: Consultancy. Nagler:Gamida Cell: Consultancy. Peled:Gamida Cell: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Lenalidomide Exhibits Activity in Mantle Cell Lymphoma through Increased NK Cell Mediated Cytotoxicity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 821-821 ◽  
Author(s):  
Patrick Hagner ◽  
Hsiling Chiu ◽  
Maria Ortiz-Estevez ◽  
Tsvetan Biyukov ◽  
Carrie Brachman ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Nk Cell ◽  
Employment Equity ◽  
Mantle Cell ◽  
Apoptosis Assay ◽  
Equity Ownership

Abstract Introduction: Lenalidomide (Len) is indicated for the treatment of relapsed/refractory (R/R) Mantle Cell Lymphoma (MCL) in the United States and Switzerland. Len binds to the cullin 4 ring E3 ubiquitin ligase complex resulting in ubiquitination and subsequent proteasomal degradation of lymphoid transcription factors Aiolos and Ikaros leading to stimulation of immune cells, such as T-cells. Clinical trial CC-5013-MCL-002 (NCT00875667) is a randomized open-label phase II study in R/R MCL patients in which Len was given orally at 25 mg/day on days 1-21 of each 28-day cycle until progression (N=170). The control arm consisted of investigator choice of single-agent rituximab, gemcitabine, fludarabine, chlorambucil, or cytarabine (N=84). We explored the immune effects of Len treatment in MCL patients enrolled in CC-5013-MCL-002 and further investigated our findings in in vitro MCL co-culture models. Methods: Peripheral blood samples for exploratory analysis were collected at Cycle 1 Day 1 (C1D1, pre-treatment), Cycle 1 Day 4 (C1D4), Cycle 2 Day 15 (C2D15) and at treatment discontinuation. Flow cytometric profiling of T, B and natural killer (NK) cell subsets was performed and differences were analyzed for correlation with clinical outcomes (response rate and progression free survival [PFS]). Cell dependent cytotoxicity was measured in 1) anti-CD3 stimulated peripheral blood mononuclear cells (PBMC) treated with vehicle or 1-10000 nM Len for 3 days and incubated with target tumor cells for an additional 4 hours followed by an apoptosis assay as measured by Annexin V/ToPro-3 flow cytometry and 2) negatively selected CD56+ NK cells stimulated with IL-2 and treated with Len (1 nM to 10 μM) for 18 hrs and incubated with target tumor cells for an additional 4 hours followed by apoptosis assay. Results: At baseline, no significant differences were observed in the absolute levels of immune subsets when comparing non-responders (NR) and responders (R) in either Len (NR=11, R=23) or control (NR=4, R=5) arms. However, in the Len arm, significantly elevated (adj. p < 0.05) proportions of CD3-CD56+CD16+ NK cells (difference of means = 8.73; 95%CI [4.48, 12.98]) were observed at C1D4 compared to baseline in the R (N=19) outcome sub-group compared to NR (N=11). A similar trend in levels of NK subsets was observed at C2D15, however the difference was not significant. In addition, elevated proportions of CD3-CD56+CD16+ NK cells (p≤0.016) at C1D4 relative to total lymphocytes correlated significantly to longer PFS in the Len arm. Immune subset analysis in the control arm did not show any correlation to response or PFS at any visit. The mechanism whereby NK cell modulation contributes to clinical benefit demonstrated by Len in patients was further explored in in vitro co-culture systems with MCL cell lines. Len treated PBMC co-cultured with Jeko-1, Granta-519, and Mino MCL cell lines resulted in 38-47.5% more apoptosis compared to DMSO (p≤0.001). We examined the effect of Len on Aiolos and Ikaros protein expression in CD56+ NK and CD3+ T cells within anti-CD3 antibody stimulated PBMCs treated with DMSO or various concentrations of Len (1 nM to 10 μM) for 72 hours. Degradation of both Aiolos (40%) and Ikaros (95%) was observed after drug treatment in CD56+ NK cells. Aiolos and Ikaros levels were also monitored in CD3+ T cells and showed decreased levels after Len treatment, consistent with previous reports (Gandhi, 2014; Kronke, 2014). Furthermore, purified CD56+ NK cell mediated cytotoxicity produced a similar pro-apoptotic effect as the PBMC assay in all MCL cell lines versus DMSO (p≤0.01). Supernatants from co-cultures of NK cells with MCL cell lines showed significantly elevated granzyme B levels as compared to DMSO controls (p≤0.0001), suggesting that the apoptotic effects observed are induced by granzyme B. Conclusions: Lenalidomide is an immune modulating agent and NK cell modulation in particular may play a role in its clinical activity in MCL. A significant increase in proportions of NK cell subsets (vs total lymphocytes) at C1D4 versus baseline was observed and is a potential response indicator of favorable clinical outcome in R/R MCL patients treated with Len. In vitro, Len enhances cell mediated cytotoxicity of MCL cell lines in two co-culture model systems. Understanding NK cell mediated mechanism(s) has potential to enhance guiding patient selection strategies and rational combination therapies of lenalidomide in MCL. Disclosures Hagner: Celgene: Employment, Equity Ownership. Chiu:Celgene: Employment, Equity Ownership. Ortiz-Estevez:Celgene: Employment, Equity Ownership. Biyukov:Celgene: Employment, Equity Ownership. Brachman:Celgene: Employment, Equity Ownership. Trneny:Celgene: Consultancy, Honoraria, Other: Travel, accommodations, expenses, Research Funding. Morschhauser:Genentech Inc./Roche: Other: Advisory boards. Stilgenbauer:AbbVie, Amgen, Boehringer-Ingelheim, Celgene, Genentech, Genzyme, Gilead, GSK, Janssen, Mundipharma, Novartis, Pharmacyclics, Roche: Consultancy, Honoraria, Research Funding. Milpied:Celgene: Honoraria, Research Funding. Musto:Sandoz: Consultancy; Celgene: Honoraria; Roche: Honoraria; Sanofi: Consultancy; Genzyme: Consultancy; Novartis: Honoraria; Janssen: Honoraria; Mundipharma: Honoraria. Martinelli:AMGEN: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; ROCHE: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy. Heise:Celgene: Employment, Equity Ownership. Daniel:Celgene: Employment, Equity Ownership. Chopra:Celgene: Employment, Equity Ownership. Carmichael:Celgene: Employment, Equity Ownership. Trotter:Celgene Corporation: Employment. Gandhi:Celgene: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership.

scholarly journals Lirilumab Enhances Anti-Tumor Efficacy of Elotuzumab

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4711-4711 ◽  
Author(s):  
Caroline Sola ◽  
Mathieu Blery ◽  
Cécile Bonnafous ◽  
Elodie Bonnet ◽  
Nicolas Fuseri ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Nk Cells ◽  
Tumor Cells ◽  
Therapeutic Efficacy ◽  
Nk Cell ◽  
Employment Equity ◽  
Equity Ownership ◽  
Checkpoint Receptors

Abstract Tumor cells that express reduced levels of Major Histocompatibility Complex (MHC) class I molecules may be recognized and killed by Natural Killer cells (NK cells), through a process known as “missing self” recognition. In humans, this is controlled by inhibitory receptors such as Killer Immunoglobulin-like Receptors (KIR) that recognize Human Leukocyte Antigen (HLA)-A, -B or –C. Engagement of KIR by HLA molecules results in inhibitory signaling that reduces NK cell-mediated natural killing and antibody-dependent cellular cytotoxicity (ADCC). Hence, antibodies that block interactions between inhibitory KIR and their HLA ligands are being evaluated as an anti-cancer therapeutic strategy. The anti-KIR2DL1/2/3-specific monoclonal antibody, lirilumab (BMS-986015 / IPH2102), is a fully human IgG4 that blocks binding of KIR to HLA-C, and is being developed for treating hematologic malignancies and solid tumors. Elotuzumab (BMS901608 / HuLuc63) is a humanized IgG1 anti-SLAMF7 (signaling lymphocyte activation molecule family member 7, CS-1) being developed for the treatment of Multiple Myeloma (MM). SLAMF7 is a cell surface glycoprotein highly expressed in myelomatous cells and only at low levels on normal cells. NK cell-mediated ADCC is one of the main mechanisms of action of elotuzumab, but ADCC is negatively regulated by KIR checkpoint receptors. Thus a combination of lirilumab and elotuzumab has strong scientific rationale. The aim of the present study was to assess whether lirilumab would enhance elotuzumab anti-MM activity in vitro with human peripheral blood NK cells and MM cell lines, and in vivo in a newly developed xenogenic mouse model. Two MM cell lines (OPM-2 and U266B1) were identified that express both HLA-C and SLAMF7. These MM cells were capable of activating peripheral blood NK cells from healthy donors in vitro, as assessed by three different endpoints (CD107 mobilization on NK cells surface and intracellular production of the cytokines IFN-g and TNF- a); each of these responses were significantly enhanced, in a dose-dependent manner, by both lirilumab and elotuzumab independently. Moreover, the elotuzumab-mediated functional activation of KIR2D+ NK cells could be further enhanced by the addition of increasing doses of lirilumab. The best combinatorial effect was observed in response to MM cells expressing low densities of SLAMF7. These data suggest that lirilumab treatment may increase the therapeutic efficacy of elotuzumab, particularly in MM patients with low SLAMF7 expression. In these experiments, it was not possible to clearly identify the impact of Fc receptor genotype or HLA-C genotype on the NK cell responses. To assess the therapeutic efficacy of lirilumab and elotuzumab in vivo, we generated a novel strain of double-transgenic mice expressing human KIR2DL3 as well as its ligand, HLA-cw3, on a Rag1-/- background (KIR-cw3-tgRAG mice), to allow engraftment of human MM tumor cells expressing SLAMF7. The OPM-2 MM cell line was subcutaneously engrafted in these mice and when high tumor volumes were reached, mice were treated with lirilumab, elotuzumab or a combination of both. As monotherapy, each of monoclonal antibody had some therapeutic effect while the combination of both resulted in a significantly stronger anti-tumor effect and increased survival of the mice. Median survival of mice treated with huIgG control was 38 days, 41 days with lirilumab, 42 days with elotuzumab and 51 days with both mAbs in combination (10 mice per group). In conclusion, we demonstrate that blockade of KIR checkpoint receptors with lirilumab was able to augment elotuzumab mediated ADCC in vitro and synergized with elotuzumab to mediate potent anti-MM activity in vivo. Taken together, these data provide a rationale for clinical trials to test combination treatment of lirilumab and elotuzumab in MM patients. Disclosures Sola: InnatePharma: Employment, Equity Ownership. Blery:Innate Pharma: Employment, Equity Ownership. Bonnafous:Innate Pharma: Employment, Equity Ownership. Bonnet:Innate Pharma: Employment, Equity Ownership. Fuseri:Innate Pharma: Employment, Equity Ownership. Graziano:Bristol-Myers Squibb: Employment; Bristol-Myers Squibb: Equity Ownership. Morel:Innate Pharma: Employment, Equity Ownership. André:Innate Pharma: Employment, Equity Ownership.

scholarly journals Development and Scale-up of a Novel GMP Method for Enrichment and Expansion of Terminally Differentiated Adaptive Natural Killer Cells (FATE-NK100) with Enhanced Anti-Tumor Function

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1225-1225 ◽  
Author(s):  
Frank Cichocki ◽  
Bahram Valamehr ◽  
Dhifaf Sarhan ◽  
Bin Zhang ◽  
Sarah Cooley ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Nk Cells ◽  
Cell Expansion ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Vehicle Control ◽  
Employment Equity ◽  
Nk Cell Differentiation ◽  
Equity Ownership

Abstract Natural killer (NK) cells are innate lymphoid cells that mediate immune responses against pathogens and cancer. Human NK cells are distinguished by the surface phenotype CD3-CD56+ and differential expression of the CD56 surface antigen defines subsets. CD56bright NK cells are presumed to be precursors of CD56dim NK cells, and terminal maturation of CD56dim NK cells is associated with acquisition of CD57. Rather than being an immunosenescence marker CD57 acquisition represents a shift toward greater effector function, including increased CD16 signaling (Fc receptor responsible for triggering antibody-dependent cellular cytotoxicity), enhanced cytotoxicity and decreased responsiveness to interleukin (IL)-12 and IL-18 stimulation. Cytomegalovirus (CMV) infection is uniquely associated with expansion of CD57+ NK cells expressing the activating receptor NKG2C.We have reported that in vivo expanded of CD57+NKG2C+ NK cells (referred to as adaptive NK cells) persist for over one year and are directly associated with reduced leukemia relapse after reduced intensity hematopoietic cell transplantation. Ex vivo expansion to enrich the subset of cells with the adaptive NK cell phenotype represents a new strategy to obtain high numbers of NK cells with enhanced effector function for use in adoptive transfer to treat cancer patients. The main challenge in enriching for CD57+ NK cells using current ex vivo expansion protocols is that IL-15, the cytokine that drives NK cell proliferation and is critical for NK cell survival preferentially expands less mature NK subsets that fail to terminally differentiate in culture. Our group has developed a novel NK cell expansion method that overcomes this barrier. Peripheral blood mononuclear cells from CMV seropositive donors are depleted of CD3+ T cells and CD19+ B cells and cultured for 7-9 days with IL-15 and a small molecule inhibitor of glycogen synthase kinase 3-beta (GSK3β), a multifunctional kinase downstream of the PI(3)K pathway. Compared to vehicle control, addition of the GSK3β inhibitor led to a substantial increase (2.2-fold ± 0.19, n=23, p<0.0001) in the CD57+ NK cell population. NK cells were highly enriched (90.9% ± 2.2) relative to the pre-cultured population post CD3/CD19 depletion (23.3% ± 2.5) (p<0.0001) Sorting experiments where purified NK cell subsets were cultured with monocytes obtained from the same donor and either vehicle or GSK3β inhibitor clearly demonstrated that the GSK3β inhibitor enhanced NK cell maturation. Importantly, NK cells expanded in the presence of the GSK3β inhibitor exhibited enhanced interferon (IFN)-γ production relative to the vehicle control in response to leukemia cells in vitro (2.15-fold ± 0.60, n=7, p=0.0002)and were superior in their ability to control tumor growth out to at least one month in a xenogeneic adoptive transfer model. To determine whether inhibition of GSK3β acted directly or indirectly on NK cells to drive terminal differentiation, purified CD56+ NK cells were cultured with or without monocytes in the presence or absence of the GSK3β inhibitor.The presence of differentiated monocytes was required for effective terminal NK cell differentiation, suggesting a monocyte-dependent, indirect effect of GSK3β (Figure 1A). Monocytes cultured with the GSK3β inhibitor exhibited markedly higher surface expression of an array of markers associated with monocyte maturation including HLA-DR, IL-15Rα, CD80, CD83 and CCR7 (Figure 1B). Thus, our data demonstrate that efficient NK cell differentiation is dependent upon the maturation state of the co-cultured monocytes.We have scaled our process to manufacture a GMP product (referred to as FATE-NK100) for clinical use. Using a representative apheresis product from a CMV seropositive donor containing 19.8 x 108 CD57+ NK cells and 1.7 x 108 CD57+NKG2C+ adaptive NK cells, we achieved 6.4-fold NK cell expansion resulting in a final GMP-grade product containing 142.2 x 108 CD57+ NK cells and 15.8 x 108 CD57+NKG2C+ adaptive NK cells.The cytotoxicity of activity of these ex vivo expanded adaptive NK cells in response to tumor targets is superior that of CD3/CD19-depleted haploidentical NK cells activated overnight with either IL-2 or IL-15 in the NK products in current clinical trials.These data have been presented to the FDA in preparation for a clinical trial of FATE-NK100 in lymphodepleted patients with advanced AML anticipated for Q1 2017. Disclosures Cichocki: Fate Therapeutics, Inc: Research Funding. Valamehr:Fate Therapeutics, Inc: Employment. Cooley:Fate Therapeutics: Research Funding. Bjordahl:Fate Therapeutics, Inc: Employment. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Rogers:Fate Therapeutics, Inc: Employment. Green:Fate Therapeutics, Inc: Employment. McKenna:Fate Therapeutics, Inc: Research Funding. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Wolchko:Fate Therapeutics: Employment. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

scholarly journals Combining Immunocytokine and Ex Vivo Activated NK Cells as a Platform for Enhancing Graft-Versus-Tumor Effects Against GD2+ Murine Neuroblastoma

2021 ◽  
Vol 12 ◽  
Author(s):  
Paul D. Bates ◽  
Alexander L. Rakhmilevich ◽  
Monica M. Cho ◽  
Myriam N. Bouchlaka ◽  
Seema L. Rao ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Tnf Α

Management for high-risk neuroblastoma (NBL) has included autologous hematopoietic stem cell transplant (HSCT) and anti-GD2 immunotherapy, but survival remains around 50%. The aim of this study was to determine if allogeneic HSCT could serve as a platform for inducing a graft-versus-tumor (GVT) effect against NBL with combination immunocytokine and NK cells in a murine model. Lethally irradiated C57BL/6 (B6) x A/J recipients were transplanted with B6 bone marrow on Day +0. On day +10, allogeneic HSCT recipients were challenged with NXS2, a GD2+ NBL. On days +14-16, mice were treated with the anti-GD2 immunocytokine hu14.18-IL2. In select groups, hu14.18-IL2 was combined with infusions of B6 NK cells activated with IL-15/IL-15Rα and CD137L ex vivo. Allogeneic HSCT alone was insufficient to control NXS2 tumor growth, but the addition of hu14.18-IL2 controlled tumor growth and improved survival. Adoptive transfer of ex vivo CD137L/IL-15/IL-15Rα activated NK cells with or without hu14.18-IL2 exacerbated lethality. CD137L/IL-15/IL-15Rα activated NK cells showed enhanced cytotoxicity and produced high levels of TNF-α in vitro, but induced cytokine release syndrome (CRS) in vivo. Infusing Perforin-/- CD137L/IL-15/IL-15Rα activated NK cells had no impact on GVT, whereas TNF-α-/- CD137L/IL-15/IL-15Rα activated NK cells improved GVT by decreasing peripheral effector cell subsets while preserving tumor-infiltrating lymphocytes. Depletion of Ly49H+ NK cells also improved GVT. Using allogeneic HSCT for NBL is a viable platform for immunocytokines and ex vivo activated NK cell infusions, but must be balanced with induction of CRS. Regulation of TNFα or activating NK subsets may be needed to improve GVT effects.

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