CB-derived Natural Killer Cells Research Articles

Ex Vivo Expanded Cord Blood Natural Killer Cells Combined with Rituximab and High-Dose Chemotherapy and Autologous Stem Cell Transplantation for B Cell Non-Hodgkin Lymphoma

Relapse is the major cause of failure of high-dose chemotherapy (HDC) with autologous stem cell transplantation (ASCT) for B cell non-Hodgkin lymphomas (B-NHL). Improvement strategies include use in combination with effective immunotherapies. We hypothesized that the combination of rituximab/HDC/ASCT with expanded cord blood (CB)-derived natural killer (NK) cells is safe and active in B-NHL. Patients with B-NHL age 15 to 70 years and appropriate ASCT candidates were eligible for the study. The CB units were selected without considering HLA match with the recipient. The CB NK cells were expanded from day -19 to day -5. Treatment included rituximab on days -13 and -7, BEAM (carmustine/etoposide/cytarabine/melphalan) on days -13 to -7, lenalidomide on days -7 to -2, CB NK infusion (108/kg) on day -5, and ASCT (day 0). The primary endpoint was 30-day treatment-related mortality (TRM); secondary endpoints included relapse-free survival (RFS), overall survival (OS), and persistence of CB NK cells. We enrolled 20 patients. CB NK cells were expanded a median of 1552-fold with >98% purity and >96% viability. We saw no adverse events attributable to the CB NK cells and 0% 30-day TRM. At median follow-up of 47 months, the RFS and OS rates were 53% and 74%, respectively. CB NK cells were detectable in blood for 2 weeks, independent of HLA-mismatch status. CD16 expression in donor NK cells was correlated favorably with outcome, and homozygosity for the high-affinity CD16 variant (158 V/V) in CB, but not recipient, NK cells was correlated with better outcomes. Our data indicate that the combination of expanded and highly purified CB-derived NK cells with HDC/ASCT for B-NHL is safe. CD16 expression in donor NK cells, particularly if homozygous for the high-affinity CD16 variant, was correlated with better outcomes.

Innate Cell Engager (ICE®) AFM13 Combined with Preactivated and Expanded (P+E) Cord Blood (CB)-Derived Natural Killer (NK) Cells for Patients with Refractory CD30-Positive Lymphomas: Final Results

CONCLUSIONS Pts with refractory Hodgkin (HL) and other CD30+ lymphomas have few effective therapies available and targeted NK cell immunotherapy is an active area of research. Transfer of non-targeted NK cells has shown limited clinical benefit as target recognition of cancer cells by NK cells constitutes a barrier. The innate cell engager AFM13 is a first-in-class CD30/CD16A bispecific antibody construct that induces selective killing of CD30+ tumor cells by engaging and activating NK cells. As a single agent, AFM13 has limited clinical activity against HL, likely due to the reduced effector function of autologous NK cells in these pts, which provides the rationale for combining AFM13 with allogeneic NK cells. Our prior preclinical work identified a promising combination of CB-derived NK cells that were first IL-12/IL-15/IL-18-preactivated followed by ex vivo expansion (P+E) with engineered K562 feeder cells expressing membrane-bound IL-21, 4-1BBL and CD48 and exogenous IL-2, and subsequently complexed with AFM13 just before infusion; these cytokine-induced memory-like NK cells presented chimeric antigen receptor (CAR)-NK-like features and increased in vitro and in vivo antitumor activity compared to either non-AFM13-precomplexed P+E NK cells or to AFM13 alone (Kerbauy et al, Clin Cancer Res 2021). Building on this work we wished to study this CB NK-cell therapy in pts with refractory CD30+ lymphomas. This investigator-initiated single-center phase I-II trial (NCT04074746) evaluated the safety and activity of P+E AFM13-precomplexed CB NK cells followed by systemic AFM13. Pts ages 15-75 with CD30+ lymphomas refractory to brentuximab vedotin were enrolled. The goals were to identify the recommended phase 2 dose (RP2D) of NK cells, estimate the overall (ORR) and complete response (CR), event-free (EFS) and overall survival (OS) rates, and study NK cell activation and persistence. Each treatment cycle consisted of fludarabine/cyclophosphamide (days −5 to −3) followed by infusion (day 0) of the AFM13-precomplexed CB NK cells, cultured for 14 days as described above, and three weekly IV infusions of AFM13 (200 mg, days 7, 14 and 21). Two cycles were planned for pts 1-19 and up to 4 cycles from patient 20 on. Response was evaluated on day 28 of each cycle. Enrollment proceeded at 3 dose levels (DL) of 10 6, 10 7 and 10 8 NK/Kg, respectively. Each patient received the same NK dose in all cycles. Between 09/20-12/22 we enrolled 42 pts (37 HL, 5 NHL), pretreated with a median 7 lines, at DL1 (N=3), DL2 (N=3) and DL3 (N=36), with 1 (N=2), 2 (N=21), 3 (N=3) or 4 cycles (N=16). All had active progressive disease at enrollment and no bridging therapy was given. The cords used for each of the 117 cycles were selected without consideration for HLA match, which was 0/6 (N=53), 1/6 (N=46), 2/6 (N=14), 3/6 (N=3) and 4/6 (N=1). The product infused consisted of >94% NK cells (expanded >2,700 fold), with only 0.02% T cells. The NK cells were 96% viable, 91% bound to AFM13, and 97.6% CD16+. The products were infused fresh. There was 1 grade 2 infusion-related reaction (IRR) in 115 infusions of AFM13-NK (0.8%) and 27 IRR infusion related reactions in 350 infusions of AFM13 (7.7%) (1 G3, 26 G2). We saw no cases of CRS, ICANS or GVHD of any grade. DL3 (10 8 NK/Kg) was established as the RP2D. The ORR and CR were 92.8% and 66.7%, respectively (94.4% and 72.2%, respectively, in 36 pts treated at the RP2D). Nine pts had a response consolidated with SCT (5 allo, 4 auto). At median follow-up of 14 (6-34) months, the EFS/OS rates are 31%/76%; median EFS/OS are 8 months/not reached. No relapses were associated with antigen loss. In the subset of pts planned for 4 cycles at RP2D, 15 of 23 pts remained event free at 6 mo (4 pts with and 11 without SCT consolidation), for 6-month EFS/OS rates of 65%/83%. Overall, 8 of the 9 pts (89%) receiving a consolidation SCT are event free at 10 to 34 months. AFM-bound CB NK cells were detected in blood from day 1 post infusion for up to 3 weeks. Donor NK levels followed similar patterns after each cycle, which argues against a sensitization after the first NK infusion. CyTOF studies showed increased expression of AFM13 and activation markers in donor NK cells. We confirmed trafficking of AFM13-bound donor NK cells to tumor sites shortly after infusion. CB-derived cytokine-induced memory-like NK cells precomplexed with AFM13 have excellent tolerability and activity for pts with heavily pretreated and refractory CD30+ lymphoma.

Innate Cell Engager AFM13 Combined with Preactivated and Expanded Cord Blood-Derived NK Cells for Patients with Double Refractory CD30+ Lymphoma

Patients with multiple relapsed or refractory (R/R) Hodgkin lymphoma (HL) and other CD30+ lymphoma have few effective therapies available and targeted adoptive natural killer (NK) cell immunotherapy is an active area of investigation. Transfer of non-targeted NK cells has shown limited clinical benefit as target recognition of cancers by NK cells remains a substantial barrier. The innate cell engager (ICE®) construct, AFM13, is a first-in-class CD30/CD16A bispecific antibody construct that induces specific and selective killing of CD30+ tumor cells by engaging and activating NK cells. As a single agent, AFM13 has limited clinical activity against HL, likely due to the reduced effector function of autologous NK cells in these patients, which provides the rationale for the combination of AFM13 with allogeneic NK cells. Our prior preclinical work identified a promising combination of cord blood (CB)-derived NK cells that were first IL-12/IL-15/IL-18-preactivated followed by expansion (P+E) with engineered K562 feeder cells expressing membrane-bound IL-21, 4-1BB ligand and CD48 ex vivo, and subsequently complexed with AFM13 just prior to infusion (Kerbauy et al, Clin Cancer Res 2021; 27:3744-56). These cytokine-induced memory-like CB-derived NK cells precomplexed with AFM13 (AFM13-NK) presented chimeric antigen receptor (CAR)-NK cell-like features and showed an increased in vitro and in vivo antitumor activity compared to either P+E NK or AFM13 alone. Building on this work we wished to clinically study adoptive AFM13-NK cell therapy in pts with R/R CD30+ lymphomas. METHODS: This single-center phase I-II trial (NCT04074746) evaluates the safety and activity of AFM13-NK, followed by AFM13 monotherapy. Patients aged 15-75 years with R/R CD30+ lymphomas refractory to brentuximab vedotin are enrolled. The trial goals are to identify the recommended phase 2 dose (RP2D) of AFM13-NK cells, estimate the overall (ORR) and complete response (CR) rates, event-free (EFS) and overall survival (OS) rates, and studies of NK cell activation and persistence. Each treatment cycle consisted of fludarabine/cyclophosphamide (days −5 to −3), followed by infusion of the AFM13-NK cells, cultured for 14 days as described above (day 0), and three weekly IV infusions of AFM13 (200 mg, days 7, 14 and 21). Two cycles were administered to patients 1-19 and 4 cycles were given starting with patient 20. Response was evaluated on day 28 of each cycle. Patients were enrolled at 3 dose levels: DL1 (106 NK/Kg), DL2 (107NK/Kg) and DL3 (108 NK/Kg). Each patient received the same NK dose in all cycles. RESULTS: As of 7/31/22, 30 pts have been enrolled and treated (Table) at DL1 (N=3), DL2 (N=3) and DL3 (N=24), with 1 (N=1), 2 (N=24), 3 (N=3) or 4 cycles (N=3). All patients had active progressive disease at enrollment and no bridging therapy was given. The cords used for each of the 69 cycles were selected with no consideration for HLA match, which was 0/6 (N=31), 1/6 (N=31), 2/6 (N=5), 3/6 (N=1) and 4/6 (N=1). The product infused consisted of >99% NK cells (expanded >2,700 fold), with only 0.02% T cells. The NK cells were >98% viable and 92% of them were bound to AFM13. The products were infused fresh. There were no infusion-related reactions (IRR) after infusing AFM13-NK and 11 IRR in 182 infusions of AFM13 alone (6%) (1 grade 3, 10 grade 2). We saw no cases of CRS, ICANS or GVHD of any grade. DL3 (108 NK/Kg) was established as the RP2D. The ORR in the entire study is 97% with 63% CRs. All 24 patients treated at the RP2D responded (100% ORR) with 70.8% (17/24) CRs. Five patients had a response consolidated with a SCT. At median follow-up of 8 (range 1-23) months, the EFS and OS rates of all 30 patients are 57% and 83%, respectively. AFM-NK cells were detected in serum from day 1 post infusion and persisted for 3-4 weeks. Donor NK levels followed similar patterns after each cycle, which argues against a sensitization effect in the patient after the first NK infusion. Donor NK cells showed by CyTOF increased binding to AFM13 and expression of activation markers. CONCLUSIONS: This is the first clinical trial to date using an ICE® construct precomplexed with cytokine-induced memory-like CB-NK cells to treat patients with CD30+ R/R HL and NHL. Our preliminary results indicate that this ICE® precomplexed CB-NK cell therapy has an excellent tolerability profile and is highly active in patients with heavily pretreated R/R CD30+ lymphomas and warrants further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparable transforming growth factor beta-mediated immune suppression in ex vivo-expanded natural killer cells from cord blood and peripheral blood: implications for adoptive immunotherapy

T cell-based therapies like genetically modified immune cells expressing chimeric antigen receptors have shown robust anti-cancer activity in vivo, especially in patients with blood cancers. However, extending this approach to an “off-the-shelf” setting can be challenging, as allogeneic T cells carry a significant risk of graft-versus-host disease (GVHD). By contrast, allogeneic natural killer (NK) cells recognize malignant cells without the need for prior antigen exposure and have been used safely in multiple cancer settings without the risk of GVHD. However, similar to T cells, NK cell function is negatively impacted by tumor-induced transforming growth factor beta (TGF-β) secretion, which is a ubiquitous and potent immunosuppressive mechanism employed by most malignancies. Allogeneic NK cells for adoptive immunotherapy can be sourced from peripheral blood (PB) or cord blood (CB), and the authors’ group and others have previously shown that ex vivo expansion and gene engineering can overcome CB-derived NK cells’ functional immaturity and poor cytolytic activity, including in the presence of exogenous TGF-β. However, a direct comparison of the effects of TGF-β-mediated immune suppression on ex vivo-expanded CB- versus PB-derived NK cell therapy products has not previously been performed. Here the authors show that PB- and CB-derived NK cells have distinctive gene signatures that can be overcome by ex vivo expansion. Additionally, exposure to exogenous TGF-β results in an upregulation of inhibitory receptors on NK cells, a novel immunosuppressive mechanism not previously described. Finally, the authors provide functional and genetic evidence that both PB- and CB-derived NK cells are equivalently susceptible to TGF-β-mediated immune suppression. The authors believe these results provide important mechanistic insights to consider when using ex vivo-expanded, TGF-β-resistant PB- or CB-derived NK cells as novel immunotherapy agents for cancer.

A Multi-Omics Approach for Evaluating the Impact of Cytokines and Donor Source on NK Cell Expansion

IntroductionAs the field of cancer immunotherapy progresses, natural killer (NK) cells have become an ideal candidate for targeting multiple types of cancer. These cells are able to selectively target virally infected cells and tumor cells without damaging the healthy cells of the immune system making them an ideal candidate for treatment in AML, CML, MM, Neuroblastoma, Breast Cancer, and Renal Cell Carcinoma. While NK cells have shown their potential as immunotherapy agents, one of the biggest challenges includes expanding and harvesting enough cells for multiple transfusions when there are limited numbers of starting cells. Our group developed an NK cell expansion platform utilizing membrane bound-IL21 (mbIL-21), which allows for sustained proliferation in order to generate high numbers of cells. Continued research supported that mbIL-21 enables sustained NK cell proliferation compared to mbIL-15 due to mbIL-21 activation through STAT3 signaling. Recent shifts to umbilical cord blood have led to research using cord blood (CB) derived NK cells. However, this presents a challenge as a viable clinical option as others have shown that the expansion of these CB-derived NK cells have been shown to produce less potent NK cells. Here, we will use feeder cells expressing either mbIL15 or mbIL21 to expand NK cells (as is currently done in clinical trials), to deeply compare and contrast the impact of these two cytokines on NK cell biology and simultaneously compare and contrast the use of peripheral-derived NK cells and CB-derived NK cells.MethodsNK cells were obtained from healthy donor leukopacks (n =4) and isolated using RosetteSep human NK cell enrichment cocktail. These samples were stimulated with either mbIL-21 or mbIL-15 irradiated feeder cells (IFC) and cultured in AIM-V media supplemented with 100 I/U of IL-2 and immune cell serum replacement (ICSR). Cord Blood NK cells were obtained from OrganaBio (n=4) and stimulated with mbIL-21 IFC . The cells were cultured in AIM-V media supplemented with 100 I/U of IL-2 and ICSR (complete media). All NK cell expansions were stimulated at day 0 and day 7, as previously described, and cell cultures were assessed every 2-3 days keep cells at an optimal concentration by supplementing with complete media. Cells were collected at day 14 to capture a picture before mbIL-15 expanded cells became senescent and were immediately used for cytokine production assays, RNA collected, ATAC-seq collection, and CyTOF fixation. The remaining cells were frozen for cytotoxicity assays, mitochondrial function assays, and degranulation assays. For mitochondrial function and cytotoxicity assays, cells were thawed and cultured in complete media for two days before use.ResultsOverall, the peripheral blood NK cells expanded with mbIL21 showed an increase in expansion at day 14 when compared to those expanded with mbIL15. NK cells expanded with mbIL21 demonstrated a more consistent cytotoxicity profile against CHLA-136, a neuroblastoma cell line, compared to mbIL21 expanded NK cells when cultured at a 0.5:1 ratio (E:T). While the results were not statistically significant at 14 days, previous data demonstrates that we see increased changes at 21 days. Of note, we had an overall more reproducible serial killing ability in the IL21 expanded samples compared to the variability observed in IL15-expanded samples. We saw a significant difference (p < 0.0001) in peripheral blood NK cells expanded with mbIL21 compared to cb-NK cells expanded with mbIL21 with a 0.5:1 ratio (E:T) against CHLA136. mbIL-21 expanded peripheral blood NK cells had a higher maximum respiration capacity than those expanded with mbIL-15 when measured under mitochondrial stress via seahorse assay (p= 0.016).ConclusionsOur data showed that the cells began to show a different phenotypic profile at day 14 depending on the type of NK cell and the type of cytokine used for stimulation. By day 14 data supported that mbIL-21 expanded cells can better survive under low glucose conditions such as tumor microenvironments than mbIL15 expanded cells. The cytotoxicity data demonstrated that peripheral derived NK cells have improved serial killing ability than the cb-NK cells and we will continue to test their ability against additional tumor cell lines that have various HLA-typing. To further elucidate these differences, we will use our RNA-seq, ATAC-seq, and CyTOF data to understand what is causing the different profiles. [Display omitted] DisclosuresLee: 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.

129 Development of AB-201, a novel allogeneic anti-HER2-specific CAR-NK cell therapy for the treatment of HER2+ tumors

BackgroundHuman Epidermal Growth Factor Receptor 2 (HER2), is a receptor tyrosine kinase that is highly expressed on the surface of many solid tumors. While many patients derive meaningful benefit from the approved HER2-directed therapies, most will eventually suffer relapse or progression of their disease highlighting the need for additional treatment options. Currently there are no FDA-approved cellular therapies targeting HER2. Over the past decade, however, cellular therapy has been shown to be a viable treatment option in different cancer types. Here we present AB-201, an off-the-shelf, cryopreserved cord blood (CB)-derived HER2 chimeric antigen receptor (CAR)-natural killer (NK) cell therapy as a safe, active, and readily available option for patients with HER2+ solid tumors.MethodsAB-201 is comprised of ex vivo expanded allogeneic CB-derived NK cells that have been genetically modified to express a HER2-directed CAR and presented as a cryopreserved infusion-ready product. The manufacturing process utilizes a feeder-cell line engineered to express factors specifically identified as supportive to NK cell expansion and a lentiviral transduction step to introduce the HER2 CAR construct. In vitro characterization of AB-201 included evaluation of the purity and expression of cell surface markers by flow cytometry and short- (4 hour) and long-term (over 5 days) cytotoxicity assays in the presence of HER2+ tumor cell lines at various effector to target ratios. In addition, AB-201 efficacy was assessed in vivo in established ovarian (intraperitoneal, SKOV-3), breast (intraperitoneal, HCC1954) and gastric (subcutaneous, N87) xenograft models in NSG mice.ResultsHER2 CAR expression was detected in 93.1% of AB-201 cells. AB-201 is 97.9% CD3-/CD56+ cells and 94.6% CD56+/CD16+. Further characterization of AB-201 demonstrated high expression of NK activating receptors such as NKG2D, NKp30, NKp46, and DNAM-1 and expression of the chemokine receptor, CXCR3. AB-201 demonstrated concentration-dependent and HER2 targeted short-term cytotoxic activity and sustained long-term cell killing against the tumor cell lines SKOV-3, HCC1954, and NCI-N87. Efficacy, as evidenced by a significant reduction in bioluminescent signal or tumor volume, was observed in all xenograft models. A significant survival benefit over non-transduced NK cells or trastuzumab controls was demonstrated in the HCC1954 model.ConclusionsData presented herein suggests that AB-201, a highly pure and readily expandable HER2-directed CAR NK cell product, has potential to be an effective therapy in the treatment of HER2+ tumors.Ethics ApprovalThe animal studies were conducted in accordance with an Institutional Animal Care and Use Committee-approved protocol and with the approval of an IACUC committee at each center where the studies took place

KIR Genotype and KIR Ligand Phenotypes That Improve the in Vitro Cord Blood or Autologous NK Cell Mediated Cytotoxicity Against Myeloma Marrow Plasma Cells

<h3>Instruction</h3> Killer Immunoglobulin-like Receptor (KIR) inhibition is known to influence the cytotoxic effects of natural killer (NK) cells against cancer cells. KIRs can be protective against relapse in myeloma (MM) (Gabriel et al Blood 2010, Kröger et al leukemia 2011) by NK cell licencing <i>or through</i> blocking of the inhibitory KIR receptors such as NKG2A (Mahaweni et al, 2018). <h3>Objectives</h3> In this study, the role of KIR geno/phenotypes and their ligands on autologous and cord blood (CB) derived NK cell mediated cytotoxicity against MM plasma cells (PC) were investigated. <h3>Methods</h3> Peripheral blood NK cells and marrow PCs were isolated from 20 patients with relapsed MM. PC were isolated from fresh BM aspirates using EasySep Human CD138 Positive Selection Kit (StemCell Technologies) and were subjected to cytotoxicity assays with either autologous or CB derived NK cells isolated (Human NK cell Enrichment Cocktail, Stemcell Technologies) and cultured in the presence of IL-15 (10ng/ml) for 2-8 days. KIR/KIR ligand genotyping were performed by Olerup SSP KIR Genotyping Kit (Olerup, Stockholm, Sweden) in order to detect KIR genotypes; KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5A/B, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, and KIR ligands; HLA-ABw4+, HLA-BBw4+Thr80, HLA-BBw4+Ile80, HLA-BBw4+Asp77,Thr80, HLA-CAsn80, and HLA-CLys80. Effector cells were stained with antibodies against NKG2A (CD159a) and CD158i (KIR2DS4) and analyzed using a Beckman Coulter Flow Cytometer. <i>Cytotoxicity assays</i>: Using <i>3,3</i> (DiO) as an indicator and 1:10 target:effector cell ratio PC death was evaluated after 120 mins of incubation, using flow cytometry. <h3>Results</h3> 20 MM patients were tested in vitro. Following incubation of PC wo NK cells, spontaneous PC death ratio was found to be 22.0% (11.4%-51.2%). While autologous NK mediated cytotoxicity was median 17.3% (11.4%-43.6%), CB-NK cell mediated cytotoxicity was 23.2% (range: 11.5%-51.2%). Highest cytotoxicity was achieved with CB-NK cells (CB7) with a KIR AB haplotype, carrying 8 inhibitory KIR genes against PC from MM12. Effector cells with the iKIR gene number above 6 were found to cause higher cytotoxicity (13/19; p=0.031). Additionally, higher (>30%) expression of NKG2A on CB-NK cells were found to be associated with lack of cytotoxicity. <h3>Conclusion</h3> CB-derived NK cells were found to be able to induce higher PC death than autologous PB-NK cells. Also higher number of iKIR genotypes and lacking KIR2DS4 receptor was found to be associated with higher cytotoxicity. In our current study we are reporting KIR NKG2A which binds with HLA-E to play an additional role. Allogeneic NK cell mediated immunotherapies are promising due their clinical advantages ie no need for HLA matching. Furthermore certain KIR haplotypes (ie KIR2DS4⁺ or NKG2A⁻) may offer better therapeutic efficacy if confirmed by others as well.

Medulloblastoma rendered susceptible to NK-cell attack by TGFß neutralization

Background & Aim Medulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in patients with high-risk disease, and traditional therapies often cause significant permanent morbidity. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy because they recognize malignant cells without the need for a known target, they are readily available from multiple banks, and they expand to significant numbers. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment, such as Transforming Growth Factor β (TGF-β). Methods, Results & Conclusion To overcome the detrimental effects of TGF-β, we modified CB-derived NK cells to express a dominant negative TGF-β receptor II (DNRII) by retroviral transduction and evaluated their ability to kill a TGF-β-secreting MB cell line (Daoy). We observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence ofmedulloblastoma-conditioned, TGF-β-richmedia (decreasing to 14.9±2.1% killing at an E:T ratio of 5:1 from 25.1±2.9%, n=10, p Hence, CB NK cells expressing a TGF-β DNRII may have a functional advantage over unmodified NK cells in the presence of TGF-β-rich MB, warranting further investigation as a novel therapeutic for the treatment of patients with high-risk medulloblastoma.

KIR 2DS4 May Influence Autologous and Cord Blood(CB) Natural Killer (NK)Cell Mediated in Vitro Cytotoxicity Against Freshly Isolated Human Bone Marrow Myeloma Plasma Cells and Cell Lines

Background and Aim: NK cell effects are mediated by Killer Immunoglobulin-like Receptor (KIR) inhibition. There are few reports on the protective role of KIRs against relapse in myeloma(MM) (Gabriel et al Blood 2010, Kröger et al leukemia 2011). However, which NK cells or for whom will be more effective and the role of individual differences in this mechanism is indefinite. In this study we aimed to accomplish cytotoxicity against both MM cells and human myeloma cell lines (HMCL) and to compare the cytotoxicity achieved with CB versus autologous NK cells as well as to investigate the influence of NK cell KIR geno/phenotypes.Patients and Methods: A total of 18 patients with refractory/relapsed MM were included in this study.Cell culturing: CD138+ PC isolated from fresh bone marrow aspirates of relapsed MM patients or HMCL (U266, RPMI8226 and H929) using RosetteSep™ Human Multiple Myeloma Cell Enrichment Cocktail (StemCell Technologies) were subjected to cytotoxicity assays with CB derived or autologous PB derived NK cells expanded in the presence of IL-15 (10ng/ml) for 2-8 days.Phenotyping: Effector cells (autologous or CB-NK) were analyzed by flow cytometry using CD45, CD3, CD16, CD56, CD158a (KIR2DL1), CD158i (KIR2DS4) and CD158b1/b2 (KIR 2DL2/2DL3) monoclonal antibodies.Cytotoxicity assays:3,3 dioctadecyloxacarbocyanine perchlorate (DiO) dye was used to distinguish target cells (PC) from effector cells (NK). PC and NK cells were mixed at an 1:10 target: effector cell ratio and inoculated with Propidium Iodide (PI) in order to counter stain the dead cells. Naive PC were used as controls. Following 120 mins of incubation, PC death was evaluated using flow cytometry. After incubation of target cells (PC) with effector cells (NK cells), cytotoxic effects were determined by subtraction of NK mediated PC death from spontaneous PC death.Results: Following IL-15 expansion NK cells were found to express the CD16+56+3- phenotype. Eighteen MM patient PCs and six different HMCL samples were used in cytotoxicity assays(Table 1). In vitro PC viability was less than 45% in four samples and could not be analyzed in the study. Following incubation of PC wo NK cells, spontaneous PC death ratio was found to be 22.6% (min-max: 3%-42.3%). Twelve cytotoxicity assays targeting six MM PC were performed with autologous and CB-derived NK cells. Autologous NK and CB NK mediated cytotoxicity median rates were found to be 19.0% (range: 1.4% - 43.6%) and 31.3% (range: 2.4% - 51.2%), respectively. Additionally, CB-NK cells were evaluated against HMCL samples (four U266, one RPMI8226 and one H929). Median cytotoxicity against HMCL was 27.4% (range: 5.6% - 52.1%). Highest cytotoxicity against MM PC (MM-9) and HMCL (H929) were achieved by CB NK cells (51.2% and 52.1 % respectively). There was no cytotoxic effect against PCs of patients MM6 and MM10 (Figure 1-2).Flow-cytometric phenotyping of effector NK cells showed consistency (10/13) with genotypes. Since only one (CB19) with 19% CD158i positivity was PCR negative, a %20 cut-off rate was chosen. Both CD158a (KIR2DL1) and CD158b1/b2 (KIR 2DL2/2DL3) were positive among all patients and no significant correlation with cytotoxic effects was detectable. However out of five high cytotoxic reactions three NK cells were 2DS4 (+). While out of eight assays which resulted with low cytotoxicity only three NK cells were positive for the activating KIR 2DS4. When we analyzed CB NK cells against HMCL U266, three CB NK cells two of which were 2DS4(+) showed high cytotoxicity, while the 2DS4(-) CB NKs did not. The only assay targeting RPMI-8226 which is known to be a NK resistant HMCL, KIR2DS4(+) (CB17) derived NK cells were not sufficiently cytotoxic.Conclusion: Allogeneic CB NK cells were able to induce higher cell kill than autologous PB-derived NK cells. KIR types of autologous NK cells were not predictive for cytotoxic efficacy. Lacking KIR2DS4 in CB NK cells was correlated with less cytotoxicity. Additionally, KIR2DS4(+) CB NK cells were found to be highly cytotoxic against both PC and HMCL. NK cells may be an effective alternative due to its profound clinical advantages such as matching for HLA or KIR ligand is not required. Furthermore, if confirmed by others, certain KIR haplotypes (ie Haplotype A and KIR2DS4 +) may offer better therapeutic potential.Acknowledgment: The study has been supported by Scientific and Research Council of Turkey (TUBITAK grant no: 115S579) and Turkish Academy of Sciences. [Display omitted] DisclosuresBeksac:Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen,Janssen-Cilag,Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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

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. [Display omitted] DisclosuresChamplin:Sanofi: Research Funding; Otsuka: Research Funding. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

Generating suppression-resistant natural killer cells as an enhanced immunotherapeutic for neuroblastoma

Abstract Refractory neuroblastoma (NB) has a poor prognosis and there is a need to explore novel therapies. Natural killer (NK) cells serve as a promising immunotherapy for NB, however they are impaired by the immunosuppressive TGFb-rich NB tumor microenvironment. Critically, TGFβ impairs NK cell proliferation and cytolytic activity. To overcome these detrimental effects, we genetically modified NK cells to express a variant TGFβ receptor – composed of a truncated extracellular TGFbRII domain fused to a synthetic Notch-like receptor coupled to RELA – which may be able to associate with exogenous TGFβ and convert downstream signals to enhance NK cell function. NK cells were isolated from umbilical cord blood (CB; universal donor source) and expanded ex vivo. The modified TGFβ receptor was cloned into an SFG vector backbone. Expanded CB-derived NK cells were transduced with the modified TGFβ receptor (42.5±5.5%). Transduction did not negatively affect the endogenous expression of the activating receptors NKG2D, NKp44, or NKp30 (p&amp;gt;0.05) or NK cell proliferative capacity (p&amp;gt;0.05). Non-transduced (NT) NK cells were cytotoxic against K562 target cells in a dose-dependent manner (45.2±4.69% killing at 40:1), however NK cell cytolytic activity was impaired following exposure to TGFβ (p&amp;lt;0.05). In contrast, transduced-NK cells exhibited dose-dependent cytotoxicity (40.2±4.42% killing at 40:1), which was unaffected following exposure to TGFβ (p&amp;gt;0.05). Administration of transduced-NK cells in a xenograft model of human NB delayed tumor progression by &amp;gt;14 days and significantly prolonged survival compared to control NK cells (p=0.04). In summary, this novel immunotherapeutic may translate as a potent treatment modality for patients with NB.

Ex Vivo-expanded Natural Killer Cells Derived From Long-term Cryopreserved Cord Blood are Cytotoxic Against Primary Breast Cancer Cells.

With over 600,000 units of umbilical cord blood (CB) stored on a global scale, it is important to elucidate the therapeutic abilities of this cryopreserved reservoir. In the advancing field of natural killer (NK) cell cancer immunotherapy, CB has proven to be a promising and noninvasive source of therapeutic NK cells. Although studies have proven the clinical efficacy of using long-term cryopreserved CB in the context of hematopoietic stem cell transplantations, little is known about its use for the ex vivo expansion of effector immune cells. Therefore, our group sought to derive ex vivo-expanded NK cells from long-term cryopreserved CB, using an artificial antigen presenting cell-mediated expansion technique. We compared the expansion potential and antitumor effector function of CB-derived NK (CB-NK) cells expanded from fresh (n=4), short-term cryopreserved (<1-year old, n=5), and long-term cryopreserved (1-10-year old, n=5) CB. Here, we demonstrated it is possible to obtain an exponential amount of expanded CB-NK cells from long-term cryopreserved CB. Ex vivo-expanded CB-NK cells had an increased surface expression of activating markers and showed potent antitumor function by producing robust levels of proinflammatory cytokines, interferon-γ, and tumor necrosis factor-α. Moreover, expanded CB-NK cells (n=3-5) demonstrated cytotoxicity towards primary breast cancer cells (n=2) derived from a triple-negative breast cancer and an estrogen receptor-positive/progesterone receptor-positive breast cancer patient. Long-term cryopreservation had no effect on the expansion potential or effector function of expanded CB-NK cells. Therefore, we propose that long-term cryopreserved CB remains clinically useful for the ex vivo expansion of therapeutic NK cells.

Abstract A36: The cytolytic and anti-leukemic potential of ex vivo differentiated NK cells is confined in the CD94 positive population

Abstract Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against acute myeloid leukemia (AML). We developed a cytokine-based culture method for the generation of NK cells derived from CD34+hematopoietic progenitor cells (HPC) isolated from fresh umbilical cord blood (UCB). Immuno-phenotyping of ex vivo expanded NK cells showed typical inhibitory and activating NK receptors except for CD16 and the KIR receptors. CB-derived NK cells displayed good cytolytic activity and phenotypic analysis showed that the lytic potential of these ex vivo differentiated NK was associated with their expression level of CD94. We further characterized UCB-derived NK cells by investigating in vivo their toxicity, biodistribution, and anti-leukemic potential. For this purpose, immune deficient mice were engrafted with a patient derived AML strain. We showed that in vivo only the CD94+ NK cells survive and form conjugate with leukemic blasts. The characterization of CD94+ cells and the involvement of CD94 and its ligand in the regulation of these ex vivo differentiated allogenic NK cells lytic activity will be discussed. Our findings suggest that CD94+ NK selection could be a pertinent approach to select highly functional expanded NK cells before therapeutic use. Citation Format: Salem Chouaib, Nadine Saleh Khalifé, Meriem Hasmim, Yanyan Zhang, Manale Doldur, Jean Henri Bouhris, Fawzia Louache. The cytolytic and anti-leukemic potential of ex vivo differentiated NK cells is confined in the CD94 positive population [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr A36.

Cord blood NK cells engineered to express IL-15 and a CD19-targeted CAR show long-term persistence and potent antitumor activity.

Chimeric antigen receptors (CARs) have been used to redirect the specificity of autologous T cells against leukemia and lymphoma with promising clinical results. Extending this approach to allogeneic T cells is problematic as they carry a significant risk of graft-versus-host disease (GVHD). Natural killer (NK) cells are highly cytotoxic effectors, killing their targets in a non-antigen-specific manner without causing GVHD. Cord blood (CB) offers an attractive, allogeneic, off-the-self source of NK cells for immunotherapy. We transduced CB-derived NK cells with a retroviral vector incorporating the genes for CAR-CD19, IL-15 and inducible caspase-9-based suicide gene (iC9), and demonstrated efficient killing of CD19-expressing cell lines and primary leukemia cells in vitro, with marked prolongation of survival in a xenograft Raji lymphoma murine model. Interleukin-15 (IL-15) production by the transduced CB-NK cells critically improved their function. Moreover, iC9/CAR.19/IL-15 CB-NK cells were readily eliminated upon pharmacologic activation of the iC9 suicide gene. In conclusion, we have developed a novel approach to immunotherapy using engineered CB-derived NK cells, which are easy to produce, exhibit striking efficacy and incorporate safety measures to limit toxicity. This approach should greatly improve the logistics of delivering this therapy to large numbers of patients, a major limitation to current CAR-T-cell therapies.

Cord blood natural killer cells expressing a dominant negative TGF-β receptor: Implications for adoptive immunotherapy for glioblastoma

Cord blood (CB) natural killer (NK) cells are promising effector cells for tumor immunotherapy but are currently limited by immune-suppressive cytokines in the tumor microenvironment, such as transforming growth factor (TGF-β). We observed that TGF-β inhibits expression of activating receptors such as NKG2D and DNAM1 and decreases killing activity against glioblastoma tumor cells through inhibition of perforin secretion. To overcome the detrimental effects of TGF-β, we engrafted a dominant negative TGF-β receptor II (DNRII) on CB-derived NK cells by retroviral transduction and evaluated their ability to kill glioblastoma cells in the presence of TGF-β. After manufacture using Good Manufacturing Practice–compliant methodologies and transduction with DNRII, CB-derived DNRII-transduced NK cells expanded to clinically relevant numbers and retained both their killing ability and their secretion of interferon-γ upon activation. More important, these cells maintained both perforin expression and NKG2D/DNMA1 expression in the presence of TGF-β allowing for recognition and killing of glioblastoma tumor cells. Hence, NK cells expressing a DNRII should have a functional advantage over unmodified NK cells in the presence of TGF-β-secreting tumors and may be an important therapeutic approach for patients with cancer.

Cord Blood Derived Natural Killer Cells Engineered with a Chimeric Antigen Receptor Targeting CD19 and Expressing IL-15 Have Long Term Persistence and Exert Potent Anti-Leukemia Activity

Natural killer (NK) cells are a major component of the innate immune system, possessing the ability to lyse their targets without the need for prior sensitization or specificity for antigen. Besides their classical role in providing potent antitumor and antiviral immunity, NK cells can reduce the risk of graft-versus-host disease (GVHD) by targeting host antigen-presenting cells, as well as activated alloreactive donor T cells, indicating that NK-mediated graft-versus-leukemia (GVL) responses may occur in the absence of GVHD. Although most groups have relied on autologous or adult peripheral blood donor-derived NK cells, we have studied umbilical CB as a potential source of NK cells because of their availability as an "off-the-shelf" frozen product and their potent preclinical activity against leukemia cells. To overcome the obstacle of limited numbers of NK cells in a single CB unit, we have established GMP-compliant conditions for the ex vivo expansion of clinically relevant doses of such cells. By using GMP grade K562-based artificial antigen-presenting cells (aAPCs), which express membrane-bound IL-21 (clone 9.mbIL21), to numerically expand highly functional and mature CB-derived NK cells.To further enhance the GVL effect independent of KIR-ligand mismatch, we have genetically modified human CB-derived NK cells with a retroviral vector, CAR19-CD28-zeta-2A-IL15 (CAR19/IL15), which incorporates the genes for CAR-CD19, IL-15 to enhance proliferation and survival, and the inducible caspase-9 molecule. CB-NK cells could be stably transduced with CAR19/IL15, proliferated efficiently in vitro and maintained superior effector function against CD19-expressing leukemia cell lines and primary CLL cells. Moreover, the effector functions of both NK-CAR and nontransduced NK cells against K562 were comparable, indicating that the genetic modification of CB-NK cells does not alter their intrinsic cytotoxicity against NK-sensitive targets. Because of concerns over autonomous, uncontrolled NK cell growth due to autocrine production of IL15, we also incorporated into our construct a suicide gene based on the inducible caspase-9 (IC9) gene. The addition of as little as 10 nM of the small molecule dimerizer CID AP20187 to cultures of iC9/CAR19/IL15+ NK cells induced apoptosis/necrosis of >60% of transgenic cells within 4 hours as assessed by annexin-V-7AAD staining. The infusion of CAR.CD19.IL15-transduced CB-NK cells into a NOD-SCID-gamma null model of lymphoblastic lymphoma (Raji model) resulted in impressive anti-tumor responses (Fig. 1). Moreover, adoptively infused CAR-transduced CB NK persisted for up to 70 days post-infusion (Fig. 2), supporting our hypothesis that IL-15 enhances the proliferation and survival of the engineered CB-NK cells. Based on these promising data, we now propose to manufacture a GMP-grade CAR19-CD28-zeta-2A-IL15 vector for a phase 1 dose escalation trial in patients with high risk B-cell leukemia. [Display omitted] [Display omitted] DisclosuresWierda:Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Research Funding. Rezvani:Pharmacyclics: Research Funding.

Cellular engineering and therapy in combination with cord blood allografting in pediatric recipients.

Cord blood (CB) transplantation is an alternate source of human hematopoietic progenitor cells for allogeneic stem cell transplantation in children and adolescents with both malignant and nonmalignant diseases. Current limitations included delay in hematopoietic reconstitution, increased incidence of primary graft failure and slow cellular immunoreconstitution. These limitations lead to a significant increase in primary graft failure, infectious complications and increased transplant-related mortality. There is a number of experimental approaches currently under investigation including cellular engineering to circumvent these limitations. In this review, we summarize the recent findings of utilizing ex vivo CB expansion with Notch1 ligand Delta 1, mesenchymal progenitor cells, the use of human placenta-derived stem cells and CB-derived natural killer cells. Early and preliminary results suggest some of these experimental cellular strategies may in part ameliorate the incidence of primary graft failure, delays in hematopoietic reconstitution and/or slowness in cellular immune reconstitution following unrelated CB transplantation.

Antigen Presenting Cell-Mediated Expansion of Human Umbilical Cord Blood Yields Log-Scale Expansion of Natural Killer Cells with Anti-Myeloma Activity

Natural killer (NK) cells are important mediators of anti-tumor immunity and are active against several hematologic malignancies, including multiple myeloma (MM). Umbilical cord blood (CB) is a promising source of allogeneic NK cells but large scale ex vivo expansion is required for generation of clinically relevant CB-derived NK (CB-NK) cell doses. Here we describe a novel strategy for expanding NK cells from cryopreserved CB units using artificial antigen presenting feeder cells (aAPC) in a gas permeable culture system. After 14 days, mean fold expansion of CB-NK cells was 1848-fold from fresh and 2389-fold from cryopreserved CB with >95% purity for NK cells (CD56+/CD3−) and less than 1% CD3+ cells. Though surface expression of some cytotoxicity receptors was decreased, aAPC-expanded CB-NK cells exhibited a phenotype similar to CB-NK cells expanded with IL-2 alone with respect to various inhibitory receptors, NKG2C and CD94 and maintained strong expression of transcription factors Eomesodermin and T-bet. Furthermore, CB-NK cells formed functional immune synapses with and demonstrated cytotoxicity against various MM targets. Finally, aAPC-expanded CB-NK cells showed significant in vivo activity against MM in a xenogenic mouse model. Our findings introduce a clinically applicable strategy for the generation of highly functional CB-NK cells which can be used to eradicate MM.

Ex Vivo IL-2 Expansion of CB-NK Cells Promotes Synergistic LFA-1 and CD2 Engagement at the NK Cell Lytic Immune Synapse; Implications for Adoptive CB-NK Cell Therapy in Acute Myeloid Leukemia.

Abstract 3029Poster Board II-1005Donor peripheral blood (PB) natural killer (NK) cell have shown clinical promise in cancer immunotherapy. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. Umbilical cord blood (CB) represents an important alternative source of NK cells for adoptive immune therapy. We first demonstrated that cord blood (CB) derived NK cells have poor cytolytic activity and deficiency in the formation of the F-actin immunological synapse with HLA class I deficient target K562 cells and primary AML blasts compared to PB-NK cells. In this study, we explored the cellular mechanism of these dysfunctions. We hypothesized that adhesion and signaling molecules may be defective in unmanipulated CB NK cells. Activating receptor Both CD2 and the integrin lymphocyte function-associated antigen (LFA-1) play important roles in both T lymphocyte and NK cell immune synapse formation and their trafficking to the immune synapse regulates both T and NK cell function. We now show that unmanipulated CB NK cells exhibit reduced LFA-1 mediated adhesion to mobilized ICAM-1 compared to IL-2 expanded CB NK cells (CB NK 29.7+/- 3.2 %, vs expanded CB NK 78.5+/- 6.1%, n=6). Moreover, unmanipulated CB-NK cells demonstrated reduced surface expression of CD2, and high affintyLFA-1 detected by the specific antibody (MHM24). There was decreased recruitment of CD2 and LFA-1 to the NK cell immune synapse site as quantified by confocal microscope analysis (RRI CD2 CB NK 2.02 vs PB NK 4.98, n=3). Furthermore, defective LFA-1 trafficking lead to a decrease in downstream cytotoxic granules that traffic to the immunological synapse as demonstrated by decreased perforin trafficking to the CB-NK synapse site (> 60% reduction).We next wanted to confirm that CD2 or LFA-1 play a role in restoring the immune synapseformation for IL-2 expanded CB NK cells. We incubated expanded CB NK cells with blocking antibodies specific for LFA-1 or CD2 prior to conjugation to the K562 target cells. After CD2 or LFA-1 blocking there was decreased synapse formation, with a resultant decrease in cytotoxic function. When monoclonal antibodies against both CD2 and LFA-1 were used there was significant blockade of the formation of the immune synapse, and a marked reduction of CB NK cell cytolytic activity (Mean specific lysis of K562 targets at E:T ratio 20:1 was 81% IgG control vs 22% with anti-CD2; and 29% with anti-LFA-1, n=6, P<0.001). This data shows that CD2 and LFA-1 are defective in unmanipulated CB NK cells resulting in impaired immune synapse formation. In contrast, ex vivo IL-2 expansion of CB-NK cells enhanced lytic synapse formation with the synergistic repair of CD2 and LFA-1 localization and activity. We believe our results provide important mechanistic insights for the potential use of IL-2 expanded CB-derived NK cells for adoptive immune therapy in leukemia. DisclosuresNo relevant conflicts of interest to declare.

Ex Vivo Expansion of Cord Blood NK Cell Have In Vivo Efficacy Against Leukemia.

Introduction Allogeneic stem cell transplantation has demonstrated the ability to prevent leukemic relapse via an immune-mediated graft-vs.-leukemia effect. Natural killer (NK) cells have been shown to comprise a significant component of this anti-leukemia effect and have been reported to enhance engraftment and reduce graft-vs.-host disease. Ex-vivo expansion of peripheral blood-derived NK cells has been demonstrated. Cord blood (CB) is a promising alternate source of NK cells with enhanced cytokine/antigen-responsiveness, proliferation and increasing availability. We studied expansion conditions for the generation of CB NK cells for clinical use, and evaluated them in vivo in NOD-SCID/IL2Rgnull mice engrafted with human leukemia.Methods Two expansion protocols were compared:1.CD56+ NK cells were isolated and cultured with irradiated CD56− feeder cells in RPMI-1640 supplemented with 100ng/mL IL-2 for 21 days.2.CB mononuclear cells were depleted of CD3+ cells prior to culture.In vivo NK cytolytic activity was measured via chromium release assays on leukemia cell lines (K562, Nalm6) and fresh patient leukemia blasts. Functionality was confirmed in vivo as well by injecting 106 K562 cells or 2.5 ×104 Nalm6 cells into NOD-SCID/IL2Rgnull mice. 2 ×106 CB-expanded NK cells were administered on day +1, and an additional 5×106 CB NK cells were administered on day +7. NK killing of leukemic targets was then confirmed by flow cytometry.Results Feeder cells mediated >30-fold expansion of CB NK cells, generating a nearly pure population that contained 97% CD3−CD56+ cells. Expanded CB NK could lyse >90% of K562 targets at an E:T ratio of 10:1, as well as >40% of patient leukemia blast. In CD3− depleted cultures, similar expansions were observed, however only 55% of these cells were CD3−CD56+ with the remainder being 10% CD3+CD56+ NK/T and 35% CD3+ T lymphocytes. In the NOD-SCID~/IL2Rgnull model, ex vivo expanded CB NK cells demonstrated the ability to reduce the leukemia burden of both AML (K562) and ALL (Nalm6) cells >50%.Conclusions We have demonstrated the feasibility of expanding CB NK ex vivo to clinically-relevant doses with minimal manipulation. These expanded CB NK cells demonstrated cytotoxic activity in vitro and in vivo against a variety of human leukemic cell lines and patient leukemia blasts. These results provide rationale for immunotherapy of leukemia with CB-derived NK cells.