Cord Blood 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.

Abstract 3191: Genetically modified TGF-β dominant negative receptor and IL15 enhance Natural Killer (NK) cell mediated cytolytic activity in glioblastoma

Abstract Introduction: Glioblastoma (GBM) presents with a poor prognosis and dismal survival rates. While cell-based immune therapies have shown success in other malignancies, there is currently little evidence that GBM is as responsive to current approaches. Novel therapies for these patients are therefore urgently needed. The antitumor effect may be limited by GBM’s rapid progression, its extensive intertumoral heterogeneity, and its immune suppressive microenvironment. For cell-based therapies to be effective, they need to be manufactured rapidly or in advance, recognize multiple targets on GBM, and resist immune suppression. We therefore sought to develop “off-the-shelf” cord blood-derived NK cell therapies capable of recognizing multiple stress ligands on tumor cells but are protected from the microenvironment by expression of a dominant negative receptor to TGFβ (abrogating the negative effects of the cytokine) and secretion of IL15 (overcoming negative signals from the microenvironment). We investigate the therapeutic efficacy of NK cells engineered to express IL15-and/or TGFβ-DNR in glioblastoma model in-vitro and in-vivo. Methods: NK cells were isolated from umbilical cord blood by magnetic selection, and subsequently modified to express IL-15, and/or TGFβ-DNR. Engineered NK cells were expanded with feeder cells. Retroviral transduction efficiency was confirmed by flow cytometry. IL15 expression was quantified by ELISA. To determine the cytotoxicity activity of our engineered NK cells against glioblastoma, we measured lysis of the U87MG cell line. To determine in vivo efficacy of our engineered NK cells, we used xenograft NSG mouse models that were orthotopically engrafted with U87MG in the brain. Tumor growth was measured by bioluminescence. Results: CAR-NK cells were successfully transduced (with a transduction efficiency ranging from 66-83% (mean 74.55% + 10.6, n>3) from umbilical cord blood. NK cells engineered to secrete IL15 secreted a mean of 288.3 pg/mL. After exposure to TGFβ in vitro, NK cells transduced with TGFβ-DNR had a increase of killing [mean 58.25 % + 4.51 at 20:1 (E:T) 48.57% + 11.55 without TGFβ, n=3] compared to un-transduced NK cells [mean 11.98 % + 4.51 at 20:1 (E:T) 49.04% + 5.34 without TGFβ, n=3]. Mice injected with IL15 and TGFβ DNR showed better control of tumor (3.99 x 105 + 0.0007 bioluminescence Vs 1.673 x 107 + 0.160 in mice given mock-transduced NK cells and 8.44 x 109 + 28.62 in control mice). Conclusion: Our preliminary results show that cord blood NK cells modified to secrete IL15 and express TGFβ-DNR can recognize and lyse GBM cells in vitro and orthotopically implanted GBM tumors in vivo. Citation Format: Nethaji Muniraj, Kajal Chaudhry, Joshua Luke Terao, Vipin Suri, Luke Barron, Catherine Bollard, Conrad Russell Cruz. Genetically modified TGF-β dominant negative receptor and IL15 enhance Natural Killer (NK) cell mediated cytolytic activity in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3191.

Comparative Study of the Two High-Efficient Strategies for in vitro Generation of Human Umbilical Cord Blood-derived Natural Killer Cells

To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies. Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy. After a 14-day's culture, the contents of CD3-CD56+ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3+CD4+ T cells and CD3+CD56+ NKT cells in M-NK group decreased significantly. The percentages of CD16+, NKG2D+, NKp44+, CD25+ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a+ NK cells in M-NK group were higher under the same effector-target ratio (E∶T) (P<0.05). The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.

Abstract LB102: Off-the-shelf cord blood FLT3 CAR-NK cells for immunotherapy of acute myeloid leukemia

Abstract Despite the unprecedented clinical success of autologous chimeric antigen receptor (CAR) T cell therapy in B cell leukemia and lymphoma patients, CAR T cell therapy for acute myeloid leukemia (AML) patients has lagged, in part due to the time required for autologous CAR T cell preparation in the face of the rapid relapse of AML following remission, and the possibility of on-target off-tumor hematopoietic toxicity. In this study we investigated the functional activity of an allogeneic, off-the-shelf FLT3 CAR natural killer (NK) cells expressing soluble (s) IL-15 (FLT3 CAR_s15 NK) to treat FLT3+ AML. Cord blood NK cells were first transduced with FLT3 CAR intracellular signaling constructs containing either CD28/CD3ζ or 2B4/CD3ζ. Each displayed similar cytotoxicity against FLT3+ AML in vitro (p &amp;lt; 0.05) and in vivo (p &amp;lt; 0.01) when compared to mock transduced NK cells. Cord blood NK cells transduced with either soluble (s) IL-15 (s15 NK cells) or membrane (m) bound IL-15 showed comparable extended survival in vivo and superior to NK cells transduced with sIL-15/IL-15Rα (p &amp;lt; 0.01) or mIL-15/IL-15Rα (p &amp;lt; 0.01). However, only those NK cells transduced with soluble IL-15 were able to activate neighboring non-transduced (NT) NK cells and T cells in a paracrine fashion. The FLT3 CAR_s15 NK cells were expanded ex-vivo to over 1500-fold in 16 days with ~50% of the NK cells expressing both the FLT3 CAR and sIL-15. These FLT3 CAR_s15 NK cells were then subjected to a cytotoxicity assay against the FLT3+ AML cell line (MOLM-13) and were 10 times more potent than NT NK cells (p &amp;lt; 0.01) and 1.7 times more potent than s15 NK cells (p &amp;lt; 0.05). Likewise, when co-cultured with the FLT3+ AML cell line, FLT3 CAR_s15 NK cells produced a greater amount of IFN-γ compared to NT NK cells (p &amp;lt; 0.01) or compared to s15 NK cells (p &amp;lt; 0.05). Furthermore, following cryopreservation, our thawed FLT3 CAR_s15 NK cells demonstrated high recovery (~90%) and viability (&amp;gt; 85%), maintaining their FLT3 CAR expression at ~50% with a phenotype and cytotoxicity that was nearly equivalent to fresh FLT3 CAR_s15 NK cells. Armed with these data we assessed the efficacy of our allogeneic, off-the-shelf FLT3 CAR_s15 NK cells in vivo using immunodeficient mice engrafted with the MOLM-13 FLT3+ cell line. Repeated infusions of FLT3 CAR_s15 NK cells provided an improved median survival compared to placebo group (36 vs 24 days; p &amp;lt; 0.001) or compared to identical infusions of s15 NK cells (36 vs 31 days; p &amp;lt; 0.05) with similar results obtained in a patient-derived xenograft model. Finally, to assess toxicity against normal peripheral blood mononuclear cells (PBMC), including FLT3+ dendritic cells, and against hematopoietic stem cells (HSCs) we performed in vitro and in vivo studies, respectively, using our FLT3 CAR_s15 NK cells. We did not observe an untoward cytotoxicity of PBMC in vitro when compared to NT or s15 NK cells and no disruption of HSC differentiation in vivo when compared to placebo group. In summary, our in vitro and in vivo studies with allogeneic off-the-shelf cord blood derived FLT3 CAR_s15 NK cells demonstrated enhanced cytotoxicity and IFN-γ secretion against FLT3+ AML in vitro and enhanced the survival of mice engrafted with FLT3+ AML without any evidence of PBMC or HSC toxicity. We believe these data collectively support a rationale for investigating this novel form of allogeneic, off-the-shelf FLT3 CAR_s15 NK cell therapy in patients stricken with FLT3+ AML. Citation Format: Anthony G. Mansour, Kun-Yu Teng, Zhiyao Li, Zheng Zu, Hanyu Chen, Aliya Ali, Jianying Zhang, Ting Lu, Shoubao Ma, Michael A. Caligiuri, Jianhua Yu. Off-the-shelf cord blood FLT3 CAR-NK cells for immunotherapy of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB102.

Abstract LB211: Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) remains one of the major causes of cancer related mortality worldwide, yet clinical advances with checkpoint inhibitors suggest that an anti-tumor immune response can be harnessed to prolong survival in NSCLC patients. We recently described the identification of a tumor reactive natural killer (NK) cell population by its in vivo upregulation of PD-L1 in the presence of tumor, referred to here as TRACK-NK cells. PD-L1(+) NK cells are more potent than PD-L1(-) NK cells against tumor cell targets. Further, the administration of anti-PD-L1 antibody (atezolizumab) functions as an immune agonist on TRACK-NK cells resulting in significantly enhanced degranulation (P &amp;lt; 0.001) and secretion of IFN-gamma (P &amp;lt; 0.001) in the presence of tumor. Here we investigated the functional activity and product safety of allogeneic, off-the-shelf TRACK-NK cells retrovirally transduced to express soluble (s) IL-15 to treat NSCLC without or with the addition of atezolizumab. TRACK-NK cells derived from umbilical cord blood NK cells were expanded over 1,000-fold ex-vivo in 17 days with ~50% of the final product engineered to express sIL15 and cytokine-activated to express PD-L1. The phenotype of the final TRACK-NK cell product showed a significant increase in the expression of PD-L1, CD25 and CD69 when compared to non-transduced (P &amp;lt; 0.01) or NK cells transduced to express sIL15 without cytokine activation (s15 NK cells) (P &amp;lt; 0.01). Following cryopreservation, our thawed TRACK-NK cell product demonstrated high recovery (&amp;gt;85%) and viability (&amp;gt;80%). In a Real Time Cell Analysis (RTCA) in vitro cytotoxicity assay against a human NSCLC cell line (A549), TRACK-NK cells were 10 times more potent than non-transduced (NT) NK cells (P &amp;lt; 0.001) and 2 times more potent than s15 NK cells (P &amp;lt; 0.01). To assess the efficacy of our frozen, allogeneic off-the-shelf TRACK-NK cells in vivo, we utilized immunodeficient mice bearing human A549 metastatic NSCLC. Intravenous infusion of TRACK-NK cells resulted in significantly more suppression of luciferase-A549 NSCLC growth in mice compared to mice treated with NT NK cells (P &amp;lt; 0.05) or s15 NK cells (P &amp;lt; 0.01). In terms of dose response, 1 × 107 TRACK-NK cells per dose delivered every other day for a total of 4 doses showed significantly better tumor control compared to a dose of 5 × 106 TRACK-NK cells (P &amp;lt; 0.001) but similar control compared to 2 × 107 TRACK-NK cells. The combination of TRACK-NK cells plus atezolizumab showed better control of NSCLC growth in vivo compared to TRACK-NK cells alone (P &amp;lt; 0.01). Finally, we evaluated the safety of our TRACK-NK product in immunodeficient mice without tumor engraftment and did not observe any significant change in body weight, body temperature, liver enzymes, kidney function, or blood counts compared to mice treated with vehicle control. No cytokine release syndrome was observed. In summary, our studies using human allogeneic off-the-shelf TRACK-NK cells proved to be safe and non-toxic, demonstrated enhanced cytotoxicity against NSCLC in vitro and enhanced control of tumor growth in vivo, further improved with atezolizumab. As the intravenous infusion of activated NK cells naturally traffic to lung, our TRACK-NK platform will move forward to the clinic for the treatment of relapsed and refractory NSCLC patients. Citation Format: Ting Lu, Anthony G. Mansour, Rui Ma, Christian Bustillos, Zhiyao Li, Shoubao Ma, Zhenlong Li, Hanyu Chen, Kun-Yu Teng, Jianying Zhang, Michael A. Caligiuri, Jianhua Yu. Tumor-reactive and anti-PD-L1 co-stimulated killer cells (TRACK-NK) for immunotherapy of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB211.

IPSC-Derived Natural Killer Cell Therapies - Expansion and Targeting.

Treatment of cancer with allogeneic natural killer (NK) cell therapies has seen rapid development, especially use against hematologic malignancies. Clinical trials of NK cell-based adoptive transfer to treat relapsed or refractory malignancies have used peripheral blood, umbilical cord blood and pluripotent stem cell-derived NK cells, with each approach undergoing continued clinical development. Improving the potency of these therapies relies on genetic modifications to improve tumor targeting and to enhance expansion and persistence of the NK cells. Induced pluripotent stem cell (iPSC)-derived NK cells allow for routine targeted introduction of genetic modifications and expansion of the resulting NK cells derived from a clonal starting cell population. In this review, we discuss and summarize recent important advances in the development of new iPSC-derived NK cell therapies, with a focus on improved targeting of cancer. We then discuss improvements in methods to expand iPSC-derived NK cells and how persistence of iPSC-NK cells can be enhanced. Finally, we describe how these advances may combine in future NK cell-based therapy products for the treatment of both hematologic malignancies and solid tumors.

Cord Blood Natural Killer Cells Inhibit Sepsis Caused by Feces-Induced Acute Peritonitis via Increasing Endothelium Integrity

Sepsis is associated with acute peritonitis, which can be induced by lipopolysaccharide exposure and feces. Generally, lipopolysaccharide induces mono-microbial peritonitis, whereas feces cause poly-microbial peritonitis; the latter is a more complicated and closer to the clinical diseases. Although several reports have discussed the mechanism of immune response in peritonitis-induced sepsis, however, the role of natural killer (NK) cells in sepsis, especially the relationship between NK cells and stabilization of the vascular endothelial barrier, is still unclear. Accordingly, in this study, we assessed the roles of NK cells in an acute sepsis model in mice. NK cells were injected via the tail vein into mice with acute sepsis, and nitric oxide (NO), anti-inflammatory cytokine, and angiogenic factors were tested to explore the effects of NK cells on sepsis. The survival rate of septic model mice infused with NK cells was significantly improved compared with the control group. Interestingly, the levels of NO, interleukin-10, and vascular endothelial growth factor (VEGF) decreased in NK cells therapy group. After the injection of NK cells, CD31 positive endothelial cells significantly increased in the kidneys and liver, although the expression of VEGF, ANGPT-1, and ET-1 was downregulated. Consistent with our hypothesis, the transfusion of NK cells into mice with sepsis blocked inflammation and increased endothelium integrity. Overall, these findings suggest that NK cells may block sepsis by modulating the VEGF pathway.

Construction and evaluation of dual-effect cord blood natural killer cells expressing highaffinity PD-1 and chimeric antigen CD19 receptor

To obtain novel dual-effect cord blood natural killer cells (CBNKCs) expressing high-affinity PD-1 (HAPD1) and chimeric antigen CD19 receptor (CAR) to improve the effect of CAR-based immunotherapy. A dual-effect lentiviral vector expressing both HAPD1 and CAR targeting CD19 was constructed. CBNKCs were infected with the vector to obtain HAPD1 CAR19 CBNKCs. The surface markers of the cells including CD3-/CD16+CD56+, CD3+/CD16+CD56+, CD3+/CD4+, and CD3+/CD8+ were tested during cell proliferation. The cytotoxicity of CBNKCs, CAR19 CBNKCs and HAPD1 CAR19 CBNKCs incubated with CD19-positive target cells at the effector-target ratios of 5∶1, 10∶1 and 20∶1 was tested on days 7, 9, 12, and 15 of cell culture. The cytotoxicity of the cells against the target cells was also tested in NPG mice. CBNKCs were successfully transduced with T-cell designed CAR19 and HAPD1 CAR19 with an efficiency of (18.63±1.88)%. Infection with the lentiviral vector significantly reduced the cell expansion efficiency of the CBNKCs (10.97±2.77 vs 24.84±3.17, P < 0.05) but did not significantly affect the expressions of the surface markers (P>0.05). HAPD1 CAR CBNKCs showed stronger anti-tumor effect than CAR19 CBNKCs [(68.38±8.08)% vs (49.65±13.60)% at the effector-target ratios of 5∶1 and (79.11±7.42)% vs (59.78 ± 9.32)% at 10∶1; P < 0.05]. The infected CBNKCs showed the strongest cytotoxicity at 9 and 12 days after lentivirus infection. In the mouse models, transplantation of the dual-effect cells resulted in a significantly lower percentage of tumor cells in white blood cells than transplantation CAR-CBNKCs [(19.21 ± 3.07%) vs (29.08 ± 3.15)%, P < 0.05]. We obtained a novel dual-effect CBNKC co-expressing HAPD1 and CAR. The cells show strong cytotoxicity against the target tumor cells both in vitro and in vivo, which sheds light on a new strategy of immunotherapy against tumor cells.

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.

Development of CD19 CAR Engineered Human Placental CD34 +-Derived Natural Killer Cells (CAR19-CYNK) As an Allogeneic Cancer Immunotherapy

Development of CD19 CAR Engineered Human Placental CD34 +-Derived Natural Killer Cells (CAR19-CYNK) As an Allogeneic Cancer Immunotherapy

Cord Blood NK Cells Engineered to Express a Humanized CD123-Targeted Chimeric Antigen Receptor (CAR) and IL-15 As Off-the-Shelf Therapy for Acute Myeloid Leukemia

The outcomes of patients with AML remain disappointing, with only fewer than half of all adults (and <10% of adults 60 years or older) achieving long-term remissions. Thus, novel therapies are urgently needed. Chimeric antigen receptors (CARs) have been used extensively to redirect the specificity of T cells in patients with lymphoid malignancies. These infusions have been primarily restricted to the autologous setting, but generation of autologous products for each patient is logistically cumbersome and not scalable. A banked allogeneic product could overcome these limitations; but, allogeneic T-cells (even if HLA-matched) carry a major risk of graft-versus-host disease (GVHD). Natural killer (NK) cells provide an attractive alternative to T-cells for CAR therapy as they do not cause GVHD. Cord blood (CB) is a readily available “off-the-shelf” source of allogeneic NK cells that can be expanded to large, highly functional doses for CAR therapy.CD123 (IL3-receptor α chain) is a potential immunotherapeutic target in AML due to its overexpression on leukemic stem cells and AML blasts, rather than normal hematopoietic stem cells. Thus, we reprogrammed CB-NK cells to target CD123+ve tumor cells by retroviral transduction using synthetic constructs producing either murine-derived (iC9-2A-CD123CAR-CD28-CD3z-2A-IL15) or humanized (iC9-2A-hCD123CAR-CD28-CD3z-2A-IL15) scFv domains. We humanized the ScFv derived from murine antibody clone 26292 to avoid human anti-mouse antibody responses, which could potentially limit the persistence and efficacy of the CAR-NK cells when infused in patients. These constructs also express exogenous soluble IL-15 to support NK cellular survival and proliferation, as well as an inducible caspase-9 (iC9) suicide gene as a safety precaution to ablate CAR+ve NK cells if needed.Retroviral transduction with murine (CD123CAR) and humanized (hCD123CAR) (n=12) resulted in a median CAR positivity of 75% and 76%, respectively, 14 days post transduction and viability of >90%. Notably, CAR expression was stable over time. iC9/CAR.123/IL15+ and iC9/hCAR.123/IL15+ NK cells expanded for 2 weeks showed no signs of exhaustion, such as downregulation of eomesodermin and T-bet, or upregulation of KLRG1, and exhibited a mature phenotype, similar to ex vivo expanded non-transduced NK cells (NT-NK) using 22 markers. In a 4 hour 51Cr release assay, murine CD123CAR+ve and hCD123CAR+ve NK cells were equally effective at killing CD123+ve AML cell lines (THP-1 and MOLM-14), and exhibited significantly higher cytotoxicity when compared to NT-NK cells at all tested effector to target cell ratios (20:1; 10:1; 5:1 and 1:1). Notably, CAR123 and hCAR123-transduced NK cells were as equally efficient as NT-NK cells in killing CD123 negative targets such as K562 (positive control) and KARPAS (negative control), indicating that the enhanced killing of CD123+ targets by the transduced cells is mediated by the CAR receptor and not due to a non-specific enhancement in NK cytotoxicity. In addition, CD123CAR+ve and hCD123CAR+ve NK cells showed significantly higher production of IFN-γ, TNF-α, and CD107a degranulation in response to MOLM14 and THP in comparison to NT-NK cells. Indeed, using confocal microscopy, we showed significantly higher accumulation of CAR molecules at the immunologic synapse (IS) formed between CD123CAR+ve and hCD123CAR+ve CB-NK cells and CD123+ve targets compared to the diffuse presence of CARs at the IS with K562 target cells, indicating that CAR molecules participate in IS formation in a CD123-dependent manner.We validated elimination of iC9/CAR.123/IL-15 transduced NK via activation of iC9 in vitro. The addition of 10nM of the small molecule dimerizer AP1903 (Bellicum Pharmaceuticals, Inc) to cultures of iC9/CAR123/IL-15 or iC9/hCAR123/IL-15-transduced NK cells induced apoptosis of transduced NK cells within 4 hours but had no effect on the viability of NT-NK cells. Studies to test the in vivo anti-tumor efficacy of iC9/CAR123/IL-15 or iC9/hCAR123/IL-15-transduced NK cells in an NSG mouse model of MOLM14 are under way.In summary, our data demonstrate the capacity of CD123CAR+ve and hCD123CAR+ve CB-NK cells to kill CD123+ targets and support the use of CD123-specific CAR NK cells in the clinic for patients with relapsed AML as a means to decrease disease burden, possibly prior to consolidative therapies such as allogeneic transplantation. DisclosuresNo relevant conflicts of interest to declare.

Optimizing NK Cell-Based Immunotherapy in Myeloid Leukemia: Abrogating an Immunosuppressive Microenvironment.

Natural killer (NK) cells are prominent cytotoxic and cytokine-producing components of the innate immune system representing crucial effector cells in cancer immunotherapy. Presently, various NK cell-based immunotherapies have contributed to the substantial improvement in the reconstitution of NK cells against advanced-staged and high-risk AML. Various NK cell sources, including haploidentical NK cells, adaptive NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, chimeric antigen receptor NK cells, cytokine-induced memory-like NK cells, and NK cell lines have been identified. Devising innovative approaches to improve the generation of therapeutic NK cells from the aforementioned sources is likely to enhance NK cell expansion and activation, stimulate ex vivo and in vivo persistence of NK cells and improve conventional treatment response of myeloid leukemia. The tumor-promoting properties of the tumor microenvironment and downmodulation of NK cellular metabolic activity in solid tumors and hematological malignancies constitute a significant impediment in enhancing the anti-tumor effects of NK cells. In this review, we discuss the current NK cell sources, highlight ongoing interventions in enhancing NK cell function, and outline novel strategies to circumvent immunosuppressive factors in the tumor microenvironment to improve the efficacy of NK cell-based immunotherapy and expand their future success in treating myeloid leukemia.

Combining AFM13, a Bispecific CD30/CD16 Antibody, with Cytokine-Activated Blood and Cord Blood-Derived NK Cells Facilitates CAR-like Responses Against CD30+ Malignancies.

Natural killer (NK)-cell recognition and function against NK-resistant cancers remain substantial barriers to the broad application of NK-cell immunotherapy. Potential solutions include bispecific engagers that target NK-cell activity via an NK-activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL12/15/18 cytokine pre-activation. We assessed single-cell NK-cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL12/15/18 pre-activation of blood and cord blood-derived NK cells was investigated in vitro and in vivo. We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK-cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from patients with Hodgkin lymphoma. IL12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30+ lymphoma targets directed by AFM13, compared with cNK cells. Cord-blood NK cells preactivated with IL12/15/18 and ex vivo expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK-cell effector function. AFM13-NK complex cells exhibited enhanced responses to CD30+ lymphomas in vitro and in vivo. We identify AFM13 as a promising combination with cytokine-activated adult blood or cord-blood NK cells to treat CD30+ hematologic malignancies, warranting clinical trials with these novel combinations.

Umbilical cord blood NK cells offer multiple advantages for cancer immunotherapy: lessons learned from Glycostem’s orphan drug oNKord®

Adoptive transfer of natural killer (NK) cells has been demonstrated as an efficacious and safe treatment option in patients with cancer. For clinical use, these potent immune cells can be directly isolated or generated from several sources using a variety of protocols. Increasing evidence indicates that NK cell products prepared from hematopoietic stem and progenitor cells isolated from umbilical cord blood have many advantages. These include a highly cytotoxic activity against hematologic and solid malignancies, a minimal risk of adverse events, including graft-versus-host disease, and wide availability and applicability. The objective of this review is to provide an oversight of the various NK cell products and to discuss their advantages and disadvantages for cancer immunotherapy.

Umbilical Cord Blood and iPSC-Derived Natural Killer Cells Demonstrate Key Differences in Cytotoxic Activity and KIR Profiles.

Natural killer (NK) cells derived or isolated from different sources have been gaining in importance for cancer therapies. In this study, we evaluate and compare key characteristics between NK cells derived or isolated from umbilical cord blood, umbilical cord blood hematopoietic stem/progenitor cells, peripheral blood, and induced pluripotent stem cells (iPSCs). Specifically, we find CD56+ NK cells isolated and expanded directly from umbilical cord blood (UCB56) and NK cells derived from CD34+ hematopoietic stem/progenitors in umbilical cord blood (UCB34) differ in their expression of markers associated with differentiation including CD16, CD2, and killer Ig-like receptors (KIRs). UCB56-NK cells also displayed a more potent cytotoxicity compared to UCB34-NK cells. NK cells derived from iPSCs (iPSC-NK cells) were found to have variable KIR expression, with certain iPSC-NK cell populations expressing high levels of KIRs and others not expressing KIRs. Notably, KIR expression on UCB56 and iPSC-NK cells had limited effect on cytotoxic activity when stimulated by tumor target cells that express high levels of cognate HLA class I, suggesting that in vitro differentiation and expansion may override the KIR-HLA class I mediated inhibition when used across HLA barriers. Together our results give a better understanding of the cell surface receptor, transcriptional, and functional differences between NK cells present in umbilical cord blood and hematopoietic progenitor-derived NK cells which may prove important in selecting the most active NK cell populations for treatment of cancer or other therapies.

Exploring the NK cell platform for cancer immunotherapy.

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that are capable of killing virally infected and/or cancerous cells. Nearly 20 years ago, NK cell-mediated immunotherapy emerged as a safe and effective treatment approach for patients with advanced-stage leukaemia. Subsequently, the field of NK cell-based cancer therapy has grown exponentially and currently constitutes a major area of immunotherapy innovation. In general, the development of NK cell-directed therapies has two main focal points: optimizing the source of therapeutic NK cells for adoptive transfer and enhancing NK cell cytotoxicity and persistence in vivo. A wide variety of sources of therapeutic NK cells are currently being tested clinically, including haploidentical NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, NK cell lines, adaptive NK cells, cytokine-induced memory-like NK cells and chimeric antigen receptor NK cells. A plethora of methods to augment the cytotoxicity and longevity of NK cells are also under clinical investigation, including cytokine-based agents, NK cell-engager molecules and immune-checkpoint inhibitors. In this Review, we highlight the variety of ways in which diverse NK cell products and their auxiliary therapeutics are being leveraged to target human cancers. We also identify future avenues for NK cell therapy research.

Redirection of Cord Blood T Cells and Natural Killer Cells for Elimination of Autologous HIV-1-Infected Target Cells Using Bispecific DART® Molecules.

Mother-to-child transmission of HIV-1 remains a major global health challenge. Currently, HIV-1-infected infants require strict lifelong adherence to antiretroviral therapy to prevent replication of virus from reservoirs of infected cells, and to halt progression of disease. There is a critical need for immune interventions that can be deployed shortly after infection to eliminate HIV-1-infected cells in order to promote long-term remission of viremia, or to potentially cure pediatric HIV-1-infection. Bispecific HIV × CD3 DART® molecules able to co-engage the HIV-1 envelope protein on the surface of infected cells and CD3 on cytolytic T cells have been previously shown to eliminate HIV-1 infected cells in vitro and are candidates for passive immunotherapy to reduce the virus reservoir. However, their potential utility as therapy for infant HIV-1 infection is unclear as the ability of these novel antibody-based molecules to work in concert with cells of the infant immune system had not been assessed. Here, we use human umbilical cord blood as a model of the naïve neonatal immune system to evaluate the ability of HIV x CD3 DART molecules to recruit and redirect neonatal effector cells for elimination of autologous CD4+ T cells infected with HIV-1 encoding an envelope gene sequenced from a mother-to-child transmission event. We found that HIV × CD3 DART molecules can redirect T cells present in cord blood for elimination of HIV-infected CD4+ T cells. However, we observed reduced killing by T cells isolated from cord blood when compared to cells isolated from adult peripheral blood—likely due to the absence of the memory and effector CD8+ T cells that are most cytolytic when redirected by bispecific DART molecules. We also found that newly developed HIV × CD16 DART molecules were able to recruit CD16-expressing natural killer cells from cord blood to eliminate HIV-infected cells, and the activity of cord blood natural killer cells could be substantially increased by priming with IL-15. Our results support continued development of HIV-specific DART molecules using relevant preclinical animal models to optimize strategies for effective use of this immune therapy to reduce HIV-1 infection in pediatric populations.

Medulloblastoma rendered susceptible to NK-cell attack by TGF\u03b2 neutralization

BackgroundMedulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in a subset of patients with high risk disease, or at recurrence, where current therapies are ineffective. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy due to their recognition of malignant cells without the need for a known target, ready availability from multiple banks, and their potential to expand exponentially. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment including Transforming Growth Factor β (TGF-β). Here, we address this challenge in in vitro models of MB.MethodsCB-derived NK cells were modified to express a dominant negative TGF-β receptor II (DNRII) using retroviral transduction. The ability of transduced CB cells to maintain function in the presence of medulloblastoma-conditioned media was then assessed.ResultsWe observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence of TGF-β-rich, medulloblastoma-conditioned media (21.21 ± 1.19% killing at E:T 5:1 in the absence vs. 14.98 ± 2.11% in the presence of medulloblastoma-conditioned media, n = 8, p = 0.02), but was unaffected in CB-derived DNRII-transduced NK cells (21.11 ± 1.84% killing at E:T 5:1 in the absence vs. 21.81 ± 3.37 in the presence of medulloblastoma-conditioned media, n = 8, p = 0.85. We also observed decreased expression of CCR2 in untransduced NK cells (mean CCR2 MFI 826 ± 117 in untransduced NK + MB supernatant from mean CCR2 MFI 1639.29 ± 215 in no MB supernatant, n = 7, p = 0.0156), but not in the transduced cells. Finally, we observed that CB-derived DNRII-transduced NK cells may protect surrounding immune cells by providing a cytokine sink for TGF-β (decreased TGF-β levels of 610 ± 265 pg/mL in CB-derived DNRII-transduced NK cells vs. 1817 ± 342 pg/mL in untransduced cells; p = 0.008).ConclusionsCB 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 on its potential applications for patients with medulloblastoma.

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.