Expanded Natural Killer Research Articles

Sequential Exposure to IL21 and IL15 During Human Natural Killer Cell Expansion Optimizes Yield and Function.

Natural killer (NK) cells are frequently expanded for the clinic using irradiated, engineered K562 feeder cells expressing a core transgene set of membrane-bound (mb) IL15 and/or mbIL21 together with 41BBL. Prior comparisons of mbIL15 to mbIL21 for NK expansion lack comparisons of key attributes of the resulting NK cells, including their high-dimensional phenotype, polyfunctionality, the breadth and potency of cytotoxicity, cellular metabolism, and activity in xenograft tumor models. Moreover, despite multiple rounds of K562 stimulation, studies of sequential use of mbIL15- and mbIL21-based feeder cells are absent. We addressed these gaps and found that using mbIL15- versus mbIL21-based feeder cells drove distinct phenotypic and functional profiles. Feeder cells expressing mbIL15 alone drove superior functionality by nearly all measures, whereas those expressing mbIL21 alone drove superior yield. In combination, most attributes resembled those imparted by mbIL21, whereas in sequence, NK yield approximated that imparted by the first cytokine, and the phenotype, transcriptome, and function resembled that driven by the second cytokine, highlighting the plasticity of NK cell differentiation. The sequence mbIL21 followed by mbIL15 was advantageous in achieving significant yields of highly functional NK cells that demonstrated equivalent in vivo activity to those expanded by mbIL15 alone in two of three xenograft models. Our findings define the impact of mbIL15 versus mbIL21 during NK expansion and reveal a previously underappreciated tradeoff between NK yield and function for which sequential use of mbIL21-based followed by mbIL15-based feeder cells may be the optimal approach in many settings.

Comparison of the different anti-CD16 antibody clones in the activation and expansion of peripheral blood NK cells

Natural killer (NK) cells are promising tool for cancer treatment. Methods have been developed for large-scale NK cell expansion, including feeder cell-based methods or methods involving stimulation with NK cell activating signals, such as anti-CD16 antibodies. Different clones of anti-CD16 antibodies are available; however, a comprehensive comparison of their differential effects on inducing NK cell activation and expansion has not been conducted among these various clones under the same experimental conditions. Herein, we found that the NK cell expansion rate differed depending on the various anti-CD16 antibodies (CB16, 3G8, B73.1, and MEM-154) coated on microbeads when stimulated with genetically engineered feeder cells, K562‑membrane-bound IL‑18, and mbIL‑21 (K562‑mbIL‑18/-21). Only the CB16 clone combination caused enhanced NK cell expansion over K562‑mbIL‑18/-21 stimulation alone with similar NK cell functionality. Treatment with the CB16 clone once on the initial day of NK cell expansion was sufficient to maximize the combination effect. Overall, we developed a more enhanced NK expansion system by merging a feeder to effectively stimulate CD16 with the CB16 clone.

243 - Significantly Targeting Acute Myeloid Leukemia with the Combination of Anti-CD123 CAR Modified Expanded Natural Killer Cells and Romidepsin

243 - Significantly Targeting Acute Myeloid Leukemia with the Combination of Anti-CD123 CAR Modified Expanded Natural Killer Cells and Romidepsin

Expanded NK cells used for adoptive cell therapy maintain diverse clonality and contain long-lived memory-like NK cell populations

Multiple clinical trials exploring the potential of adoptive natural killer (NK) cell therapy for cancer have employed exvivo expansion using feeder cells to obtain large numbers of NK cells. We have previously utilized the rhesus macaque model to clonally track the NK cell progeny of barcode-transduced CD34+ stem and progenitor cells after transplant. In this study, NK cells from barcoded rhesus macaques were used to study the changes in NK cell clonal patterns that occurred during exvivo expansion using culture protocols similar to those employed in clinical preparation of human NK cells including irradiated lymphoblastoid cell line (LCL) feeder cells or K562 cells expressing 4-1BBL and membrane-bound interleukin-21 (IL-21). NK expansion cultures resulted in the proliferation of clonally diverse NK cells, which, at day 14 harvest, contained greater than 50% of the starting barcode repertoire. Diversity as measured by Shannon index was maintained after culture. With both LCL and K562 feeders, proliferation of long-lived putative memory-like NK cell clones was observed, with these clones continuing to constitute a mean of 31% of the total repertoire of expanded cells. These experiments provide insight into the clonal makeup of expanded NK cell clinical products.

Integrated single-cell transcriptomics of cerebrospinal fluid cells in treatment-naïve multiple sclerosis

Multiple sclerosis (MS) is a chronic and often disabling autoimmune disease of the central nervous system (CNS). Cerebrospinal fluid (CSF) surrounds and protects the CNS. Analysis of CSF can aid the diagnosis of CNS diseases, help to identify the prognosis, and underlying mechanisms of diseases. Several recent studies have leveraged single-cell RNA-sequencing (scRNA-seq) to identify MS-associated changes in CSF cells that are considerably more altered than blood cells in MS. However, not all alterations were replicated across all studies. We therefore integrated multiple available scRNA-seq datasets of CSF cells from MS patients with early relapsing–remitting (RRMS) disease. We provide a searchable and interactive resource of this integrated analysis (https://CSFinMS.bxgenomics.com) facilitating diverse visualization and analysis methods without requiring computational skills. In the present joint analysis, we replicated the known expansion of B lineage and the recently described expansion of natural killer (NK) cells and some cytotoxic T cells and decrease of monocytes in the CSF in MS. The previous observation of the abundance of Th1-like Th17 effector memory cells in the CSF was not replicated. Expanded CSF B lineage cells resembled class-switched plasmablasts/-cells (e.g., SDC1/CD138, MZB1) as expected. Our integrative analysis thus validates increased cell type diversity and B cell maturation in the CSF in MS and improves accessibility of available data.

Higher-Dose Donor Derived Ex Vivo Expanded Natural Killer Cell Infusion Prior to Haploidentical Stem Cell Transplantation on Acute Myeloid Leukemia: Efficacy and Safety

Adoptive immunotherapy with donor-derived natural killer (NK) cell was deemed as a promising therapeutic in hematological malignancies with anti-cancer, anti-graft-versus-host disease (GvHD), and anti-viral potential. However, the clinical usage was limited by lacking evidence on optimal dosage and timing. Here, we conducted a retrospective cohort study of compassionate use of higher dose (1x10^9/kg) NK cell infusion prior to haploidentical stem cell transplantation on acute myeloid leukemia. NK cells were obtained by 50ml donor peripheral blood through purifying, and ex vivo expanding with interleukin (IL)-2 and IL-15. Twelve patients enrolled in this cohort receiving an average dose of 1 x 109/kg (0.8 - 1.8 x 109/kg) NK cell infusion 12 hours prior to day 0. All patients achieved hematologic reconstitution. I° acute GVHD (aGVHD) appeared in three patients (25%). No chronic GVHD (cGVHD) occurred. With a median follow-up of 12.5 months (range 9-62 months), eight patients are still alive and in disease free survival status. Minimal residual disease (MRD) in alive patient was sustaining negative on day +60, +180 +360 and +720. Three patients relapsed six months posttransplant and one patient died from late bone marrow failure. Our small cohort study implied that infusion of high doses ex vivo expanded donor-derived NK cell infusion prior to haploidentical stem cell transplantation was safe and could potentially have a role to decrease relapse of patients with high-risk AML.

Zoledronic acid mediated differential activation of NK cells in different organs of WT and Rag2-/- mice; stark differences between the bone marrow and gingivae

We have previously shown that natural killer (NK) cells expand, and increase their function after interaction with cells that exhibit a number of different knock-down genes. We hypothesized that deletion or knockdown of a variety of key genes such as RAG may cause de-differentiation of the cells which could lead to increased NK expansion and function since we have shown previously that NK cells are activated and expanded by less differentiated cells. When comparing the function of NK cells from bone marrow (BM), spleen, pancreas, adipose tissue, and gingiva from WT mice to those from Rag2-/- mice, we observed a significant increase in IFN-γ secretion in all tissues of Rag2-/- mice versus in WT mice, with the exception of the gingivae in which similar levels were observed. After injecting WT mice with zoledronic acid (ZOL) and tooth extraction, immune cells from BM, spleen, and purified NK cells from spleen exhibited very high induction of IFN-γ and NK cell-mediated cytotoxicity with the exception of gingiva in which immune cells exhibited the opposite. In Rag2-/- mice, ZOL injection and tooth extraction stimulated IFN-γ secretion from BM immune cells but inhibited IFN-γ secretion from both spleen and gingivae. In both WT and Rag2-/- mice, immune cells from gingivae exhibited decreased IFN-γ secretion when activated, indicating significant regulation of immune cell function in the gingival microenvironment. However, even though significantly lower induction of IFN-γ was observed in both WT and Rag2-/- gingival cells after ZOL injection, ZOL mediated secretion of IFN-γ was still higher in the gingivae of WT mice when compared to those of Rag2-/- gingival cells. These results suggest an important role for IFN-γ in the pathogenesis of osteonecrosis lesions observed in post-tooth extraction jawbone.

A Phase I Study to Determine the Maximum Tolerated Dose of ex Vivo Expanded Natural Killer Cells Derived from Unrelated, HLA-Disparate Adult Donors

The administration of allogeneic natural killer (NK) cells following a lymphodepleting chemotherapy regimen is emerging as a well-tolerated therapeutic approach in the management of various malignancies. Contrary to the expected complications of allogeneic T cell therapy, there remains no evidence of graft-versus-host disease (GVHD) mediated by NK cells in numerous clinical trials. On the contrary, preclinical and clinical studies suggest that NK cells do not induce GVHD and in fact may prevent its development following allogeneic hematopoietic cell transplantation (HCT). In this study, we sought to determine the maximum tolerated dose of non-HLA-matched donor NK cells derived from peripheral blood and ex vivo expanded using a novel feeder cell platform. In a single-center Phase I clinical trial using a 3×3 design, 9 subjects each received 2 infusions of NK cells 2 weeks apart following a preparative regimen of cyclophosphamide (60 mg/kg i.v.) and fludarabine (25 mg/m2/day i.v for 5 days). No exogenous cytokines were administered. NK cells were administered at 3 dose levels: 1×107/kg, 2.5×107/kg, and 5×107/kg. Three subjects had myelodysplastic syndrome (MDS) or acute myelogenous leukemia (AML), and the other 6 subjects had colorectal carcinoma. Recipients were monitored over a 4-week period for GVHD as well as other adverse events and for persistence of donor NK cells in systemic circulation. Disease assessment was started at 28 days following the first NK cell infusion and continued until postinfusion day 100 or disease progression. In all 9 study subjects, there was no occurrence of GVHD and no dose-limiting toxicities that would warrant cohort expansion at any of the 3 planned cell dose levels. Low-level donor NK cell persistence was observed up to 4 weeks after the first NK cell infusion at all dose levels. The best observed response was a complete response with incomplete platelet recovery in a MDS subject who experienced disease relapse after prior allogeneic HCT. Other responses were stable disease in 1 subject with MDS and 2 subjects with colorectal cancer up to postinfusion day 100. This off-the-shelf, third-party NK cell product can be administered safely without inducing GVHD and exhibits in vivo persistence promoted by preparative lymphodepletion alone. The observed clinical responses could be enhanced by administration of exogenous cytokine support, as well as complementary approaches that promote NK cell function in the tumor microenvironment.

A Transcriptional Signature of IL-2 Expanded Natural Killer Cells Predicts More Favorable Prognosis in Bladder Cancer.

Activation of natural killer (NK) cell function is regulated by cytokines, such as IL-2, and secreted factors upregulated in the tumor microenvironment, such as platelet-derived growth factor D (PDGF-DD). In order to elucidate a clinical role for these important regulators of NK cell function in antitumor immunity, we generated transcriptional signatures representing resting, IL-2-expanded, and PDGF-DD-activated, NK cell phenotypes and established their abundance in The Cancer Genome Atlas bladder cancer (BLCA) dataset using CIBERSORT. The IL-2-expanded NK cell phenotype was the most abundant in low and high grades of BLCA tumors and was associated with improved prognosis. In contrast, PDGFD expression was associated with numerous cancer hallmark pathways in BLCA tumors compared with normal bladder tissue, and a high tumor abundance of PDGFD transcripts and the PDGF-DD-activated NK cell phenotype were associated with a poor BLCA prognosis. Finally, high tumor expression of transcripts encoding the activating NK cell receptors, KLRK1 and the CD160–TNFRSF14 receptor–ligand pair, was strongly correlated with the IL-2-expanded NK cell phenotype and improved BLCA prognosis. The transcriptional parameters we describe may be optimized to improve BLCA patient prognosis and risk stratification in the clinic and potentially provide gene targets of therapeutic significance for enhancing NK cell antitumor immunity in BLCA.

Ex vivo expanded allogeneic natural killer cells have potent cytolytic activity against cancer cells through different receptor-ligand interactions

BackgroundRecently, allogeneic natural killer (NK) cells have gained considerable attention as promising immunotherapeutic tools due to their unique biological functions and characteristics. Although many NK expansion strategies have been reported previously, a deeper understanding of cryopreserved allogeneic NK cells is needed for specific therapeutic approaches.MethodsWe isolated CD3−CD56+ primary natural killer (pNK) cells from healthy donors and expanded them ex vivo using a GMP-compliant method without any feeder to generate large volumes of therapeutic pNK cells and cryopreserved stocks. After validation for high purity and activating phenotypes, we performed RNA sequencing of the expanded and cryopreserved pNK cells. The pNK cells were used against various cancer cell lines in 7-AAD/CFSE cytotoxicity assay. For in vivo efficacy study, NSG mice bearing subcutaneous cisplatin-resistant A2780cis xenografts were treated with our pNK cells or cisplatin. Antitumor efficacy was assessed by measuring tumor volume and weight.ResultsCompared to the pNK cells before expansion, pNK cells after expansion showed 2855 upregulated genes, including genes related to NK cell activation, cytotoxicity, chemokines, anti-apoptosis, and proliferation. Additionally, the pNK cells showed potent cytolytic activity against various cancer cell lines. Interestingly, our activated pNK cells showed a marked increase in NKp44 (1064-fold), CD40L (12,018-fold), and CCR5 (49-fold), and did not express the programmed cell death protein 1(PD-1). We also demonstrated the in vitro and in vivo efficacies of pNK cells against cisplatin-resistant A2780cis ovarian cancer cells having a high programmed death-ligand 1(PD-L1) and low HLA-C expression.ConclusionsTaken together, our study provides the first comprehensive genome wide analysis of ex vivo-expanded cryopreserved pNK cells. It also indicates the potential use of expanded and cryopreserved pNK cells as a highly promising immunotherapy for anti-cancer drug resistant patients.

P-006: Expanded natural killer cells with daratumumab, lenalidomide and dexamethasone combination potentiates antimyeloma activity in a myeloma xenograft model

<h3>Background</h3> Advances in the treatment of multiple myeloma (MM) significantly improved overall survival of patients. Nonetheless, MM still remains an incurable disease. This emphasizes urgent need for novel combination therapies for the treatment of MM. Recently, natural killer (NK) cells are becoming an attractive and safe tool in the field of immunotherapy. <h3>Methods</h3> Here, we investigated how daratumumab, lenalidomide, and dexamethasone (DRd) treatment potentiate antitumor effects of NK cells in a MM xenograft mouse model. NK cells were expanded and activated using K562-OX40 ligand and membrane-bound IL-18 and IL-21 in the presence of IL-2 and IL-15 from peripheral blood mononuclear cells of MM patients. A human MM xenograft model was established using human RPMI8226-RFP-FLuc cells in NOD/SCID IL-2Rγnull (NSG) mice. Tumor bearing mice were divided into six treatment groups: no treatment, expanded NK cells (eNK), Rd, Rd + eNK, DRd, and DRd + eNK. DRd treatment strongly enhanced the cytotoxicity of eNK by upregulating NK cell's activation ligands and effector function. <h3>Results</h3> DRd combination with eNK significantly prolonged mouse survival and reduced MM progression and serum M-protein level. Moreover, DRd treatment significantly increased eNK persistence and homing into MM sites. <h3>Conclusions</h3> Our findings suggest that DRd treatment augments the anti-myeloma effects of ex vivo expanded and activated NK cells by modulating immune responses in MM-bearing mice.

Therapeutic Effects of a Novel Fusion of ALT-803, an IL-15 Superagonist, with 4 Single-Chains of Anti-CD20 Antibody (2B8T2M) in Combination with Expanded Natural Killer Cells Against Rituximab Sensitive and Resistant Burkitt Lymphoma (BL)

Background:The prognosis of patients with relapsed or progressed B-cell (CD20+) non-Hodgkin lymphoma (B-NHL), including Burkitt lymphoma (BL), is dismal due to chemo-radiotherapy resistance (Cairo et al, JCO, 2012, Cairo et al, BJH, 2016). Rituximab has been widely used in frontline treatment of B-NHL, however, some patients retreated with rituximab relapse which limit patient treatment options (Goldman/Cairo, Leukemia, 2013). Several strategies for overcoming rituximab-resistance are currently being evaluated, including engineering immune cells with chimeric antigen receptors (Chu/Cairo et al, Can Imm Res 2015), as well as second-generation engineered anti-CD20 antibodies (Tiwari/Cairo et al BJH 2015). Nature Killer (NK) cells are bone marrow-derived cytotoxic lymphocytes that play a major role in the rejection of tumors. A variety of activating and inhibitory receptors on the NK cell surface are engaged to regulate NK cell activities. However, NK therapy is limited by several factors, including small numbers of active NK cells in unmodified peripheral blood, lack of tumor targeting specificity, and multiple mechanisms of tumor escape of NK cell immunosurveillance. Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells (Chu/Cairo, et al, Can Imm Res 2015). ALT-803 (Altor BioScience Corporation) is a superagonist of an IL-15 variant bound to an IL-15RαSu-Fc fusion with enhanced IL-15 biological activity (Zhu et al . 2009 J Immunol), longer half-life and increased potency (Han, et al . Cytokine . 2011). Recently, a new therapeutic molecule 2B8T2M was generated by fusing ALT-803 to four single-chains of the tumor-targeting monoclonal antibody, rituximab (Liu/Wong, et al, JBC, 2016). 2B8T2M displayed tri-specific binding activity through its recognition of the CD20 molecule on tumor cells, activated NK cells to enhance antibody-dependent cellular cytotoxicity (ADCC), and induced apoptosis of B-lymphoma cells (Liu/Wong, et al, JBC, 2016).Objective: To examine if 2B8T2M significantly enhances the cytotoxicity of exPBNK against rituximab-sensitive and -resistant BL cells.Method: PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells (Denman/Dean Lee, PLoS One, 2012). CD56+CD3- exPBNK cells were isolated using Miltenyi NK cell isolation kit. ALT-803 and 2B8T2M were generously provided by Altor BioScience Corporation. NK receptors expression and cytotoxicity were examined as we previous described (Chu/Cairo, et al, Can Imm Res 2015). IFN-γ and granzyme B levels were examined by standard enzyme-linked immunosorbent assays. Equal doses of Rituximab, ALT-803, Rituximab+ALT-803, obinutuzumab (obinu) were used for comparison. IgG was used as controls. Rituximab-sensitive and -resistant BL cells Raji, Raji-2R and Raji-4RH, were used as target cells.Results: Both ALT-803 and 2B8T2M at equal doses significantly enhanced NK activating receptors such as NKG2D, NKp30, NKp44, and CD16 on exPBNK cells compared to IgG controls.2B8T2M significantly enhanced exPBNK cytotoxicity against rituximab-sensitive Raji cells compared to the controls IgG, Rituximab, ALT-803, Rituximab+ALT-803, obinu (2B8T2M vs IgG vs ALT803 vs Rituximab vs ALT803+Rituximab vs obinu=98.34%+0.24% vs 56.5+1.83% vs 62.8+1.1% vs 69.52+1.79% vs 81.85+1.83% vs 81.23+1.8%, p<0.001, E:T=1:1). 2B8T2M also significantly enhanced exPBNK cytotoxicity against rituximab-resistant Raji-2R cells (2B8T2M vs IgG vs ALT803 vs Rituximab vs ALT803+Rituximab vs obinu=90.7%+1.36% vs 20.47+7.7% vs 48.18+8.07% vs 37.44+3.63% vs 51.02+1.42% vs 65.51+3.03%, p<0.001, E:T=1:1) and resistant Raji-4RH cells (p<0.001, E:T=1:1) (Fig.1). Furthermore, we confirmed the enhanced cytotoxicity by measuring IFN-γ and granzyme B production. 2B8T2M significantly enhanced IFN-γ and granzyme B production from exPBNK against rituximab-sensitive Raji and -resistant Raji-2R and Raji-4RH compared to IgG (p<0.001), Rituximab (p<0.001), ALT-803 (p<0.001), Rituximab+ALT-803 (p<0.001), and obinutuzumab (p<0.001).Conclusions: 2B8T2M significantly enhanced exPBNK activating receptor expression and in vitro cytotoxicity against rituximab-sensitive and -resistant BL cells. The in vivo functions of 2B8T2M with exPBNK against rituximab-sensitive and -resistant BL cells using humanized NSG models are under investigation. [Display omitted] DisclosuresAlter:Altor BioScience: Employment, Equity Ownership. Jeng:Altor Bioscience: Employment, Equity Ownership. Rhode:Altor BioScience: Employment, Equity Ownership. Lee:Intellia Therapeutics, Inc: Consultancy; Courier Therapeutics, Inc: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Speakers Bureau; Cyto-Sen Therapeutics, Inc: Other: Founder, Vice President, Medical Director. Wong:Altor BioScience: Employment, Equity Ownership. Cairo:Jazz Pharmaceuticals: Speakers Bureau.

Activated and Expanded Natural Killer Cells Expressing an NKG2D-CAR Efficiently Target Multiple Myeloma Cells

Background:Multiple myeloma (MM) remains an incurable disease and new therapeutic venues are required. Immune-based therapies represent a new weapon in the fight against cancer. Natural killer (NK) cells have an important role as natural control of tumor cells. Based on that, we carried out a proof-of-concept phase I clinical trial to make multiple infusions of autologous activated and expanded NK cells (NKAE) together with anti-myeloma drugs in MM (NCT02481934). In spite of the good clinical results, better responses are needed to erradicate MM. Chimeric antigen receptors (CARs) are emerging as a very good treatment strategy against MM. CAR studies have focused on their expression in T cells. However, CAR-NK cells could have lower toxicity and provides a method to redirect these cells more specifically to target refractory cancer. As our group has previously demonstrated an essential role of NKG2D receptor in NK cell cytotoxic activity, the objective of this work was to combine NK cell activation with the expression of an NKG2D-CAR in primary NK cells from MM patients.Methods:NKAE cells were generated by co-culture of peripheral blood mononuclear cell (PBMCs) with the genetically modified and previously irradiated K562 CL9 mIL21 cell line (kindly given by the Nationwide Children's Hospital) at a 1:1.5 ratio, and interleukin-2 at 100 IU/ml. NKAE cells were transduced with NKG2D CARs containing 4-1BB and CD3z signaling domains (kindly provided by the Perelman School of Medicine). The viral supernatant was produced by transient transfection of HEK293T cells with the vector genome plasmid and lentiviral packaging helper plasmids. NKAE expansion and NKG2D-CAR expression were evaluated by flow cytometry based on the percentage of the NK cell population and on the expression of the activating receptor NKG2D. The gating strategy was based on dead/live cells and doublet discrimination. NKAE-CAR in vitro cytotoxicity against MM cell lines was tested by performing 2-hour europium-TDA release assays.Results:NKAE cells were expanded during 10 days, after that transduction was performed using MOI=5. Better transduction efficiency was found on purified NKAE cells (100% purity) than on NKAEs which were not purified (purity higher than 80%). One day after transduction purified NKAE cells show an increase of NKG2D positive cells (62,6%±12,2%) when compare with untransduced NKAE cells (n=4). NKAE-CAR cells had similar viability as the untransduced NKAE cells. NKG2D-CAR expression was monitored 72 hours after transduction, 24% of NKG2D positive cells was due to episomal expression (38,5%±1,54%) but remain stable up to 7 days (36,8±11,06%). NKAE-CAR in vitro cytotoxic activity was tested at different NKAE-CAR:MM cells ratio (1:8 up to 32:1) using MM L-363 cell line as target (n=4). NKG2D CAR-expressing NKAE cells showed significantly higher cytotoxic activity against myeloma cells than untransduced NKAE cells even at low ratio. NKAE-CAR cells showed maximum cytotoxic capacity at 8:1 ratio (112%±19,2% lysed MM cells), however untransduced NKAE cells showed maximum activity at 32:1 ratio (67,5%±15,8%). At same ratio (8:1), NKAE-CAR cells were able to destroy 112% (±19,2%) MM cells, 53,75% more than untransduced NKAE cells. At very low ratio (1:8) NKG2D-CAR expressing NKAE cells could destroy 42,4% (±2,4%) MM cells.Conclusions:Primary NK cells from MM patients can be activated and transduced in vitro with an NKG2D CAR showing higher cytotoxic activity that untransduced NKAE cells. These results warrant further development of NKAEs-CAR as a new treatment modality for refractory MM. DisclosuresLee:Miltenyi: Speakers Bureau; Courier Therapeutics, Inc: Membership on an entity's Board of Directors or advisory committees; Cyto-Sen Therapeutics, Inc: Other: Founder, Vice President, Medical Director; Intellia Therapeutics, Inc: Consultancy.

The IL15 Superagonist Complex ALT-803 Potently Enhances the Proliferative Capacity and Activity of in Vitro Expanded Natural Killer Cells

IntroductionWe previously reported on the treatment of 8 relapsing high-risk multiple myeloma (HRMM) patients with ex vivo activated/expanded NK cells (ENKs). We demonstrated that interleukin-2 (IL2) provided suboptimal cytokine support post adoptive transfer of ENKs. Aiming to improve upon prior data we hypothesize that the novel IL15 superagonist complex ALT-803 will provide superior support compared to IL2 and enhance ENK activity, proliferation and persistence.MethodsTo compare the activity of IL2 and ALT-803 on ENKs, we performed long-term in vitro culture studies. Analogous to the 8-day clinical production method, healthy donor (HD) peripheral blood mononuclear cells (PBMCs) were first co-cultured with the leukemic cell line K562 modified to express both membrane-bound IL15 and the costimulatory molecule 4-1BBL, and high-dose IL2. We deliberately chose to maintain IL2 in the expansion method rather than substituting ALT-803 because our FDA-approved GMP-grade expansion was validated using IL2. On expansion Day 8 (the day clinical ENK products are infused), the ENKs were either maintained at high-dose (300U/mL) IL2, or otherwise cultured at low-dose (20U/mL) IL2 or 35ng/mL ALT-803, in all conditions without K562 stimulator cells. Proliferation (fold-change from baseline) was measured on Days 14, 21, and 28, and expression of key activating and inhibitory markers was assessed via flow cytometry. Relative cytolytic ability was determined via 4-hour chromium (Cr)-release assays against K562 and the MM cell lines OPM2, JJN3, and U266.ResultsProliferation of ALT-803-ENKs was superior to both IL2-ENK conditions at all assessed time points; on Day 28, fold-expansion of ALT-803-ENKs was 753, versus 156- and 557-fold for ENKs maintained in 20U or 300U/mL IL2, respectively, Figure 1A. Immunophenotyping studies demonstrated significant differences between ENKs cultured in 20U/mL IL2 versus ALT-803-treated ENKs, including activation (CD26, CD69, DNAM1, NKp30) and functional (CD16, perforin, granzyme, TRAIL) molecules. ALT-803-treated ENKs maintained an activated phenotype, whereas IL2 ENKs (20U/mL) exhibited reduced expression of several markers by Day 14, with further decreases noted on Days 21 and 28. Representative examples are depicted in Figure 1B. Morphologically, IL2-ENKs acquired apoptotic features after extended culture, whereas ALT-803-ENKs did not. Though cytolytic ability was variable on Days 14 and 21, the activity of ALT-803-ENKs exceeded that of IL2-ENKs (20U/mL) against K562, OPM2, JJN3, and U266 cell lines on Day 28. High-dose IL2-ENKs exhibited a similar phenotype and cytolytic ability compared to ALT-803-ENKs.ConclusionsWe compared clinically-relevant doses of IL2 and ALT-803 to determine whether ALT-803 is superior at maintaining ENK activity and proliferative capacity. ALT-803 ENKs exhibited robust proliferation through Day 28 and sustained expression of key activation and functional markers, highlighting the superiority of ALT-803 in this setting. After four weeks of continuous culture, ALT-803-ENKs maintained greater cytolytic ability against MM cell lines. Though high-dose IL2 is capable of sustaining ENK activation state in vitro, this dose is not achievable in humans. Unlike IL2, ALT-803 does not induce expansion of T regulatory cells, and has a more favorable toxicity profile. Importantly, ALT-803 stimulated and maintained expression of CD16, indicating that it would be well-suited to pair with monoclonal antibodies for the support of antibody-dependent cellular cytotoxicity. Further studies are in progress to study the effects of ALT-803 on ENKs in the presence of suppressive cytokines/cells to determine if ALT-803 is able to overcome the effects of the tumor microenvironment. [Display omitted] DisclosuresWong:Altor BioScience: Employment, Equity Ownership. Jeng:Altor Bioscience: Employment, Equity Ownership. Shrestha:Altor Bioscience: Employment, Equity Ownership. Davies:Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Morgan:Bristol Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Expanded natural killer cells augment the antimyeloma effect of daratumumab, bortezomib, and dexamethasone in a mouse model.

The use of natural killer (NK) cells is a promising and safe immunotherapeutic approach in the field of cancer immunotherapy. However, combination treatments are required to enhance the effector functions and therapeutic efficacy of NK cells. In this study, we investigated the potential of daratumumab (Dara), bortezomib, and dexamethasone (Dvd) to augment the antitumor effects of NK cells in a multiple myeloma (MM) xenograft mouse model. NK cells were expanded and activated using the K562-OX40 ligand and membrane-bound IL-18 and IL-21 in the presence of IL-2 and IL-15 from peripheral blood mononuclear cells from MM patients. A human MM xenograft model was established using human RPMI8226-RFP-FLuc cells in NOD/SCID IL-2Rγnull (NSG) mice. Tumor-bearing mice were divided into six treatment groups: no treatment, expanded NK cells (eNKs), Dara, Dara + eNKs, Dvd, and Dvd + eNKs. Dvd treatment strongly enhanced the cytotoxicity of eNKs by upregulating expression of NK cell activation ligands, downregulating expression of NK cell inhibitory ligands, and promoting antibody-dependent cellular cytotoxicity. The combination of eNKs with Dvd significantly prolonged mouse survival and reduced the tumor burden and serum M-protein level. Furthermore, Dvd pretreatment significantly increased eNK persistence and homing to MM sites. Our findings suggest that Dvd treatment potentiates the antimyeloma effects of NK cells expanded and activated ex vivo by modulating immune responses in MM-bearing mice.

Heterodimeric IL-15 in Cancer Immunotherapy.

Simple SummaryThe rapidly expanding field of cancer immunotherapy uses diverse technologies, including cytokines, T cells, and antibody administration, with the aim to induce effective immune responses leading to tumor control. Interleukin-15 (IL-15), a cytokine discovered in 1994, supports the homeostasis of cytotoxic immune cells and shows promise as an anti-tumor agent. Many studies have elucidated IL-15 synthesis, regulation and biological function and explored its therapeutic efficacy in preclinical cancer models. Escherichia coli-derived single-chain IL-15 was tested in the first in-human trial in cancer patients. Its effects were limited by the biology of IL-15, which in vivo comprises a complex of the IL-15 chain with the IL-15 receptor alpha (IL-15Rα) chain, together forming the IL-15 heterodimer (hetIL-15). Currently, single-chain IL-15 and several heterodimeric IL-15:IL-15Rα variants (hetIL-15, N-803 and RLI) are being tested in clinical trials. This review presents a summary of contemporary preclinical and clinical research on IL-15.Immunotherapy has emerged as a valuable strategy for the treatment of many cancer types. Interleukin-15 (IL-15) promotes the growth and function of cytotoxic CD8+ T and natural killer (NK) cells. It also enhances leukocyte trafficking and stimulates tumor-infiltrating lymphocytes expansion and activity. Bioactive IL-15 is produced in the body as a heterodimeric cytokine, comprising the IL-15 and the so-called IL-15 receptor alpha chain that are together termed “heterodimeric IL-15” (hetIL-15). hetIL-15, closely resembling the natural form of the cytokine produced in vivo, and IL-15:IL-15Rα complex variants, such as hetIL-15Fc, N-803 and RLI, are the currently available IL-15 agents. These molecules have showed favorable pharmacokinetics and biological function in vivo in comparison to single-chain recombinant IL-15. Preclinical animal studies have supported their anti-tumor activity, suggesting IL-15 as a general method to convert “cold” tumors into “hot”, by promoting tumor lymphocyte infiltration. In clinical trials, IL-15-based therapies are overall well-tolerated and result in the expansion and activation of NK and memory CD8+ T cells. Combinations with other immunotherapies are being investigated to improve the anti-tumor efficacy of IL-15 agents in the clinic.

Phase 2 Clinical Trial of Infusing Haploidentical K562-mb15-41BBL–Activated and Expanded Natural Killer Cells as Consolidation Therapy for Pediatric Acute Myeloblastic Leukemia

BackgroundAcute myeloid leukemia (AML) accounts for approximately 20% of pediatric leukemia cases; 30% of these patients experience relapse. The antileukemia properties of natural killer (NK) cells and their safety profile have been reported in AML therapy. We proposed a phase 2, open, prospective, multicenter, nonrandomized clinical trial for the adoptive infusion of haploidentical K562-mb15-41BBL–activated and expanded NK (NKAE) cells as a consolidation strategy for children with favorable and intermediate risk AML in first complete remission after chemotherapy (NCT02763475). Patients and MethodsBefore the NKAE cell infusion, patients underwent a lymphodepleting regimen. After the NKAE cell infusion, patients were administered low doses (1 × 106/IU/m2) of subcutaneous interleukin-2. The primary study endpoint was AML relapse-free survival. We needed to include 35 patients to demonstrate a 50% reduction in relapses. ResultsSeven patients (median age, 7.4 years; range, 0.78-15.98 years) were administered 13 infusions of NKAE cells, with a median of 36.44 × 106 cells/kg (range, 6.92 × 106 to 193.2 × 106 cells/kg). We observed chimerism in 4 patients (median chimerism, 0.065%; range, 0.05-0.27%). After a median follow-up of 33 months, the disease of 6 patients (85.7%) remained in complete remission. The 3-year overall survival was 83.3% (95% confidence interval, 68.1-98.5), and the cumulative 3-year relapse rate was 28.6% (95% confidence interval, 11.5-45.7). The study was terminated early because of low patient recruitment. ConclusionThis study emphasizes the difficulties in recruiting patients for cell therapy trials, though NKAE cell infusion is safe and feasible. However, we cannot draw any conclusions regarding efficacy because of the small number of included patients and insufficient biological markers.

Using Expanded Natural Killer Cells as Therapy for Invasive Aspergillosis.

Invasive aspergillosis (IA) is a major opportunistic fungal infection in patients with haematological malignancies. Morbidity and mortality rates are high despite anti-fungal treatment, as the compromised status of immune system prevents the host from responding optimally to conventional therapy. This raises the consideration for immunotherapy as an adjunctive treatment. In this study, we evaluated the utility of expanded human NK cells as treatment against Aspergillus fumigatus infection in vitro and in vivo. The NK cells were expanded and activated by K562 cells genetically modified to express 4-1BB ligand and membrane-bound interleukin-15 (K562-41BBL-mbIL-15) as feeders. The efficacy of these cells was investigated in A. fumigatus killing assays in vitro and as adoptive cellular therapy in vivo. The expanded NK cells possessed potent killing activity at low effector-to-target ratio of 2:1. Fungicidal activity was morphotypal-dependent and most efficacious against A. fumigatus conidia. Fungicidal activity was mediated by dectin-1 receptors on the expanded NK cells leading to augmented release of perforin, resulting in enhanced direct cytolysis. In an immunocompromised mice pulmonary aspergillosis model, we showed that NK cell treatment significantly reduced fungal burden, hence demonstrating the translational potential of expanded NK cells as adjunctive therapy against IA in immunocompromised patients.

Current progress in cancer immunotherapy based on natural killer cells.

One of the most common diseases in the present era is cancer. The common treatment methods used to control cancer include surgery, chemotherapy, and radiotherapy. Despite progress in the treatment of cancers, there still isno definite therapeutic approach. Among the currently proposed strategies, immunotherapy is a new approach that can provide better outcomes compared with existing therapies. Employing natural killer (NK) cells is one of the meansof immunotherapy. As innate lymphocytes, NKcells are capable of rapidly responding to cancer cells without being sensitized or restricted to the cognate antigen in advance, as compared to T cells that are tumor antigen-specific. Latest insights into thebiology of NK cells have clarified the underlying molecular mechanisms of NK cell maturation and differentiation, as well as controlling their effector functions through the investigation of the ligands and receptors engaged in recognizing cancer cells by NK cells. Elucidating the fact that NK cells recognize cancer cells could similarly show the mechanism through which cancer cells possibly avoid NK cell-dependent immune surveillance. Additionally, the expectations for novel immunotherapies by targeting NK cells have increased through the latest clinical outcomes of T-cell-targeted cancer immunotherapy. For this emerging method, researchers are still attempting to develop protocols for conferring the best proliferation and expansion medium, activation pathways, utilization dosage, transferring methods, as well as reducing possible side effects in cancer therapy. This study reviews the NK cells, their proliferation and expansion methods, and their recent applications in cancer immunotherapy.

Abstract 5046: Natural Killer (NK) human immune system (HIS) immunograft platform to evaluate the pharmacodynamics of immuno-oncology therapeutics

Abstract Background: Harnessing NK cell anti-cancer cytotoxicity has gained interest as a therapeutic strategy, and consequently improved preclinical models supporting the translation of NK cell-mediated therapies to the clinic are desired. Reproducible models with human NK engraftment into immunodeficient mice co-engrafted with cell line-derived xenograft or patient-derived xenograft tumor models have been lacking due to an inability to support NK cell engraftment and persistence. Here we evaluated IL-15-NOG mice for the engraftment and sustained survival of both ex vivo expanded and primary human NK cell isolates for establishing models that engraft effectively with both human NK cells and a PDX or CDX tumor. Materials and Methods: NK cells from normal adult peripheral blood mononuclear cells (PBMC) donors (N=3) were expanded using two different commercially available kits and evaluated for NK phenotype, expansion rates and yields. Ex vivo expanded NK cells were tested for antibody dependent cell cytotoxicity (ADCC). Titrated doses of ex vivo expanded NK cells were adoptively transferred into IL-15-NOG mice for human chimerism, and the persistence and survival of NK cells and their immunophenotype were assessed. In separate studies, naïve NK cells enriched from PBMC were also evaluated for NK cell persistence and expansion in vivo. To establish an NK ImmunoGraft, NK cells were engrafted in xenograft tumor bearing mice (CDX or PDX) and tumor growth kinetics and infiltrating NK cells phenotype were characterized. Results: Donor dependent NK expansion was observed ex vivo, with 28 to 50-fold expansion by two weeks. NK cells expanded ex vivo were CD3−CD16+CD56± and varied based on the expansion kit utilized. ADCC mediated lysis was observed with ex vivo expanded NK cells. Nearly all CD45+ cells in circulation were NK cells, and these peaked by week 2, and were maintained for up to 10 weeks in IL-15-NOG mice. Primary NK cells engrafted with slower kinetics, with peak abundance at 3-4 weeks. NK cells expressed granzyme B, and further functional studies are in progress. For all NK cell populations, cell density-dependent engraftment was observed with a largely stable NK phenotype observed across the study. In the absence of any therapeutic treatment, NK cell persistence and expansion in vivo did not inhibit tumor xenograft growth kinetics in IL-15-NOG mice. Conclusions: IL-15-NOG mice support the survival and persistence of human NK cells from both ex vivo expanded and naïve NK cells, suggesting the universality of this platform for human NK engraftment. Our preliminary studies support IL-15 NOG mouse model as a suitable system for evaluation of NK cellular therapies or NK cell-modulating therapies in the context of patient-derived or cell-line derived xenograft (PDX or CDX) mouse models. Citation Format: Bhavna Verma, Jon Weidanz, Amy Wesa. Natural Killer (NK) human immune system (HIS) immunograft platform to evaluate the pharmacodynamics of immuno-oncology therapeutics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5046.