Natural Killer Cells For Adoptive Immunotherapy Research Articles

Optimizing the Procedure for Manufacturing Clinical-grade Genetically Manipulated NK cells for Adoptive Immunotherapy

BackgroundEx vivo expanded natural killer (NK) cells hold significant potential as antitumor effector cells for adoptive immunotherapy. However, producing clinical-grade genetically modified NK cells in sufficient quantities presents a considerable challenge. MethodsWe tested RPMI 1640, KBM581, SCGM, NK MACS, X-VIVO 15, and AIM-V, each supplemented with fetal bovine serum (FBS), human AB serum, human platelet lysate (HPL), or Immune Cell Serum Replacement (SR) combined with feeder cells, to produce cytotoxic NK cells. Subsequent analyses were conducted to assess cell viability, expansion folds, cytotoxicity, immunophenotype, and transcriptome profile of NK cells under certain conditions. Furthermore, transfer plasmids varying in transgene size, promoter elements, backbones and packaging plasmids with different envelopes were used to transduce NK cells and differences in transduction efficiency were compared. Nucleofection was performed every two days from Day 0 to Day 12 to determine the optimal time window for gene editing. ResultsNK cells cultured in KBM581 medium supplemented with SR exhibited the best expansion, achieving over 5000-fold increase within two weeks and exceeding 25,000-fold expansion within three weeks. Additionally, NK cells cultured in KBM581 medium with human AB serum demonstrated the highest cytolytic activities and exhibited higher expression of NKp30, 2B4, PRF1, Granzyme B, and IL2RG. Baboon envelope (BaEV) pseudotyped lentivirus outperformed BaEV-Vesicular Stomatitis Virus type-G (VSVG) hybrid envelope lentivirus, achieving robust NK cell transduction. Additionally, efficient gene knockout efficiency was achieved in NK cells on day 4 to day 6 post feeder cell activation using LONZA DN-100 program, which can strike a balance between editing efficiency and cell expansion. ConclusionsThis research presents a Good Manufacturing Practice (GMP)-compliant protocol utilizing a feeder cell expansion system for the large-scale production of highly cytotoxic Natural Killer (NK) cells. The protocol facilitates genetic modification of these cells, positioning them as promising candidates for universal therapeutic applications in immunotherapy.

OS-113 Adoptive natural killer cell immunotherapy can increase the recurrence-free survival following transarterialchemoembolization in patients with intermediate stage hepatocellular carcinoma

OS-113 Adoptive natural killer cell immunotherapy can increase the recurrence-free survival following transarterialchemoembolization in patients with intermediate stage hepatocellular carcinoma

Venetoclax acts as an immunometabolic modulator to potentiate adoptive NK cell immunotherapy against leukemia

Natural killer (NK) cell-based immunotherapy holds promise for cancer treatment; however, its efficacy remains limited, necessitating the development of alternative strategies. Here, we report that venetoclax, an FDA-approved BCL-2 inhibitor, directly activates NK cells, enhancing their cytotoxicity against acute myeloid leukemia (AML) both invitro and invivo, likely independent of BCL-2 inhibition. Through comprehensive approaches, including bulk and single-cell RNA sequencing, avidity measurement, and functional assays, we demonstrate that venetoclax increases the avidity of NK cells to AML cells and promotes lytic granule polarization during immunological synapse (IS) formation. Notably, we identify a distinct CD161lowCD218b+ NK cellsubpopulation that exhibits remarkable sensitivity to venetoclax treatment. Furthermore, venetoclax promotes mitochondrial respiration and ATP synthesis via the NF-κB pathway, thereby facilitating IS formation in NK cells. Collectively, our findings establish venetoclax as a multifaceted immunometabolic modulator of NK cell function and provide a promising strategy for augmenting NK cell-based cancer immunotherapy.

A Killer Disarmed: Natural Killer Cell Impairment in Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) treatment remains a big challenge due to the heterogeneous nature of the disease and its ability to progress to acute myeloid leukemia (AML). The only curative option is allogeneic hematopoietic stem cell transplantation (HSCT), but most patients are unfit for this procedure and are left with only palliative treatment options, causing a big unmet need in the context of this disease. Natural killer (NK) cells are attractive candidates for MDS immunotherapy due to their ability to target myeloid leukemic cells without prior sensitization, and in recent years we have seen an arising number of clinical trials in AML and, recently, MDS. NK cells are reported to be highly dysfunctional in MDS patients, which can be overcome by adoptive NK cell immunotherapy or activation of endogenous NK cells. Here, we review the role of NK cells in MDS, the contribution of the tumor microenvironment (TME) to NK cell impairment, and the most recent data from NK cell-based clinical trials in MDS.

Improved Antitumor Effect of NK Cells Activated by Neutrophils in a Bone Marrow Transplant Model.

The licensing process mediated by inhibitory receptors of the Ly49 C-type lectin superfamily that recognizes self-major histocompatibility complex (MHC) class I in mice is essential for the proper antitumor function of natural killer (NK) cells. Several models for NK cell licensing can be exploited for adoptive immunotherapy for cancer. However, the appropriate adoptive transfer setting to induce efficient graft versus tumor/leukemia effects remains elusive, especially after hematopoietic stem cell transplantation (HSCT). In our previous experiment, we showed that intraperitoneal neutrophil administration with their corresponding NK receptor ligand-activated NK cells using congenic mice without HSCT. In this experiment, we demonstrate enhanced antitumor effects of licensed NK cells induced by weekly intraperitoneal injections of irradiated neutrophil-enriched peripheral blood mononuclear cells (PBMNCs) in recipient mice bearing lymphoma. Bone marrow transplantation was performed using BALB/c mice (H-2d) as the recipient and B10 mice (H-2b) as the donor. The tumor was A20, a BALB/c-derived lymphoma cell line, which was injected subcutaneously into the recipient at the same time as the HSCT. Acute graft versus host disease was not exacerbated in this murine MHC class I mismatched HSCT setting. The intraperitoneal injection of PBMNCs activated a transient licensing of NK subsets expressed Ly49G2, its corresponding NK receptor ligand to H-2d, and reduced A20 tumor growth in the recipient after HSCT. Pathological examination revealed that increased donor-oriented NK1.1+NK cells migrated into the recipient tumors, depending on neutrophil counts in the administered PBMNCs. Collectively, our data reveal a pivotal role of neutrophils in promoting NK cell effector functions and adoptive immunotherapy for cancer.

Targeting immune checkpoints: how to use natural killer cells for fighting against solid tumors.

Natural killer (NK) cells are unique innate immune cells that mediate anti-viral and anti-tumor responses. Thus, they might hold great potential for cancer immunotherapy. NK cell adoptive immunotherapy in humans has shown modest efficacy. In particular, it has failed to demonstrate therapeutic efficiency in the treatment of solid tumors, possibly due in part to the immunosuppressive tumor microenvironment (TME), which reduces NK cell immunotherapy's efficiencies. It is known that immune checkpoints play a prominent role in creating an immunosuppressive TME, leading to NK cell exhaustion and tumor immune escape. Therefore, NK cells must be reversed from their dysfunctional status and increased in their effector roles in order to improve the efficiency of cancer immunotherapy. Blockade of immune checkpoints can not only rescue NK cells from exhaustion but also augment their robust anti-tumor activity. In this review, we discussed immune checkpoint blockade strategies with a focus on chimeric antigen receptor (CAR)-NK cells to redirect NK cells to cancer cells in the treatment of solid tumors.

Lipid-laden lung mesenchymal cells foster breast cancer metastasis via metabolic reprogramming of tumor cells and natural killer cells

While the distant organ environment is known to support metastasis of primary tumors, its metabolic roles in this process remain underdetermined. Here, in breast cancer models, we found lung-resident mesenchymal cells (MCs) accumulating neutral lipids at the pre-metastatic stage. This was partially mediated by interleukin-1β (IL-1β)-induced hypoxia-inducible lipid droplet-associated (HILPDA) that subsequently represses adipose triglyceride lipase (ATGL) activity in lung MCs. MC-specific ablation of the ATGL or HILPDA genes in mice reinforced and reduced lung metastasis of breast cancer respectively, suggesting a metastasis-promoting effect of lipid-laden MCs. Mechanistically, lipid-laden MCs transported their lipids to tumor cells and natural killer (NK) cells via exosome-like vesicles, leading to heightened tumor cell survival and proliferation and NK cell dysfunction. Blockage of IL-1β, which was effective singly, improved the efficacy of adoptive NK cell immunotherapy in mitigating lung metastasis. Collectively, lung MCs metabolically regulate tumor cells and anti-tumor immunity to facilitate breast cancer lung metastasis.

Natural Killer Cell Recognition and Control of Epithelial Cancers.

Natural killer (NK) cells possess an innate ability to recognize cancer and are key mediators of cytotoxic efficacy for anticancer antibodies. Recent advances in the ability to generate, qualify, and safely infuse NK cells have led to a wide variety of clinical trials in oncology. Although their efficacy is best established for liquid cancers, their potential application in solid cancers has received increased attention. Here, we provide general background across a disparate group of exemplary solid tumors for which there is evidence for an NK cell role, discuss NK cell recognition motifs specific to each and murine and human studies of each that are supportive of NK cell adoptive immunotherapy, and end with special considerations relevant to the solid tumor microenvironment.

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

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

Gold-seaurchin based immunomodulator enabling photothermal intervention and αCD16 transfection to boost NK cell adoptive immunotherapy

Despite huge potentials of NK cells in adoptive cell therapy (ACT), formidable physical barriers of the tumor tissue and deficiency of recognizing signals on tumor cells severely prevent NK cell infiltrating, activating and killing performances. Herein, a nano-immunomodulator AuNSP@αCD16 (CD16 antibody encoding plasmid) is explored to remodel the tumor microenvironment (TME) for improving the antitumor effects of adoptive NK cells. The as-prepared AuNSP, with a seaurchin-like gold core and a cationic polymer shell, exhibited a high gene transfection efficiency and a stable NIR-II photothermal capacity. The AuNSP could trigger mild photothermal intervention to partly destroy tumors and collapse the dense physical barriers, making a permeable TME for NK cell infiltration. What's more, the AuNSP could achieve αCD16 gene transfection to modify tumor surface with CD16 antibody, marking a unique structure on tumor cells for NK cell recognition and then lead to strong NK cell activation by CD16-mediated antibody-dependent cellular cytotoxicity (ADCC). As expected, the designed AuNSP@αCD16 induced an immune-favorable TME for NK cell performing killing functions against solid tumors, increasing the release of cytolytic granules and proinflammatory cytokines, which ultimately achieved a robustly boosted NK cell-based immunotherapy. Hence, the AuNSP@αCD16-mediated TME reconstituting strategy provides a substantial perspective for NK-based ACT on solid tumors. Statement of significanceIn adoptive cell therapy (ACT), natural killer (NK) cells exhibit greater off-the-shelf utility and improved safety comparing with T cells, but the efficacy of NK cell therapy is severely compromised by formidable physical barriers of the tumor tissue and deficiency of NK cell recognizing signals on tumor cells. Herein, a nano-immunomodulator AuNSP@αCD16, with the abilities of inducing mild photothermal intervention and modifying the tumor cell surface with αCD16, is explored to reconstruct an infiltration-favorable and activation-facilitating tumor microenvironment for NK cells to perform killing functions. Such a simple and safe strategy is believed as a very promising candidate for future NK-based ACT.

Allogeneic, CD34 +, Umbilical Cordblood-Derived NK Cell Adoptive Immunotherapy for the Treatment of Acute Myeloid Leukemia Patients with Measurable Residual Disease

Although the majority of patients with acute myeloid leukemia (AML) achieve complete remission (CR), most of them relapse due to measurable residual disease (MRD), leading to poorer prognosis. The elimination of MRD before morphological relapse in patients who achieved CR is therefore an important therapeutic strategy to achieve improved long term outcomes in AML.A promising approach is targeting MRD via natural killer (NK) cell adoptive immunotherapy. NK cells can be used without HLA matching (allogeneic), enabling the transfer of high numbers of immune effector cells, which were not exposed to cytotoxic chemotherapy. A phase I dose-escalation trial in ten elderly subjects with AML in morphologic CR after induction chemotherapy showed that a single infusion of an allogeneic NK cell product generated from umbilical cord blood-derived CD34 + cells, following cyclophosphamide/fludarabine (Cy/Flu) pre-conditioning, was safe and well tolerated, with some patients (2 of 4) achieving MRD negativity (Dolstra et al. 2017).Currently, our dose escalation and expansion trial with the “off-the shelf”, cryopreserved, allogeneic NK cell product GTA002 (NCT04632316) is ongoing in adults with AML who are in morphologic CR (including CRi) with MRD and who are not proceeding to allogeneic hematopoietic stem cell transplantation.Two patients have been dosed with a single GTA002 infusion of approximately 5 x 10 8 viable NK cells and completed 6- and 3-month follow-up, respectively. Patient #1 is a 73-year-old male, with AML with recurrent genetic abnormalities (including NPM1mut and FLT3-ITD), who initially received azacitidine and gilteritinib, leading to stable disease. Upon progression, the patient received further treatment with decitabine, followed by 3 cycles of azacitidine/venetoclax (aza/ven), which led to CRi with MRD persistence (confirmed by centralized multiparameter flow cytometry (MFC) on bone marrow (BM)). The patient then received cyclophosphamide (Cy) and fludarabine (Flu) as preconditioning regimen from day -6 to day -3 (Cy: 900 mg/m 2; Flu:15 mg/m 2/day; Flu adjusted for renal impairment), followed by a single intravenous infusion of GTA002 on day 0. The patient experienced a serious adverse event (SAE) on day 5 (grade 3 febrile neutropenia) deemed related to Cy/Flu, which fully resolved on day 16. MRD results by MFC on BM showed that patient #1 converted to MRD negativity (<0.1%) on day 0, which was sustained at 1, 2, 3 and 6 months. Interestingly, NPM1 MRD was detectable by next generation sequencing (MRD-NGS) up to month 1 in peripheral blood (PB), but was cleared by month 2, 3 and 6 in PB (<0.01%VAF). In line with the PB results, NPM1 MRD was detectable in BM at month 1 and was cleared at months 3 and 6.Patient #2 is a 78 year old male with a history of MDS with pancytopenia and IDH2, SRSF2 and PTPN11 mutations that evolved into AML with myelodysplasia-related changes. The patient achieved CR after receiving aza/ven, with MRD positivity after 3 cycles, leading to clinical trial inclusion. He was preconditioned with an adjusted dose of Cy/Flu (Cy 300 and Flu 30 mg/m 2/day from day -5 to day -3), with no SAEs reported. Analysis of BM by MFC showed MRD positivity at screening and on day 0, which turned to MRD negativity at month 1, turning positive again at month 2 and 3. Follow up in PB and BM by MRD-NGS showed that the IDH2 and SRSF2 clones persisted after preconditioning and GTA002 infusion, but the PTPN11 clone became undetectable in PB by Day 0 and in BM by month 2 and month 3.Taken together, these cases describe clearance of MRD by MFC and NGS in AML with a single dose infusion of allogeneic, CD34+ cord blood derived NK cells in one patient and clearance of the PTPN11 mutated clone in another one, reconfirming the excitement to further investigate this treatment strategy for disease control. The delayed clearance of MRD could suggest an ongoing immunotherapeutic effect that will be further investigated. DisclosuresHeuser: Karyopharm: Research Funding; Daichi Sankyo: Honoraria, Research Funding; Bayer AG: Honoraria, Research Funding; BMS/Celgene: Research Funding; Jazz Pharmaceuticals: Honoraria, Research Funding; BergenBio: Research Funding; Astellas: Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Roche: Research Funding; Tolremo: Honoraria; AbbVie: Honoraria; Janssen: Honoraria. Thol: Abbvie: Honoraria; Astellas: Honoraria; Novartis: Honoraria; Jazz: Honoraria; Pfizer: Honoraria; BMS/Celgene: Honoraria, Research Funding. Schwarzer: Bantam Pharmaceutical: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Pinkernell: Medigene AG: Ended employment in the past 24 months; Glycostem Therapeutics: Current Employment. Ganser: Jazz Pharmaceuticals: Honoraria; Novartis: Honoraria; Celgene: Honoraria.

New Orders to an Old Soldier: Optimizing NK Cells for Adoptive Immunotherapy in Hematology.

NK (Natural Killer) cell-mediated adoptive immunotherapy has gained attention in hematology due to the progressing knowledge of NK cell receptor structure, biology and function. Today, challenges related to NK cell expansion and persistence in vivo as well as low cytotoxicity have been mostly overcome by pioneering trials that focused on harnessing NK cell functions. Recent technological advancements in gene delivery, gene editing and chimeric antigen receptors (CARs) have made it possible to generate genetically modified NK cells that enhance the anti-tumor efficacy and represent suitable “off-the-shelf” products with fewer side effects. In this review, we highlight recent advances in NK cell biology along with current approaches for potentiating NK cell proliferation and activity, redirecting NK cells using CARs and optimizing the procedure to manufacture clinical-grade NK and CAR NK cells for adoptive immunotherapy.

Reversing Hypoxia with PLGA-Encapsulated Manganese Dioxide Nanoparticles Improves Natural Killer Cell Response to Tumor Spheroids.

The adoptive transfer of natural killer (NK) cells, which can recognize and obliterate cancer cells, provides a practical alternative to current treatment modalities to improve cancer patients' survival. However, translating NK cell therapies to treat solid tumors has proven challenging due to the tumor microenvironment (TME). Hypoxia in the TME induces immunosuppression that inhibits the cytotoxic function of NK cells. Thus, reversing hypoxia-induced immunosuppression is critical for effective adoptive NK cell immunotherapy. In this study, we use manganese dioxide nanoparticles (MnO2 NPs) to catalyze the degradation of tumor-produced hydrogen peroxide, thereby generating oxygen. For improved biocompatibility and modulation of oxygen production, the MnO2 NPs were encapsulated into poly(lactic-co-glycolic) to produce particles that are 116 nm in size and with a ζ-potential of +17 mV (PLGA-MnO2 NPs). The PLGA-MnO2 NPs showed first-order oxygen production and sustained high oxygen tension compared to equivalent amounts of bare MnO2 NPs in the presence of H2O2. The PLGA-MnO2 NPs were biocompatible, reduced hypoxia after penetration into the core of cancer spheroids, and decreased hypoxia-induced factor 1 α expression. Reducing hypoxia in the spheroid resulted in a decrease in the potent immunosuppressors, adenosine, and lactate, which was confirmed by electrospray ionization mass spectroscopy (ESI-MS). ESI-MS also showed a change in the metabolism of the amino acids aspartate, glutamine, and glutamate after hypoxia reduction in the cancer cells. Notably, the spheroids' microenvironment changes enhanced NK cells' cytotoxicity, which obliterated the spheroids. These results demonstrate that reducing hypoxia-induced immunosuppression in tumors is a potent strategy to increase the potency of cytotoxic immune cells in the TME. The developed NPs are promising new tools to improve adoptive NK cell therapy.

NK Cell Homing and Persistence in Bone Marrow after Adoptive Immunotherapy Correlates with Therapy Response

Background: Natural killer (NK) cells are innate lymphoid cells capable of killing leukemic cells without prior sensitization. Adoptive immunotherapy with allogeneic NK cells has been used at the University of Minnesota and was shown to be effective in a subset of patients with acute myeloid leukemia refractory to therapy. The factors determining clinical success of adoptive NK cell immunotherapy are largely unknown. We hypothesized that NK cell homing and persistence in the bone marrow niche may be predictive for the clinical outcomes.Methods: We retrospectively analyzed bone marrow biopsy findings 14 days after NK cell infusion in 109 patients treated at our institution between 2003-2014. To evaluate the homing and persistence of NK cell in the bone marrow, multiple methods were used. Immunohistochemical staining for the NK cell marker CD56 was performed on the core biopsy in available cases. Flow cytometry data performed on aspirate material at the time of the biopsy was re-evaluated to enumerate the NK cell percentage. Finally the percentage of Large Granular Lymphocytes was determined in a semi-quantitative manner from Wright-Giemsa stained aspirate smears. CD56 immunohistochemical stain was quantified by counting the number of CD56 positive cells per microscopic field of view (High Power Field, HPF) (400 x magnifications) and averaging over the 10 microscopic fields with the highest density of CD56 positive cells. Cases with obvious CD56 staining of blasts on the trephine core were excluded from further analysis.Results: 95 bone marrow biopsy cases could be stained for CD56, but 6 cases were excluded from analysis due to CD56 staining leukemic blast population. For the whole study group the average CD56+ cell density per High Power Field (HPF) was 10.1 (SD=17). A control group composed of 10 patients with bone marrow biopsy shortly after chemotherapy, but who did not receive NK cell infusion had an average of 3.8 CD56+ cells per HPF (SD=3.4).Patients who had a higher density of NK cells in the bone marrow compared to the control group (more than 3.8 cells/HPF) were less likely to have persistence of acute leukemia as defined by ≥5% blasts, than patients with an NK cell density similar to the control group (Figure 1, Chi-square test, p=0.037). As well, patients with <5% leukemic blasts had more CD56 positive cells per HPF (Median 5.6, range 0.1-95.8 Versus median 1.8, range 0.1-28.1, p=0.009, Mann-Whitney test) and had a greater proportion of NK cells by flow cytometry (p=0.004, Mann-Whitney test).The clinical factors which may affect NK cell homing and persistence were also evaluated. Total Body Irradiation (TBI) at a dose 200 cGy for 2 days was applied as part of pre-infusion conditioning in a subset of the patients. Patients who received TBI (32 with TBI Vs 55 without) had a significantly higher density of CD56 positive cells (Figure 2, Median 4.75Vs 2.8, p=0.04, Mann-Whitney test). Among those patients who did not receive TBI conditioning, a subset received an anti-CD25 immunotoxin agent to reduce regulatory T cells (Denileukin Difitox, Ontak). The density of NK cells was significantly higher in the patients who received anti-CD25 immunotoxin (Figure 3, Median 9.8 Vs 2.5, p=0.02, Mann-Whitney test).Conclusions: Homing and survival/expansion of adoptively transferred NK cells in the bone marrow 14 days after infusion correlates with morphologic remission of acute leukemia. While the factors influencing NK cell homing and persistence in the BM niche remain to be elucidated, our results suggest a role for TBI as part of the conditioning and anti-CD25 immunotoxin in promoting the persistence of adoptively transferred NK cells in the bone marrow niche. [Display omitted] DisclosuresMiller:Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding; Celegene: Consultancy.

Adoptive Natural Killer Cell Immunotherapy for Canine Osteosarcoma.

Osteosarcoma is the most common primary bone tumor in both humans and dogs. It is a highly metastatic cancer and therapy has not improved significantly since the inclusion of adjuvant chemotherapy into disease treatment strategies. Osteosarcoma is an immunogenic tumor, and thus development of immunotherapies for its treatment, especially treatment of microscopic pulmonary metastases might improve outcomes. NK cells are lymphocytes of the innate immune system and can recognize a variety of stressed cells, including cancer cells, in the absence of major histocompatibility complex (MHC)-restricted receptor ligand interactions. NK cells have a role in controlling tumor progression and metastasis and are important mediators of different therapeutic interventions. The core hypothesis of adoptive natural killer (NK) cell therapy is there exists a natural defect in innate immunity (a combination of cancer-induced reduction in NK cell numbers and immunosuppressive mechanisms resulting in suppressed function) that can be restored by adoptive transfer of NK cells. Here, we review the rationale for adoptive NK cell immunotherapy, NK cell biology, TGFβ and the immunosuppressive microenvironment in osteosarcoma, manufacturing of ex vivo expanded NK cells for the dog and provide perspective on the present and future clinical applications of adoptive NK cell immunotherapy in spontaneous osteosarcoma and other cancers in the dog.

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

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

CRISPR Gene-Editing of Chemokine Receptors As a Novel Strategy to Redirect NK Cell Trafficking In Vivo

Allogeneic and autologous natural killer (NK) cell-based therapies have proven to be safe in patients with hematological malignancies across multiple clinical trials. However, at present, only a small percentage of patients who have received NK cell based therapy have achieved complete remissions. NK cells must first traffic into the tumor microenvironment in order to effectively lyse tumor targets in vivo. Many hematological malignancies reside in or metastasize through bone marrow (BM) compartments. Recent studies in both murine and macaque models evaluating NK cell distribution following intravenous (i.v.) infusion unexpectedly revealed preferential trafficking into liver tissue over BM niches, which for hematological malignancies is undesirable. Therefore, we evaluated surface expression and mRNA transcript abundance of critical chemotactic receptors and adhesion molecules in primary fresh and ex vivo expanded NK cells. NK cells from six healthy donor PBMC samples were expanded for 14 days ex vivo, using two different irradiated feeder cell lines (either EBV-infected LCL or K562 cells with membrane-bound IL-21 and 41BBL). mRNA from fresh and expanded NK cells was sequenced and analyzed for differential gene expression. We observed the transcriptome of NK cells expanded with the two different feeder cell lines were similar, with only 13 differentially expressed genes. However, we observed the transcriptional landscape between fresh and expanded NK cells to be vastly different (Figure 1A). As expected, amongst the thousands of differentially expressed genes, mitotic phase transitions and DNA replication were pathways that were strongly enriched in expanded compared to fresh NK cells. Further, expanded NK cells had a robust amplification in mRNA transcription of known surface cytotoxicity and activation receptors, as well as enhanced mRNA transcription of chemotactic ligands that are implicated in bridging the innate and adaptive immune responsesincluding XCL1, XCL2, CCL5, CXCL16, and CXCL8. Remarkably, we found expanded NK cells had upregulated mRNA transcription and surface expression of CCR1, CCR5, CXCR3, and CXCR6, coupled with a substantial decrease in mRNA transcription and surface expression of CXCR4 (Figure 1B). We postulate that this particular shift in molecular expression may have the net effect of routing adoptively transferred NK cells out of the circulation into liver and other inflammatory environments, ultimately impeding cellular trafficking into the bone marrow (BM). To test this hypothesis, we used CRISPR/Cas9 to disrupt the CCR5 gene which has upregulated expression in expanded NK cells. Five days following ex-vivo expansion, NK cells were electroporated with a mix of 3 sgRNAs targeting CCR5, complexed with Cas9. Electroporated cultures harvested on day 14 maintained their proliferative capacity and had a substantial reduction in CCR5 expression (22%) compared to non-electroporated control NK cells (88%; Figure 1C). Both expanded NK cell populations were then injected i.v. into NSG mice, with blood, BM, lungs, and livers being harvested 24 hours following infusion. Disrupting CCR5 in NK cells significantly reduced cellular trafficking into the liver compared to control NK cells (p&amp;lt;.01). Moreover, the percentage of infused CCR5 CRISPR/Cas9 disrupted NK cells in the circulation was increased compared to control NK cells (Figure 1D). In conclusion, these collective data reveal NK cells undergo profound transcriptional changes when cultivated ex vivo. Many chemotactic receptors that were previously unknown to be impacted by ex vivo expansion were discovered to have a shift in transcriptional regulation which would be predicted to compromise NK cell homing to the bone-marrow. Importantly, we show for the first time that CRISPR gene-editing of chemokine receptors can be used as a novel strategy to redirect NK cell trafficking in vivo, which could bolster the effectiveness of adoptive NK cell immunotherapy for hematological malignancies and other cancers. Figure 1 Disclosures No relevant conflicts of interest to declare.

A Phase I Clinical Trial Testing the Safety of IL-21-Expanded, Off-the-Shelf, Third-Party Natural Killer Cells for Relapsed/Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome

Background: Allogeneic transplantation (allo-HCT) is an effective treatment for many patients with Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS). With HCT the long term disease free survival (DFS) rate is approximately 60% for patients transplanted in first remission. After relapse, the rate falls to approximately 40% if the patients are in remission at the time of HCT. However, many patients present with refractory chemo-resistant disease, relapse during or shortly after induction therapy, or develop complicating comorbidities due to prolonged induction. For most such relapse-refractory (R/R) AML patients, allo-HCT is not an option. These patients have a dire prognosis with only 5-10% long term DFS. Natural Killer (NK) cells are cytotoxic lymphocytes that are able to identify tumors and exert an anti-tumor effect in an MHC-I independent manner. However, NK cells from patients with cancer can be dysfunctional and reduced in number. To overcome that, NK cells expanded ex vivo to yield high numbers of cells could provide a cellular therapeutic option for cancer patients, including patients with AML/MDS. The therapeutic potential of ex vivo membrane-bound IL-21 expanded NK cells (FC21-NK) from a haploidentical donor has been established in patients with poor prognosis AML/MDS undergoing a haploidentical HCT. Besides obtaining sufficient cell numbers, having them readily available for patients is another major obstacle for adoptive NK cell immunotherapy. Patients with aggressive disease need prompt intervention yet the manufacture of patient-specific NK cells exceeds three weeks. As NK alloreactivity plays a critical role in mediating anti-tumor effects, we identified KIR and HLA-mismatched 'ideal' donors (selected through "Be The Match Biotherapies"). Using lymphocytes from these donors, we have established a third-party NK cell bank to ensure readily-available immune cell therapies that allows scalable, affordable mass-production of large numbers of NK cells suitable for banking &amp; immediate 'off-the-shelf' administration to a broad population of recipients. This trial is to determine the safety of FC21 expanded Off-the-shelf (OTS), Third-party donor-derived NK cells for relapsed/refractory AML patients. Methods: This phase 1 study follows a 3+3 design to investigate the safety of FC21-expanded, third-party, OTS NK cells for treatment of patients with primary refractory or relapsed AML or myelodysplastic syndrome. Active GvHD is excluding. Patients aged ≥18 or ≤80 years are enrolled into two cohorts: those &amp;lt;60 yrs &amp; able to tolerate intensive chemo &amp; sensitive to Cytarabine will receive Fludarabine 30 mg/m2/day (days -6 to -2) &amp; Cytarabine 2 g/m2/day (days -6 to -2). Patients &amp;gt;60 yrs or &amp;lt;60 yrs &amp; unable/unwilling to tolerate intensive chemo or disease insensitive to Cytarabine will receive Fludarabine 30 mg/m2/day (days -5 to -2) &amp; Decitabine 20 mg/m2/day (days -6 to -2). All patients subsequently receive a total of 6 infusions of NK cells administered thrice weekly for two weeks (between day 0-21). Three NK cell dose-levels: 1x107, 3x107 &amp; 1x108 cells/kg/dose will be explored to determine MTD (maximal tolerated dose). Between 3-18 patients/cohort/dose for MTD determination, plus an additional 10 patients/dose in an expansion phase may be enrolled (maximum 28/cohort = 56 total subjects). Patients will be followed up to day 56 from first NK cell infusion. Primary objectives are to determine the recommended phase 2 dose and overall response rate (CR, CRi &amp; MLFS). Secondary objectives will explore PFS, OS &amp; MRD negativity, cell counts, infectious complications, and patients proceeding to transplant. Enrollment in both cohorts is ongoing. Clinical trial information: NCT04220684. Disclosures Vasu: Kiadis Inc: Other: Kiadis has obtained exclusive licensing requirements from The OHio State University; Janssen: Membership on an entity's Board of Directors or advisory committees; Omeros: Membership on an entity's Board of Directors or advisory committees. Bhatnagar:KaryoPharm Therapuetics: Research Funding; Cell Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; KITE: Membership on an entity's Board of Directors or advisory committees. Blachly:AbbVie, AstraZeneca, KITE Pharma: Consultancy. O'Donnell:Kiadis Pharma: Other: Licensing of intellectual property. Lee:Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties.

FT538: Preclinical Development of an Off-the-Shelf Adoptive NK Cell Immunotherapy with Targeted Disruption of CD38 to Prevent Anti-CD38 Antibody-Mediated Fratricide and Enhance ADCC in Multiple Myeloma When Combined with Daratumumab

Monoclonal antibody (mAb) treatment is an effective therapeutic strategy for many cancer types, though there remains meaningful opportunity to improve mAb efficacy by optimizing the interaction with natural killer (NK) cells to enhance antibody-dependent cellular cytotoxicity (ADCC). NK cells are an ideal effector cell for combined use with tumor-targeting mAbs, as NK cells effect both innate tumoricidal capacity and ADCC. CD38-targeting mAbs, such as daratumumab, are effective in treating multiple myeloma (MM) and achieve their efficacy through multiple mechanisms, including ADCC. However, because activated NK cells express high levels of CD38, daratumumab induces NK cell depletion through fratricide, potentially reducing treatment effectiveness. Adoptive NK cell immunotherapy therefore has the potential to augment daratumumab's ADCC activity if fratricide can be reduced or prevented. FT538 is an off-the-shelf adoptive NK cell immunotherapy product candidate designed for enhanced cellular persistence and ADCC while avoiding anti-CD38 mAb induced fratricide. It is derived from induced pluripotent stem cells (iPSC) engineered to lack CD38 expression, which we have previously shown to eliminate daratumumab-induced fratricide among iPSC-derived NK cells, resulting in enhanced long-term daratumumab-mediated ADCC. FT538 is engineered to express an IL-15 receptor alpha fusion protein (IL-15RF; IL-15 tethered to IL-15 receptor α) to enhance persistence and a high-affinity non-cleavable CD16 (hnCD16, FcRγIII) to increase ADCC. To support the clinical translation of FT538, and to enable the repeatable and scalable cell production to support off-the-shelf availability of a uniform NK cell product, a clinical-grade master pluripotent stem cell line was developed. The FT538 master pluripotent stem cell line was created by reprogramming donor fibroblasts into iPSCs using our non-integrating cellular reprogramming platform, and cells were further genetically edited by targeting IL-15RF and hnCD16 to the CD38 locus. Clonal iPSC lines were generated and screened for precise knock-in and knock-out edits at the CD38 locus and a lack of off-target genome integration (15% total success rate for CD38-/-IL-15RF+CD16+). Selected engineered iPSC clones were confirmed to be free of reprogramming transgenes and to maintain genomic stability. Engineered iPSC clones were additionally tested for their NK cell differentiation potential and function, and a single clone was selected to serve as the renewable starting material for cGMP manufacturing and clinical development. Upon differentiation and expansion FT538 demonstrated a mature NK cell phenotype with expression of NK cell receptors including NKp30, NKp46, NKG2D, KIR, NKG2A, and DNAM-1. The functional impact of CD38 knockout on FT538 NK cells was confirmed in an in vitro fratricide assay, where peripheral blood (PB)-NK cells exhibited fratricide at a frequency of 33% after 3 hr culture with increasing daratumumab concentrations. In contrast, FT538 cells were entirely resistant (&amp;lt;1% specific cytotoxicity) to daratumumab-induced fratricide. In vitro cytotoxic re-stimulation assays showed that repeat exposure of PB-NK cells to daratumumab plus MM target cells resulted in a loss of cytotoxic capacity (from 74% to 58% upon re-stimulation), and a similar effect was seen for non-engineered iPSC-derived NK cells. In contrast, FT538 NK cells maintained robust ADCC in during primary and secondary exposure to MM target cells and daratumumab. FT538 with daratumumab resulted in 86% cytotoxicity against MM target cells upon first exposure and 92% cytotoxicity upon re-stimulation, with a 20-fold increase in viable NK cells at the conclusion of the assay compared to non-engineered iPSC-derived NK cells. Additionally, the combined survival benefit of IL-15RF expression and fratricide resistance mediated by the CD38 knockout as well as the enhanced hnCD16-mediated ADCC allowed for greater cytotoxicity of FT538 against MM tumor spheroids. Together, these preclinical data support the clinical translation of FT538, an off-the-shelf adoptive NK cell immunotherapy product engineered for uniform hnCD16 and IL-15RF expression with CD38 elimination for enhanced ADCC in combination with daratumumab and other anti-CD38 mAbs for the treatment of MM. Disclosures Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Cichocki:Fate Therapeutics, Inc: Research Funding. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Fong:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Kaufman:FATE Therapeutics: Consultancy, Research Funding. Malmberg:Fate Therapeutics, Inc.: Consultancy, Research Funding; Vycellix: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:CytoSen: Membership on an entity's Board of Directors or advisory committees; Moderna: Membership on an entity's Board of Directors or advisory committees; OnKImmune: Membership on an entity's Board of Directors or advisory committees; GT BioPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dr. Reddys Laboratory: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc: Consultancy, Research Funding. Valamehr:Fate Therapeutics, Inc: Employment.

FT576: A Novel Multiplexed Engineered Off-the-Shelf Natural Killer Cell Immunotherapy for the Dual-Targeting of CD38 and Bcma for the Treatment of Multiple Myeloma

Multiple myeloma (MM) is a B cell neoplasm that originates from the malignant transformation of plasma cells, with treatment strategies that include chemotherapeutic agents and immunomodulatory drugs. Recently, significant effort has been applied to the development of monoclonal antibody (mAb) and chimeric antigen receptor (CAR) T cell therapies for the treatment of advanced MM. Anti-CD38 mAb therapy is at the forefront of these efforts, with clearly demonstrated clinical benefit and availability of a FDA-approved mAb in daratumumab. Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism of action of CD38-targeted mAbs; however, high CD38 expression on natural killer (NK) cells results in fratricide, which depletes the NK cells necessary for ADCC. In addition to CD38, targeting of other MM-associated cell-surface proteins has been explored. Of these antigens, the TNF-superfamily member BCMA is among the most researched and is under development by multiple groups as a CAR target. Several clinical trials in MM have shown promising initial results targeting BCMA with CAR T cells, however there remains significant opportunity to improve both relapse rates and treatment of relapsed patients. Collectively, clinical data would suggest that combinatorial targeting of both CD38 and BCMA may improve clinical efficacy compared with targeting either antigen alone. We have developed a multiple-target, adoptive NK cell immunotherapy approach for the treatment of MM. The strategy utilizes our off-the-shelf NK cell platform with four engineered attributes: 1) an anti-BCMA CAR for direct MM targeting, 2) high affinity non-cleavable CD16 (hnCD16) for enhanced ADCC in combination with anti-CD38 mAbs, 3) CD38 deletion for resistance to anti-CD38 mAb induced NK cell depletion, and 4) IL-15/IL-15 receptor α fusion protein (IL-15RF; IL-15 fused to IL-15Rα) for enhanced NK cell persistence. The anti-BCMA CAR consists of a unique single chain variable fragment (scFv) targeting domain with a BCMA binding affinity in the low nanomolar range, providing high functional avidity and efficacy in disease settings where BCMA antigen density is low. Our approach utilizes NK cells derived from a genetically engineered, clonally-derived master pluripotent stem cell line with uniform expression of anti-BCMA CAR, IL-15RF, hnCD16, and CD38 bi-allelic knockout. The engineered master pluripotent stem cell line serves as the starting material for consistent and repeatable manufacture of off-the-shelf NK cells that contain all described attributes in a homogenous manner (termed FT576) and that can be produced at a scale to support multi-dose treatment strategies and on-demand dose availability. In preclinical studies, FT576 NK cells exhibited uniform expression of CD16, CAR, and IL15-RF and did not express CD38 (&lt;1%). In an in vitro fratricide assay, FT576 NK cells were entirely resistant to daratumumab-induced fratricide, with no detectable specific cytotoxicity when exposed to increasing concentrations of daratumumab. Conversely, peripheral blood NK cells were sensitive to daratumumab-induced fratricide (up to 33% cytotoxicity within 3 hrs of daratumumab exposure). FT576 NK cells demonstrated enhanced cytotoxicity against the MM1.S MM cell line during a long-term cytotoxicity assay compared with control NK cells that lacked CAR expression (62% cytotoxicity for FT576 vs 26% for control). In addition, cellular persistence was greater than NK cells lacking the IL-15RF protein, and FT576 NK cells demonstrated the unique ability to expand in vitro absent of exogenous cytokine support (61-fold expansion vs. 4-fold for IL-15RF negative). Importantly, FT576 NK cells remained ADCC competent, as combination with daratumumab enhanced cytotoxicity against MM cell lines in a 2D cytotoxicity assay. Additionally, FT576 mediated direct cytotoxicity against RPMI-8226 MM spheroids, leading to &gt;99% cytotoxicity in a 3D-spheroid culture model. Preclinical studies are ongoing to support the advancement of FT576 as the first-of-kind cellular therapeutic for the combination of anti-BCMA CAR and mAb-directed targeting of MM. Disclosures Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Goodridge:FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Valamehr:Fate Therapeutics, Inc: Employment.