Natural Killer Cell Therapy Research Articles

Synergistic treatment strategy: combining CAR-NK cell therapy and radiotherapy to combat solid tumors.

Immunotherapy, notably chimeric antigen receptor (CAR) modified natural killer (NK) cell therapy, has shown exciting promise in the treatment of hematologic malignancies due to its unique advantages including fewer side effects, diverse activation mechanisms, and wide availability. However, CAR-NK cell therapies have demonstrated limited efficacy against solid tumors, primarily due to challenges posed by the solid tumor microenvironment. In contrast, radiotherapy, a well-established treatment modality, has been proven to modulate the tumor microenvironment and facilitate immune cell infiltration. With these observations, we hypothesize that a novel therapeutic strategy integrating CAR-NK cell therapy with radiotherapy could enhance the ability to treat solid tumors. This hypothesis aims to address the obstacles CAR-NK cell therapies face within the solid tumor microenvironment and explore the potential efficacy of their combination with radiotherapy. By capitalizing on the synergistic advantages of CAR-NK cell therapy and radiotherapy, we posit that this could lead to improved prognoses for patients with solid tumors.

Unlocking Glioblastoma Secrets: Natural Killer Cell Therapy against Cancer Stem Cells.

Glioblastoma (GBM) represents a paramount challenge as the most formidable primary brain tumor characterized by its rapid growth, aggressive invasiveness, and remarkable heterogeneity, collectively impeding effective therapeutic interventions. The cancer stem cells within GBM, GBM stem cells (GSCs), hold pivotal significance in fueling tumor advancement, therapeutic refractoriness, and relapse. Given their unique attributes encompassing self-renewal, multipotent differentiation potential, and intricate interplay with the tumor microenvironment, targeting GSCs emerges as a critical strategy for innovative GBM treatments. Natural killer (NK) cells, innate immune effectors recognized for their capacity to selectively detect and eliminate malignancies without the need for prior sensitization, offer substantial therapeutic potential. Harnessing the inherent capabilities of NK cells can not only directly engage tumor cells but also augment broader immune responses. Encouraging outcomes from clinical investigations underscore NK cells as a potentially effective modality for cancer therapy. Consequently, NK cell-based approaches hold promise for effectively targeting GSCs, thereby presenting an avenue to enhance treatment outcomes for GBM patients. This review outlines GBM's intricate landscape, therapeutic challenges, GSC-related dynamics, and elucidates the potential of NK cell as an immunotherapeutic strategy directed towards GSCs.

IPSC-derived natural killer cells expressing the FcγR fusion CD64/16A can be armed with antibodies for multitumor antigen targeting

BackgroundAntibody therapies can direct natural killer (NK) cells to tumor cells, tumor-associated cells, and suppressive immune cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). This antigen-specific effector function of human NK...

Engineering Superaffinity Antibody Dependent Cellular Cytotoxcity Receptors into iPSC-Derived NK Cells As Next-Generation Immunotherapies for Cancer

Engineered immune cells have provided an important new approach to treating cancer. Most of the progress so far has focused on autologous CAR-T cell therapy, although this approach has been met with several limitations including high-cost and complexity of manufacturing, donor-to-donor variability, and severe toxicities in some patients. Allogenic natural killer (NK) cell therapies have also been shown to mount potent responses against hematologic malignancies but are less likely to cause high-grade toxicities and can be mass produced for off-the-shelf usage. We have developed an induced pluripotent stem cell (iPSC) derived NK (iNK) platform amenable to engineering multiple features in immune cells to improve efficacy against both solid and liquid tumors. Our previous studies showed that knocking out the CISH gene, which encodes a key regulator of activation, in iNK cells significantly improves their anti-cancer activity, in vivo persistence, metabolic fitness, polyfunctional cytokine production, and resistance to cell exhaustion. In these CISH Knockout (KO) iNK cells, we have screened a library of CAR constructs containing domains from diverse immune cell signaling receptors including NK cell activating receptors, cytokine receptors, and integrins. Our screen identified SLNK12, a CAR signaling domains that performed better than both T cell CARs and previously reported NK cell CARs in the context of CISH KO iNK cells. In this study, we describe the development of iNK cells with enhanced antibody dependent cellular cytotoxicity (ADCC) activity. NK cells normally bind antibodies via their CD16A receptor, which has medium-to-low affinity for the antibody Fc domain and is also typically expressed at a low level in unmodified iNKs. We have constructed a recombinant Fc-receptor that combines a high-affinity Fc-binding domain (to improve antibody binding) with our NK cell optimized SLNK12 CAR signaling domain to enhance ADCC-mediated killing. When co-cultured with multiple NC cell resistant tumor lines such as BT-474 (breast cancer) or FaDu (squamous cell carcinoma), these SuperAffinity ADCC receptor CISH KO iNK cells possess potent anti-cancer activity in combination with several different therapeutic monoclonal antibodies, including anti-EGFR (Cetuximab) and anti-HER2 (Trastuzumab) amongst others (Figure 1). We have assessed efficacy in a variety of different 2D and 3D spheroid cytotoxicity assays, as well as cytokine release and CAR activation assays. In addition, we demonstrate that our SuperAffinity receptor mediates tumor cell killing at lower antibody concentrations than do CD16A variants. Furthermore, introduction of serum IgG inhibits, but does not stop ADCC killing for both our SuperAffinity ADCC receptor and the CD16A receptor. Taken together, our results show that engineering CISH KO iNK cells with NK-optimized CARs for enhanced ADCC improves anti-tumor activity against a variety of solid tumor target lines. Future work will focus on advancing the new cellular therapies into clinical development.

CD38 KO/CD38-CAR Human Primary Natural Killer Cells Enhance Cytotoxicity Against CD38-Expressing Primary Lymphoma, Leukemia, and Myeloma

Natural killer (NK) cells have strong cancer immunotherapeutic potential that can be further augmented by engineering with a chimeric antigen receptor (CAR). CD38, a transmembrane glycoprotein involved in signal transduction and cellular adhesion, is highly expressed on the surface of the hematologic malignancies multiple myeloma (MM), acute myeloid leukemia (AML), Burkitt Lymphoma (BL) and T-cell acute lymphoblastic leukemia (T-ALL). FDA approved CD38 monoclonal antibodies (mAb) daratumumab and isatuximab are used to treat MM in patients, and have been tested to treat other CD38 expressing malignancies. NK cells play an imperative role in the efficacy of these antibodies and have natural antitumor activity against these cancers. However, because NK cells also highly express CD38, anti-CD38 mAb can cause NK cell fratricide, limiting their efficacy. Here, we generated multiple CD38 scFv based on isatuximab sequences and generated fratricide-resistant primary CD38-CAR NK cells using CRISPR/Cas9 genome editing and AAV6 gene delivery. Using this approach, we inserted the CD38-CAR gene into the CD38 locus to efficiently generate simultaneous CD38 KO and CD38-CAR KI with a mean expression level of %60. The CAR-expressing NK cells could be expanded on mbIL21-expressing feeder cells to a clinically relevant number of cells with no decline in CAR-expression level during two weeks of expansion confirming the lack of CAR mediated fratricide. We evaluated transduction efficiency and expression of CD38-CAR, and compared the cytotoxic function of two constructs with CD8 transmembrane (TM)/stalk and 41BB signaling domains and reverse heavy and light chains against AML, MM, T-ALL and BL immortalized cell lines and primary patient samples. We enhanced CD38 expression in target cancer cells by pre-treatment with all-trans retinoic acid (ATRA), a metabolite of vitamin A that pushes cells to maturation, consequently upregulating CD38. The sequential treatment of target cells with 10nM ATRA for 48 hours enhanced the cytotoxic activity of the CD38 KO/CD38-CAR-NK cells against primary MM, BL and AML samples. This would be particularly important for patients with low expression of CD38 on their cancers. The enhanced anti-tumor activity of the CAR was also studied and observed by CyTOF. Additionally, to better understand the therapeutic potential of CD38-CAR NK cells compared to CD38-CAR T cells we generated CD38 KO/CD38-CAR T cells using the same engineering approach and comparing their cytotoxic function. We showed that both CD38-CAR-T and CD38-CAR-NK cells have comparable anti-tumor activity compare to their paired non-edited wild type cells. Finally, we investigated the need for a CD38 KO to develop a fratricide-resistant therapy by comparing the cytolytic and metabolic functions of CD38 +/AAVS1 KO and CD38 KO CD38-CAR NK cells. We conclusively report a CD38-CAR NK cell therapy with enhanced metabolism and cytotoxicity toward a variety of hematologic malignancies, which can be further augmented by combination treatment with ATRA to target CD38-low malignancies.

Trials in Progress: A Phase I/II Clinical Trial of Off the Shelf Universal Donor Natural Killer Cells Combined with Chemotherapy for Treatment of Relapsed/Refractory Acute Myeloid Leukemia (KARMA)

Background and Significance: Natural killer (NK) cells are cytotoxic lymphocytes that play a key role in recognizing malignant cells. In AML, qualitative and quantitative dysfunction of innate NK cells portends a poor prognosis and adoptive NK cell therapy has been shown to be safe and effective with minimal risk for GVHD (Ciurea et al. 2022; Silla et al. 2021). To circumvent cost and logistics restraints of per patient manufacture of NK cells, we developed a universal donor (UD) NK cell bank for “off-the-shelf” NK cells (UD FC21-NK cells). Donors with optimal NK cell characteristics are identified through the National Marrow Donor Program, and peripheral blood NK cells are expanded for two weeks by co-culture with irradiated K562 feeder cells expressing 4-1BB ligand and membrane bound IL-21 as previously described (Denman et al. 2012). We seek to study the safety and efficacy of multiple doses of UD FC21-NK cells combined with fludarabine and cytarabine (FLA) chemotherapy in patients with relapsed or refractory AML. Study Design and Methods: This open label, single-center phase I/II study will include a dose escalation phase to establish safety and determine the recommended phase II dose followed by an expansion phase to estimate efficacy (complete response rate and MRD negative response rate). Up to 20 patients ages 18-25 with relapsed or refractory AML, including isolated extramedullary disease, will be enrolled. Exclusion criteria include active GVHD, uncontrolled infections, hematopoietic stem cell transplant within 3 months of enrollment, and donor lymphocyte infusion within 30 days of enrollment. Patients will undergo FLA chemotherapy followed by six doses of UD FC21-NK cells given thrice weekly for two weeks (Figure 1). Dose levels include 1 x 10 7 UD FC21-NK cells/kg/dose, 3 x 10 7 UD FC21-NK cells/kg/dose, and 1 x 10 8 UD FC21-NK cells/kg/dose. Patients may receive up to 2 cycles if they do not achieve a CR after cycle 1 or if necessary to bridge to transplant. Peripheral blood samples will be drawn weekly for immune correlatives including in vivo NK cell expansion and persistence. The trial is currently open to enrollment to adult patients with the intent to expand to pediatrics after demonstrating preliminary safety. Clinical trial information: NCT05503134

Epigenetic Scarring Leads to Irreversible NK Cell Dysfunction in Myeloid Malignancies

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) belong to the same spectrum of myeloid malignancies and have extremely poor outcomes in the relapsed/refractory setting. Despite advances in the understanding of the pathogenesis and molecular mechanisms of these disorders, and incremental improvements in treatment regimens, patients with MDS and AML often relapse and fail to achieve cure. These and other factors underscore the urgent need for new therapeutic alternatives that will improve the clinical outcomes of these patients. Immunotherapy using checkpoint molecule inhibitors and adoptive cell therapy using autologous immune effector cells have been mostly unsuccessful in patients with MDS and AML. This could be due to the immunosuppressive tumor microenvironment in the bone marrow niche or to intrinsic dysfunction in the immune effector cells of these patients. Natural killer (NK) cells are innate lymphocytes that play an important role in cancer immune surveillance and have distinct advantages over T cells as candidates for immunotherapy. Myeloid blasts are inherently susceptible to NK cell-mediated killing as they express many of the ligands recognized by NK cell activating receptors. However, malignant myeloid blasts are capable of adapting and developing defense mechanisms that allow them to evade NK cell-mediated cytotoxicity. Little is known about the mechanisms of NK cell immune evasion developed by myeloid blasts. Here, we show that NK cells from patients with myeloid malignancies display a global dysfunction with severely impaired secretory function and killing capacity. Single cell RNAseq and mass cytometry experiments revealed that these NK cells from MDS and AML patients display an exhausted phenotype at the transcriptomic and proteomic levels. We also show that these NK cells have an altered metabolism compared with age matched healthy control NK cells. We show that this dysfunction is mediated by a crosstalk between myeloid blasts and NK cells and cell-cell contact dependent release of transforming growth factor beta (TGF-β). This crosstalk leads to a profound epigenetic reprogramming of NK cells driven by transcription factors known to mediate exhaustion and immune suppression. Myeloid blast-induced NK dysfunction and epigenetic state of exhaustion can be prevented by pharmacologically inhibiting the TGF-β pathway or knockout of TGFBR2 in NK cells. However, our data reveal that once this dysfunction occurs it is irreversible owing to epigenetic scarring driven by the transcription factor BATF. In fact, we show that TGF-β induces the expression of BATF in NK cells, which in turn mediates a gene regulatory program leading to NK cell dysfunction by driving the expression of exhaustion and inhibitory genes (e.g. HAVCR2, ENTPD1, CTLA4, TGFBR2). Collectively, our findings reveal a novel mechanism of NK cell immune evasion manifested by stable epigenetic rewiring and inactivation of NK cells by myeloid blasts. Our data support the use of allogeneic sources for adoptive NK cell therapy in combination with strategies aiming at preventing immune suppression to treat myeloid malignancies rather than therapies aiming at reversing or rescuing the function of autologous NK cells.

Abatacept Enhances Natural Killer Cell Cytotoxicity Following Haploidentical HCT: A Novel Platform to Dissociate GVL from Gvhd

Background: Abatacept (Abata, CTLA4 Ig), has been explored for the past 3 decades for blockade of CD28-B7 costimulation pathway in organ transplantation. One of the disadvantages of Abata has been its sparing effect on natural killer (NK) cells, resulting in NK-mediated rejection in preclinical models. However, we explored this caveat of abatacept to augment NK cell mediated anti-leukemia effect without invoking T cell mediated GVHD, where unmodified donor lymphocyte infusions (DLI) were administered 6 hours following administration of abatacept in-vivo (Abata-DLI) to patients with advanced malignancies undergoing haploidentical (Haplo)-HCT on days +7, +21 and +35. An early and rapid proliferation of mature NK cells was observed, which correlated with an improved relapse-free survival, without an increase in GVHD. To explore the mechanistic pathway behind this phenomenon, we studied the effect of Abata on cytotoxicity as well as cytokine profile of NK cells both in-vitro as well as in-vivo (CTRI:REF/2021/08/046552). Methods: NK cells and monocytes were isolated from PBMC of healthy donors. NK cells ± monocytes, were incubated with IL2, IL15 and IL12/15/18 with or without Abata for 72 hours and 7 days. They were assessed for cytotoxicity and IFN-g and perforin expression against K562 cell lines at effector: target (E:T) ratio of 5:1 and 1:1 . In HCT recipients, samples were obtained at day +20 (baseline), +30, +45, +60, and 6, 12 and 18 months. The samples were cryopreserved and both cytotoxicity and cytokine assays were carried out in batches after thawing. Cytokine profile was analysed in total NK cells and in NKG2A+ and NKG2C+ subsets. Results: The study was carried out on 11 healthy donors and 20 haplo-HCT recipients (15 with malignant diseases receiving Abata-DLI and 5 with non-malignant diseases receiving Abata without DLI). NK cells from healthy donors showed significant increase in cytotoxicity at 72 hours in presence of Abata with IL2 and IL15 (Figure1, p<0.01) at E:T ratio of 5:1 as well as 1:1 at both 72 hours as well as 7 days, but not with IL12/15/18. The effect was independent of monocytes. Perforin tended to be upregulated at both 72 hours and 7 days with Abata and IL15. In patients receiving Abata-DLI, cytotoxicity of NK cells was significantly increased at all time points compared to baseline at E:T ratio of 5:1 and 1:1 (Fig 2, P < 0.01). NK-cytotoxicity was significantly higher than the corresponding donor samples at days +45 and +60 (p<0.05). This was particularly so in 11 patients without any event compared to 4 patients with relapse/GVHD. To evaluate if Abata-DLI had greater impact on NK-cytotoxicity, the data was compared with 5 patients who received Abata alone. This showed significantly greater cytotoxicity at days +45 and +60 in the Abata-DLI group, which was more pronounced, when only those without events were analysed from the Abata-DLI group (p<0.01). There was no significant change in cytokine profile in those receiving Abata alone, but in Abata-DLI group, IFN-g was significantly upregulated in the NKG2C+ subset at day +60 at 1:1 ratio. Perforin expression was significantly higher at days+45 and +60 at both 5:1 and 1:1 in the Abata-DLI cohort, for both NKG2C+ and NKG2A+ subsets. In the Abata-DLI group NKG2C+ subset showed significantly higher IFN-g at days+45 and +60 and upregulation of perforin at all time points. 3 patients transplanted with active leukemia, assessed beyond day +60, showed greater cytotoxicity compared to the corresponding donors at 6-18 months. Conclusion: In this study, Abata was shown to increase cytotoxicity of NK cells, independent of monocytes, in the presence of IL2 and IL15, but not with IL12/15/18. This was observed at both high as well as low E:T ratios, with significant upregulation of perforin. In Haplo-HCT with Abata-DLI, there was marked increase in NK-cytotoxicity with upregulation of both IFN-g and Perforin, when compared to baseline as well as in comparison to those receiving Abata without DLI. Our findings suggest that Abatacept directly augments NK-cytotoxicity and provides rationale for exploring Abata-augmented NK cell therapy as a novel approach to augment NK-mediated GVL effect without invoking T cell mediated GVHD

Azacitidine, Venetoclax and Allogeneic NK Cells in Newly Diagnosed Acute Myeloid Leukemia (ADVENT-AML): An Investigator-Initiated Multicenter Phase Ib Trial

BACKGROUND AND SIGNIFICANCE : Older/unfit patients with acute myeloid leukemia (AML) have limited treatment options. Outcomes in adverse-risk subgroups of AML are modest with CR/CRi rates of 45% to 60% and median overall survival (OS) of 5 to 12 months with current standard frontline therapy of hypomethylating agent with venetoclax (HMA-VEN).While allogeneic stem cell transplantation (allo-SCT) can significantly improve OS in AML, only 10 to 20% of older pts are able to undergo allo-SCT (Pratz et al. Blood 2019, Maiti et al. Blood 2022) Natural killer (NK) cells bridge the innate and adaptive immune system and play a major role in graft-vs-leukemia (GVL) effect responsible for the clinical benefit noted with allo-SCT. NK cell-based adoptive cellular therapies have shown good safety profiles and promising activity in AML with responses noted in 25% to 88% of patients. Furthermore, NK cells are less likely to induce graft-versus-host disease (GVHD), severe cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). We hypothesize that the combination of azacitidine, venetoclax and allogeneic NK cells will be safe and improve cure rates in AML compared to either Aza/Ven or NK cells alone. Improved clinical activity may be mediated through multiple synergistic mechanisms including reduction of disease burden by Aza/Ven, upregulation of epigenetically silenced NKG2D ligands, dual activation of extrinsic and intrinsic apoptotic cascades, mitochondrial priming of leukemia cells by venetoclax, and bypassing significant morbidity and mortality associated allo-SCT. Significance: This will be the first trial to evaluate 1) cellular therapy in the frontline setting in AML; 2) the synergy of venetoclax with adoptive NK cell therapy; and 3) Aza/Ven as a lymphodepletion strategy. The results will have significant impact on the development of cellular therapies for hematological and solid tumors. STUDY DESIGN AND METHODS: This investigator-initiated, multicenter, open-label, phase Ib study will evaluate the combination of azacitidine, venetoclax and ex vivo-expanded, off-the-shelf cryopreserved allogeneic NK cells in patients with AML (NCT05834244). Eligibility criteria: The dose escalation phase will enroll adults with relapsed or refractory AML or MDS/AML, and the dose expansion phase will enroll older/unfit pts with newly diagnosed adverse-risk AML. Eligible Pts will be ≥ 75 years, or ≥ 18 years with at least one protocol-defined comorbidity. Patients with favorable risk AML, poor end-organ function, needing immunosuppression, uncontrolled infection or CNS leukemia will be excluded. Trial design: The dose escalation phase will enroll a maximum of 12 patients at 2 dose levels to establish a recommended phase 2 dose (RP2D) using a BOIN design. The dose expansion will enroll up to 20 patients at the RP2D ( Fig. 1). Study treatment: Patients will receive Aza/Ven as standard. Two dose levels of NK cells including 0.5 x10 9 and 1 x10 9 flat dose on day 8 and 15 of the first 4 cycles will be evaluated. Subsequently, pts will continue Aza/Ven indefinitely. Objectives: The primary objective is to determine the safety of this novel triplet combination in terms of dose-limiting toxicities including acute GVHD, CRS or ICANS. Secondary objectives include overall response rate, CR/CRi by 4 cycles, rate of negative measurable residual disease, OS and relapse-free survival. Exploratory objectives include additional response and survival endpoints, the persistence of NK cells using SNP-NGS chimerism, kinetics of allorejection, remodeling of host immune microenvironment and impact on AML stem/progenitor cell hierarchies using single cell CyTOF to understand response/resistance mechanisms. Status: Trial approved by the FDA and institutional IRB. Activation planned for 2H 2023. Funding: Gateway for Cancer Research, MDACC PRIME Award, Chimeric Therapeutics, NCI Cancer Center Support Grant.

Results of Cynk-001-AML-001: A Phase I Multi-Dose Study Evaluating the Safety, Tolerability, and Persistence of Cynk-001 in Adults with De Novo or Secondary Acute Myeloid Leukemia in Morphologic Complete Remission with Minimal Residual Disease or Relapsed/Refractory AML

Introduction: Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous disease often with suboptimal outcomes. While progress has been achieved in the treatment of AML, relapse remains a significant challenge. Novel therapeutic options aimed at attaining minimal residual disease (MRD) negative complete remission (CR) are expected to reduce the incidence of relapse and prolong survival. Evidence shows that natural killer (NK) cells can exhibit potent anti-leukemia activity against AML. CYNK-001 is a CD56+CD3- enriched, off-the-shelf, unmodified allogeneic NK cells expanded from placental CD34+ cells. Aim CYNK-001-AML-001 is a phase I multi-dose study evaluating the safety, tolerability, and persistence of CYNK-001 with or without recombinant human interleukin-2 (rhIL-2) in adults with de novo or secondary acute myeloid leukemia (AML) in morphologic complete remission (CR) with minimal residual disease (MRD) or relapsed/refractory (R/R) AML. Methods This Phase I, single-arm, dose escalation clinical trial (NCT04310592) enrolled patients 18-80 years with R/R AML or patient in MRD positive CR. Two lymphodepletion regimens were assessed. Patients in cohorts 1-3 underwent standard lymphodepletion with fludarabine (25 mg/m2/day) and cyclophosphamide (300 mg/m2/day) on days -5, -4 and -3. Subsequent cohorts received enhanced lymphodepletion with fludarabine (30 mg/m2/day) and cyclophosphamide (900 mg/m2/day) on days -6, -5, -4 and -3. Escalating doses of NK cells were administered following a standard 3+3 design, with 2 separate arms (MRD+ arm and R/R arm) starting with cohort 4. A starting dose of 600 million cells on days 0, 7, and 14 was escalated to a maximum dose of 1.8 billion cells on days 0, 7, 14 and 21. The primary objective of the study was to determine the maximum tolerated dose (MTD) or maximum planned dose (MPD) of CYNK-001 and to assess the safety of multiple infusions of CYNK-001 administered using a flat, non-weight-based dose as assessed by the frequency and severity of adverse events (AE). The secondary objective was to assess the clinical efficacy of CYNK-001 in AML subjects. Results 28 patients (17 with R/R AML and 11 with MRD-positive AML) were enrolled during dose escalation, and 27 received CYNK-001. The highest dose of NK cells administered was 1.8 billion cells X 4 in cohort 6b (patients with R/R AML). Treatment was well tolerated with no dose limiting toxicities (DLTs) even at the highest dose. No cases of neurotoxicity or GvHD were reported. Four cases of grade 2 CRS were reported but there were no cases of ≥ grade 3 CRS. Two incidences of grade 4 thrombocytopenia and one grade 4 decreased white blood cell count were noted in patients who had received enhanced lymphodepletion. In R/R AML patients treated with the highest dose of CYNK-001, 2 of 4 patients achieved an objective response of Morphologic Leukemia-Free State (MLFS) on Day 28. One of these patients has maintained a response to D120 and is continuing follow up. One of 3 patients with MRD +ve AML treated with 3 doses of 1.8 billion CYNK-001 cells per dose achieved MRD negativity until day 120 at which time a blast count of 1.6% was observed. Conclusion Treatment with CYNK-001 showed biological activity as evidenced by achievement of MLFS in R/R AML patients and eradication of AML MRD positivity in AML patients in morphologic CR. Therapy was generally well-tolerated, with no dose-limiting toxicities observed with even the highest dose of CYNK-001. This trial yielded important insights into the optimal approach to lymphodepletion and the potential clinical efficacy of allogeneic NK cells in R/R AML. Based on these results, this trial is now closed to further enrollment and future development activities will focus on next generation NK cell therapies edited to optimize persistence and efficacy and limit the requirement for enhanced lymphodepletion.

Forks in the road for CAR T and CAR NK cell cancer therapies.

The advent of chimeric antigen receptor (CAR) T cell therapy has resulted in unprecedented long-term clearance of relapse/refractory hematological malignancies in both pediatric and adult patients. However, severe toxicities, such as cytokine release syndrome and neurotoxicity, associated with CAR T cells affect therapeutic utility; and treatment efficacies for solid tumors are still not impressive. As a result, engineering strategies that modify other immune cell types, especially natural killer (NK) cells have arisen. Owing to both CAR-dependent and CAR-independent (innate immune-mediated) antitumor killing capacity, major histocompatibility complex-independent cytotoxicity, reduced risk of alloreactivity and lack of major CAR T cell toxicities, CAR NK cells constitute one of the promising next-generation CAR immune cells that are also amenable as 'off-the-shelf' therapeutics. In this Review, we compare CAR T and CAR NK cell therapies, with particular focus on immunological synapses, engineering strategies and challenges.

Potentiation of natural killer cells to overcome cancer resistance to NK cell-based therapy and to enhance antibody-based immunotherapy.

Natural killer (NK) cells are cellular components of the innate immune system that can recognize and suppress the proliferation of cancer cells. NK cells can eliminate cancer cells through direct lysis, by secreting perforin and granzymes, or through antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC involves the binding of the Fc gamma receptor IIIa (CD16), present on NK cells, to the constant region of an antibody already bound to cancer cells. Cancer cells use several mechanisms to evade antitumor activity of NK cells, including the accumulation of inhibitory cytokines, recruitment and expansion of immune suppressor cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), modulation of ligands for NK cells receptors. Several strategies have been developed to enhance the antitumor activity of NK cells with the goal of overcoming cancer cells resistance to NK cells. The three main strategies to engineer and boost NK cells cytotoxicity include boosting NK cells with modulatory cytokines, adoptive NK cell therapy, and the employment of engineered NK cells to enhance antibody-based immunotherapy. Although the first two strategies improved the efficacy of NK cell-based therapy, there are still some limitations, including immune-related adverse events, induction of immune-suppressive cells and further cancer resistance to NK cell killing. One strategy to overcome these issues is the combination of monoclonal antibodies (mAbs) that mediate ADCC and engineered NK cells with potentiated anti-cancer activity. The advantage of using mAbs with ADCC activity is that they can activate NK cells, but also favor the accumulation of immune effector cells to the tumor microenvironment (TME). Several clinical trials reported that combining engineered NK cells with mAbs with ADCC activity can result in a superior clinical response compared to mAbs alone. Next generation of clinical trials, employing engineered NK cells with mAbs with higher affinity for CD16 expressed on NK cells, will provide more effective and higher-quality treatments to cancer patients.

Direct Tumor Irradiation Potentiates Adoptive NK Cell Targeting Against Parental and Stemlike Cancer in Human Liver Cancer Models

Radiation therapy (RT) has been shown to effectively induce the expression of intercellular adhesion molecule-1 (ICAM-1), which is recognized by lymphocyte function-associated antigen 1 (LFA-1) expressed on natural killer (NK) cells. However, the potential synergistic antitumor immune response of tumor irradiation and administered NK cells has not been explored in intractable human liver cancers. Furthermore, NK cell targeting against both parental and cancer stemness has never been investigated. Highly activated ex vivo NK cells were administered into the human liver tumor-bearing mice. Tumor direct RT was optimized according to tumor bearing site. HepG2 and Hep3B ICAM-1 knockout cells were generated using CRISPR/CAS9. Stemness tumor spheres were generated. NK cell cytolysis against parental and tumor sphere was evaluated using flow cytometry and real-time cytotoxicity assay. A combination of adoptive NK cell therapy with RT significantly improved therapeutic efficacy over monotherapies against subcutaneous, orthotopic, and metastatic human liver tumor models. Direct tumor irradiation potentiated NK cell recognition and conjugation against liver cancer through the LFA-1/ICAM-1 axis. Suppression of immune synapse formation on NK cells using high-affinity LFA-1 inhibitors or ICAM-1 knockout liver cancer induced "outside-in" signal blocking in NK cells, resulting in failure to eliminate liver tumor despite the combination therapy. NK cells effectively recognized and targeted triple-high epithelial cell adhesion molecule+CD133+CD24+ liver cancer expressing upregulated ICAM-1 in the irradiated tumor microenvironment, which led to prevention of the initiation of metastasis, improving survival in a metastatic model. In addition, the LFA-1/ICAM-1 axis interruption between NK cells and stemness liver tumor spheres significantly diminished NK cell cytolysis. Consistent with our preclinical data, the LFA-1/ICAM-1 axis correlated with survival outcomes in patients with metastatic cancer from the The Cancer Genome Atlas databases. NK cells in combination with tumor irradiation can provide synergistic therapeutic effects for NK cell recognition and elimination against both parental and stemlike liver cancer through LFA-1/ICAM-1.

Boosting cytotoxicity of adoptive allogeneic NK cell therapy with an oncolytic adenovirus encoding a human vIL-2 cytokine for the treatment of human ovarian cancer

Despite good results in the treatment of hematological malignancies, Natural killer (NK) cells have shown limited effectiveness in solid tumors, such as ovarian cancer (OvCa). Here, we assessed the potential of an oncolytic adenovirus expressing a variant interleukin-2 (vIL-2) cytokine, Ad5/3-E2F-d24-vIL2 (vIL-2 virus), also known as TILT-452, to enhance NK cell therapy efficacy in human OvCa ex vivo. Human OvCa surgical specimens were processed into single-cell suspensions and NK cells were expanded from healthy blood donors. OvCa sample digests were co-cultured ex vivo with NK cells and vIL-2 virus and cancer cell killing potential assessed in real time through cell impedance measurement. Proposed therapeutic combination was evaluated in vivo with an OvCa patient-derived xenograft (PDX) in mice. Addition of vIL-2 virus significantly enhanced NK cell therapy killing potential in treated OvCa co-cultures. Similarly, vIL-2 virus in combination with NK cell therapy promoted the best in vivo OvCa tumor control. Mechanistically, vIL-2 virus induced higher percentages of granzyme B in NK cells, and CD8+ T cells, while T regulatory cell proportions remained comparable to NK cell monotherapy in vivo. Ad5/3-E2F-d24-vIL2 virus treatment represents a promising strategy to boost adoptive NK cell therapeutic effect in human OvCa.

Adoptive cell immunotherapy for breast cancer: harnessing the power of immune cells.

Breast cancer is the most prevalent malignant neoplasm worldwide, necessitating the development of novel therapeutic strategies owing to the limitations posed by conventional treatment modalities. Immunotherapy is an innovative approach that has demonstrated significant efficacy in modulating a patient's innate immune system to combat tumor cells. In the era of precision medicine, adoptive immunotherapy for breast cancer has garnered widespread attention as an emerging treatment strategy, primarily encompassing cellular therapies such as tumor-infiltrating lymphocyte therapy, chimeric antigen receptor T/natural killer/M cell therapy, T cell receptor gene-engineered T cell therapy, lymphokine-activated killer cell therapy, cytokine-induced killer cell therapy, natural killer cell therapy, and γδ T cell therapy, among others. This treatment paradigm is based on the principles of immune memory and antigen specificity, involving the collection, processing, and expansion of the patient's immune cells, followed by their reintroduction into the patient's body to activate the immune system and prevent tumor recurrence and metastasis. Currently, multiple clinical trials are assessing the feasibility, effectiveness, and safety of adoptive immunotherapy in breast cancer. However, this therapeutic approach faces challenges associated with tumor heterogeneity, immune evasion, and treatment safety. This review comprehensively summarizes the latest advancements in adoptive immunotherapy for breast cancer and discusses future research directions and prospects, offering valuable guidance and insights into breast cancer immunotherapy.

Charting a killer course to the solid tumor: strategies to recruit and activate NK cells in the tumor microenvironment.

The ability to expand and activate natural Killer (NK) cells ex vivo has dramatically changed the landscape in the development of novel adoptive cell therapies for treating cancer over the last decade. NK cells have become a key player for cancer immunotherapy due to their innate ability to kill malignant cells while not harming healthy cells, allowing their potential use as an "off-the-shelf" product. Furthermore, recent advancements in NK cell genetic engineering methods have enabled the efficient generation of chimeric antigen receptor (CAR)-expressing NK cells that can exert both CAR-dependent and antigen-independent killing. Clinically, CAR-NK cells have shown promising efficacy and safety for treating CD19-expressing hematologic malignancies. While the number of pre-clinical studies using CAR-NK cells continues to expand, it is evident that solid tumors pose a unique challenge to NK cell-based adoptive cell therapies. Major barriers for efficacy include low NK cell trafficking and infiltration into solid tumor sites, low persistence, and immunosuppression by the harsh solid tumor microenvironment (TME). In this review we discuss the barriers posed by the solid tumor that prevent immune cell trafficking and NK cell effector functions. We then discuss promising strategies to enhance NK cell infiltration into solid tumor sites and activation within the TME. This includes NK cell-intrinsic and -extrinsic mechanisms such as NK cell engineering to resist TME-mediated inhibition and use of tumor-targeted agents such as oncolytic viruses expressing chemoattracting and activating payloads. We then discuss opportunities and challenges for using combination therapies to extend NK cell therapies for the treatment of solid tumors.

Adoptive NK Cell Therapy - a Beacon of Hope in Multiple Myeloma Treatment.

Major advances in the treatment of multiple myeloma (MM) have been achieved by effective new agents such as proteasome inhibitors, immunomodulatory drugs, or monoclonal antibodies. Despite significant progress, MM remains still incurable and, recently, cellular immunotherapy has emerged as a promising treatment for relapsed/refractory MM. The emergence of chimeric antigen receptor (CAR) technology has transformed immunotherapy by enhancing the antitumor functions of T cells and natural killer (NK) cells, leading to effective control of hematologic malignancies. Recent advancements in gene delivery to NK cells have paved the way for the clinical application of CAR-NK cell therapy. CAR-NK cell therapy strategies have demonstrated safety, tolerability, and substantial efficacy in treating B cell malignancies in various clinical settings. However, their effectiveness in eliminating MM remains to be established. This review explores multiple approaches to enhance NK cell cytotoxicity, persistence, expansion, and manufacturing processes, and highlights the challenges and opportunities associated with CAR-NK cell therapy against MM. By shedding light on these aspects, this review aims to provide valuable insights into the potential of CAR-NK cell therapy as a promising approach for improving the treatment outcomes of MM patients.

FT538, iPSC-Derived NK Cells Are Potent Inducers of Apoptosis in AML Cells and Their Effect Is Synergistic in Combination with Approved Therapeutic Strategies

Background: Natural killer (NK) cell-based therapies have been increasingly studied with their advantages of less treatment-related toxicity and lack of HLA restriction. Induced pluripotent stem cell (iPSC)-derived NK cells offer an opportunity for a standardized, off-the-shelf treatment with the potential to treat a wider patient population. FT538 cells are triple gene-edited iPSC-derived NKs that express a high affinity non-cleavable CD16 maintaining constant activity, a CD38 knock out preventing CD38 antibody-induced fratricide, and an IL-15/IL-15R fusion preventing the need for cytokine administration, the main source of side effects in NK cell-based therapies. Several studies have shown success in treating AML with NK cell therapy, but the effect of iPSC-derived NK cell therapy in AML is unexplored. We hypothesized that FT538 cells may be able to target high-risk AML that is traditionally resistant to most chemotherapeutic agents and synergize with Venetoclax, 5-azacytidine, Cytarabine, and Gilteritinib. Methods: To examine the effect of the FT538 cells on AML cells, we co-cultured FT538 cells at ratios of 2:1, 4:1, and 8:1 with 8 AML cell lines: OCI-AML2, OCI-AML3, THP1, U937, Molm-13, Molm-14, MV4-11, and Kasumi-1, and with cells from 11 different AML patients. We analyzed the NK cell-induced apoptosis through an Annexin-V binding assay using IncuCyte live cell imaging. As Molm-13 and MV4-11 cell lines are FLT3 mutated, to determine the combination effect of FLT3 inhibitors with FT538 cells, we treated Molm-13 and MV4-11 cells with Gilteritinib for 24 hours before seeding the FT538 cells and co-culturing overnight. The effects of the combination were then analyzed through flow cytometry with a Beckman Coulter CytoFlex. Finally, to determine the combinatorial effect of the FT538 cells with approved therapies, we treated OCI-AML3 and U937 cell lines with Venetoclax, Azacitidine, and Cytarabine. After treatment, we performed flow cytometry to measure the leukemic cell death following the combinatorial therapies. Results: We observed FT538 NK-mediated killing in all the leukemic cell lines tested. A two-way ANOVA determined that an increase in the FT538 to AML cell ratio resulted in an increase in NK cell-mediated killing of AML cells (p<0.0001). Interestingly, the number of apoptotic AML cells was 1.5 to 6.1-fold higher in the OCI-AML2, OCI-AML3, U937, and THP1 cell lines than in the Molm-13, Molm-14, MV4-11, and Kasumi-1 cell lines (p<0.0001). This could be related to the FLT3 mutations in the Molm-13, Molm-14, and MV4-11 cell lines or to the TP53 mutation in the Kasumi-1 cell line. Further, to validate this finding in primary AML cells, we observed a ratio-dependent effect on FT538-mediated killing of AML cells. However, we did not observe any statistical difference by mutation, though this could be related to the limited sample size for specific mutations. Finally, flow cytometric analysis of the effects of combination treatment demonstrated that the FT538 cells synergize with Venetoclax, or Cytarabine, or Azacitidine, or Gilteritinib (p<0.05) in the killing of AML cells. This could be because NKG2DL upregulation has been shown in AML cells treated with Venetoclax and FLT3 inhibitors and in Cytarabine-resistant AML cells, and the NKG2D/NKG2DL pathway is a main mechanism of NK cell-mediated killing. Conclusion: iPSC-derived NK therapy offers a standardized, off-the-shelf option for NK cell therapies. FT538 iPSC-derived NK cells induce apoptosis in AML cell lines and patient samples in a dose-dependent manner and show a synergistic effect with Venetoclax, Gilteritinib, Azacitidine, and Cytarabine. Therefore, iPSC-derived NK cell therapy may be a promising possibility for AML treatment, particularly for patients resistant to standard therapies.

NKX101, an Allogeneic Off-the-Shelf CAR NK Cell Therapy Targeting NKG2D-Ls, Has Potent Anti-Leukemic Activity Alone or in Combination with Ara-C

Introduction NKG2D-Ligands (NKG2D-Ls) are a family of immunomodulatory proteins that are upregulated on damaged or transformed cells but have limited expression on normal tissues. Natural killer (NK) cells target and eliminate NKG2D-L + cells through NKG2D receptor binding and subsequent activation. NKX101 is an allogeneic off-the-shelf healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D-L targeting CAR and membrane-bound IL-15 for extended persistence. In a clinical trial, NKX101 treatment combined with modified lymphodepletion incorporating cytarabine (Ara-C) has shown promise for the treatment of relapsed/refractory (r/r) AML, including generating complete responses (CR) with MRD negativity (MRD -). To support the rationale of NKX101 combined with modified lymphodepletion, we developed assays to (i) investigate the cytotoxic activity of NKX101 against individual NKG2D-Ls, (ii) determine the effect of Ara-C pre-treatment on NKG2D-L expression on AML cell lines, and (iii) evaluate the combined anti-leukemic activity of NKX101 and Ara-C. Methods NKX101 cells were generated from peripheral blood leukopaks from healthy donors. CHO-K1 cells were engineered to stably express individual NKG2D-Ls or empty vector controls and were combined with NKX101 cells for cytotoxicity experiments. AML cell lines were treated with increasing levels of Ara-C and evaluated for NKG2D-L expression by flow cytometry. To measure NKX101 cytotoxicity against AML cell lines, NucRed™-labeled target cells were pre-treated with Ara-C using IC 50 and IC 90 concentrations and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte® instrument for five days. Assessment of the contribution of each agent was determined using a dose-response matrix of increasing concentrations of NKX101 cells and Ara-C to generate specific cytotoxicity values. Combination effects were analyzed using the SynergyFinder Plus web application. Results Expression of a single NKG2D-L was sufficient to trigger NKX101 cytotoxicity against target cell lines. Flow cytometry analysis revealed that pre-treatment with Ara-C upregulated NKG2D-Ls on AML cell lines in a dose-dependent manner. NKX101 showed enhanced anti-leukemic activity against AML cells pre-treated with Ara-C compared to untreated controls, demonstrating that the combination can enhance NKX101 potency. Assessment of the combination of NKX101 and Ara-C using a dose-response matrix revealed that the two modalities combine to potently kill AML cells. Conclusions In summary, we show that NKX101 potently targets and eliminates AML cells expressing NKG2D-Ls further validating NKG2D-Ls as therapeutic targets. Additionally, we show that the combination of Ara-C and NKX101 leads to increased anti-leukemic activity, supporting continued investigation of NKX101 CAR NK cell therapy after lymphodepletion incorporating Ara-C for the treatment of r/r AML.

A Phase 1 Study of NKX101, a Chimeric Antigen Receptor Natural Killer (CAR-NK) Cell Therapy, with Fludarabine and Cytarabine in Patients with Acute Myeloid Leukemia

Introduction Relapsed or refractory (r/r) acute myeloid leukemia (AML) has a dismal prognosis. Treatment options are limited and include fludarabine (Flu) and cytarabine (Ara-C), +/- anthracyclines (e.g., FLAG-Ida). Recent progress has been limited to targeted therapies, which benefit only a subset of the population; efforts to develop cellular therapies have not been met with success. NKX101 is an NK cell therapy derived from healthy donors and engineered to express an NKG2D ligand-directed chimeric antigen receptor (CAR) to enhance killing of malignant cells, as well as a membrane bound form of interleukin (IL)-15 to promote persistence and activity. Because Ara-C is known to upregulate NKG2D ligand expression, use of Flu/Ara-C as an alternative to standard Flu/cyclophosphamide (Flu/Cy) for lymphodepletion (LD) was tested for NKX101. Methods Six patients with r/r AML were enrolled in this cohort of NKX101-101, a Phase 1 safety study. All patients had received at least one prior line of therapy, as well as approved targeted therapies for those with underlying genetic mutations. Patients received NKX101 at a dose of 1.5 billion (B) viable CAR+ cells/dose on Days 0, 7, and 14 after LD comprising Flu (30 mg/m2) and Ara-C (2 g/m2) each once daily for five days and 2 days of rest. Efficacy assessments were performed on Day 27 and included bone marrow aspiration and biopsy. Additional treatment cycles of LD and NKX101 were given to some patients to deepen or consolidate responses. Pharmacokinetic (PK) and cytokine sampling were performed throughout the treatment cycle(s). Results At baseline, five out of six patients had poor-risk genetic features, including TP53 mutation; the median bone marrow blast count was 35% (range: 20 - 86%). Patients had received a median of two prior lines of therapy, with all patients having previous venetoclax exposure (Table 1). All patients received at least three doses of NKX101 at 1.5 B cells/dose. There were no cases of cytokine release syndrome (CRS), immune cell associated neurotoxicity syndrome (ICANS), or graft-versus-host disease of any grade in any patients (Table 2). Myelosuppression and infection were the most common higher-grade toxicities. Four out of six patients had CR/CRi, with three out of six achieving CR. Three of these patients had no detectable MRD by flow, and one had 0.18% RUNX1-RUNX1T1 translocation via polymerase chain reaction testing on bone marrow. One subject was taken to consolidative hematopoietic cell transplant (HCT) and remains in CR. Another subject received a consolidative cycle of therapy and remains in CR. A third subject had three cycles of treatment with successive decrease in disease burden and remains in CRi. The fourth subject had CR after one cycle of treatment. PK profiling showed that NKX101 was consistently detected and correlated with infusion days (Figure 1). Cytokine data showed minimal post-infusion elevations above baseline; no association was observed between clinical response and elevation of serum cytokines (including IL-15, IL-6, IFNγ, IL-10, and IL-8). Data on the expression of NKG2D ligands (MICA, MICB, ULBP1, and ULBP3) in subject bone marrow samples were obtained via multiplex immunohistochemistry. This was evaluated as a composite H-score. Responders were split between those with relatively low ligand expression and those with higher ligand expression, suggesting that further factors may contribute to NK cell resistance. Conclusions Alternative LD with Flu/Ara-C followed by NKX101 shows promising early responses including MRD negativity in r/r AML. The toxicity profile of this regimen is manageable and was consistent with underlying AML and exposure to LD, without any events of CRS or ICANS. Expansion of enrollment and longer follow-up are needed. Despite being an allogeneic, non-HLA-matched cell product, NKX101 persisted for up to three weeks in PK testing. Directly comparing the data from patients treated with Flu/Cy with NKX101 versus Flu/Ara-C with NKX101 suggests that Flu/Ara-C can replace the Flu/Cy regimen common to CAR T cell therapy as LD without compromising overall NKX101 exposure.