Adoptive NK Cell Therapy: A Promising Treatment Prospect for Metastatic Melanoma

Adoptive cell therapy (ACT) represents a promising alternative approach for patients with treatment-resistant metastatic melanoma. Lately, tumor infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor (CAR)-T cell therapy have shown improved clinical outcome, compared to conventional chemotherapy or immunotherapy. Nevertheless, they are limited by immune escape of the tumor, cytokine release syndrome, and manufacturing challenges of autologous therapies. Conversely, the clinical use of Natural Killer (NK) cells has demonstrated a favorable clinical safety profile with minimal toxicities, providing an encouraging treatment alternative. Unlike T cells, NK cells are activated, amongst other mechanisms, by the downregulation of HLA class I molecules, thereby overcoming the hurdle of tumor immune escape. However, impairment of NK cell function has been observed in melanoma patients, resulting in deteriorated natural defense. To overcome this limitation, “activated” autologous or allogeneic NK cells have been infused into melanoma patients in early clinical trials, showing encouraging clinical benefit. Furthermore, as several NK cell-based therapeutics are being developed for different cancers, an emerging variety of approaches to increase migration and infiltration of adoptively transferred NK cells towards solid tumors is under preclinical investigation. These developments point to adoptive NK cell therapy as a highly promising treatment for metastatic melanoma in the future.

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799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.799 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A834-A834

Author(s):

Xue Yao ◽

Sandro Matosevic

Keyword(s):

Tumor Microenvironment ◽

Cell Therapy ◽

Nk Cells ◽

Natural Killer ◽

Solid Tumors ◽

Tumor Cells ◽

Nk Cell ◽

Killer Cell ◽

Nk Cell Therapy

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

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Small Molecule Screening Identifies Rho-Associate Protein Kinase (ROCK) As a Regulator of NK Cell Cytotoxicity Against Cancer

Blood ◽

10.1182/blood-2019-131947 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3607-3607

Author(s):

Grace Lee ◽

Sheela Karunanithi ◽

Zachary Jackson ◽

David Wald

Keyword(s):

Cell Therapy ◽

Cytotoxic Activity ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Akt Activation ◽

Nk Cell Cytotoxicity ◽

Rock Inhibition ◽

Nk Cell Therapy

NK cells are a subset of lymphocytes that directly recognize and lyse tumor cells without the limitation of antigen specific receptor recognition. In addition to behaving as cytotoxic effector cells, NK cells unlike T cells are not thought to elicit graft versus host disease. The combination of these characteristics makes NK cells a powerful tool for adoptive cell therapy. Despite the promise of NK cell therapy, key hurdles in achieving significant clinical efficacy include both generating sufficient numbers of highly tumoricidal NK cells and maintaining the cytotoxic activity of these cells in vivo despite the immunosuppressive tumor microenvironment. Our lab and others have developed several feeder cell line-based expansion modules that robustly stimulate the ex vivo proliferation of NK cells. However, strategies to enhance and sustain the activity of NK cells once administered in vivo are still limited. In order to identify strategies to enhance the cytotoxic activity of NK cells, we developed a high-throughput small molecule screen (Figure 1A) that involved a calcein-based cytotoxicity assay of ex vivo expanded and treated NK cells against ovarian cancer cells (OVCAR-3). 20,000 compounds were screened and the screen was found to be highly robust (Z'>0.59). We identified 29 hits that led to at least a 25% increase in cytotoxicity as compared to DMSO control-treated NK cells. One of the most promising hits was the pan-ROCK inhibitor, Y-27632 that led to an 30% increase in NK killing of the OVCAR-3 cells. We validated that ROCK inhibition leads to enhanced NK cell cytotoxic activity using Y-27632 (Figure 1B) as well as other well-established ROCK inhibitors such as Fasudil using a flow cytometry based killing assay. Y-27632 increased NK cell cytotoxicity in a dose- and time- dependent manner. ROCK inhibition consistently led to ~10-25% increase in NK cell cytotoxic activity directed against a variety of ovarian (Figure 1C) and other solid tumor cell lines (Figure 1D). Interestingly, we found that the NK hyperactivation persists for up to 48hrs after washing off the drug that may enable ex vivo stimulation before NK cell infusion. Our preliminary results showed that ROCK inhibition activates PI3K-dependent Akt activation (Figure 1E). We hypothesize that ROCK inhibition restores Akt activation which may be critical for NK cell activating receptor pathways and our current investigations will test these hypotheses. ROCK inhibitors, such as Y-27632 and Fasudil have been utilized in both preclinical and clinical studies for a variety of diseases such as atherosclerosis, neurodegenerative disorders, and ocular diseases. However, the consequences of ROCK inhibition in NK cells has not been thoroughly investigated. Our work shows a promising novel strategy to significantly enhance NK cell therapy against cancer that has high translational potential. Disclosures No relevant conflicts of interest to declare.

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Biological treatment of pediatric sarcomas by combined virotherapy and NK cell therapy

BMC Cancer ◽

10.1186/s12885-019-6387-5 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 5

Author(s):

Chihab Klose ◽

Susanne Berchtold ◽

Marina Schmidt ◽

Julia Beil ◽

Irina Smirnow ◽

...

Keyword(s):

Cell Therapy ◽

Nk Cells ◽

Real Time ◽

Nk Cell ◽

Sarcoma Cell ◽

Pediatric Sarcoma ◽

Pediatric Sarcomas ◽

Sarcoma Cells ◽

Human Sarcoma ◽

Nk Cell Therapy

Abstract Background In pediatric sarcomas, outcomes of established therapies still remain poor, especially due to high-grade resistances to chemotherapeutic compounds. Taking novel biological approaches into account, virotherapy was found to be efficient in many pediatric sarcoma types. Also NK cell therapy was denoted to represent a promising upcoming strategy for pediatric sarcoma patients. We here investigated a combinatorial approach employing oncolytic measles vaccine virotherapeutics (MeV) together with activated human NK cells (or PBMCs). Methods The human sarcoma cell lines A673 and HT1080 were used to evaluate the efficacy of this combinatorial treatment modality. Oncolysis was determined by measuring real-time cell proliferation using the xCELLigence RTCA SP system. Furthermore, expression of receptors on NK cells and the respective ligands on A673 cells was analyzed by flow cytometry. To measure the protein release of activated NK cells a LEGENDplex™ assay was performed. Results Monotherapy with MeV led to a time- and dose-dependent oncolytic reduction of A673 and HT1080 sarcoma tumor cell masses. Concurrently, such MeV infections did not change the expression of NK cell ligands MICA/B, ULBP1, 2, and 3, CD112, and CD155. As shown by real-time proliferation assays, infections of A673 and HT1080 sarcoma cells with MeV followed by co-culture with activated NK cells or PBMCs led to enhanced sarcoma cell destruction when compared to the respective monotherapies. In parallel, this dual therapy resulted in an increased release of granzymes, perforin, and granulysin from NK cells. In contrast, expression of activation and ontogenesis receptors on NK cells was not found to be altered after co-culture with MeV-infected A673 sarcoma cells. Conclusions Taken together, the combined treatment strategy comprising oncolytic MeV and activated NK cells resulted in enhanced oncolysis of A673 and HT1080 cells when compared to the respective monotherapies. In parallel, we observed an increased release of NK cell activation markers upon co-culture with MeV-infected A673 human sarcoma cells. These results support the onset of clinical trials combining oncolytic virotherapy with NK cell based immunotherapies.

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Natural Killer Cell Responses in Hepatocellular Carcinoma: Implications for Novel Immunotherapeutic Approaches

Cancers ◽

10.3390/cancers12040926 ◽

2020 ◽

Vol 12 (4) ◽

pp. 926 ◽

Cited By ~ 5

Author(s):

Stefania Mantovani ◽

Barbara Oliviero ◽

Stefania Varchetta ◽

Dalila Mele ◽

Mario U. Mondelli

Keyword(s):

Hepatocellular Carcinoma ◽

T Cells ◽

Nk Cells ◽

Natural Killer ◽

Immune Escape ◽

Nk Cell ◽

Ex Vivo ◽

Killer Cell ◽

Current Status ◽

Tumor Immune Escape

Hepatocellular carcinoma (HCC) still represents a significant complication of chronic liver disease, particularly when cirrhosis ensues. Current treatment options include surgery, loco-regional procedures and chemotherapy, according to specific clinical practice guidelines. Immunotherapy with check-point inhibitors, aimed at rescuing T-cells from exhaustion, has been applied as second-line therapy with limited and variable success. Natural killer (NK) cells are an essential component of innate immunity against cancer and changes in phenotype and function have been described in patients with HCC, who also show perturbations of NK activating receptor/ligand axes. Here we discuss the current status of NK cell treatment of HCC on the basis of existing evidence and ongoing clinical trials on adoptive transfer of autologous or allogeneic NK cells ex vivo or after activation with cytokines such as IL-15 and use of antibodies to target cell-expressed molecules to promote antibody-dependent cellular cytotoxicity (ADCC). To this end, bi-, tri- and tetra-specific killer cell engagers are being devised to improve NK cell recognition of tumor cells, circumventing tumor immune escape and efficiently targeting NK cells to tumors. Moreover, the exciting technique of chimeric antigen receptor (CAR)-engineered NK cells offers unique opportunities to create CAR-NK with multiple specificities along the experience gained with CAR-T cells with potentially less adverse effects.

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Ex Vivo Activated Natural Killer (NK) Cells from Myeloma Patients Kill Autologous Myeloma and Killing Is Enhanced by Elotuzumab

Blood ◽

10.1182/blood.v112.11.3666.3666 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3666-3666

Author(s):

Tarun K. Garg ◽

Susann Szmania ◽

Jumei Shi ◽

Katie Stone ◽

Amberly Moreno-Bost ◽

...

Keyword(s):

Bone Marrow ◽

Cell Therapy ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

K562 Cells ◽

Scid Mice ◽

Target Cells ◽

Recent Trial ◽

Nk Cell Therapy

Abstract Immune-based therapies may improve outcome for multiple myeloma (MM) by eradicating chemo-resistant disease. Our recent trial utilizing IL2 activated, killer immunoglobulin-like receptor-ligand mismatched NK cell transfusions from haplo-identical donors yielded (n) CR in 50% of patients. Unfortunately, after NK cell therapy, 2/10 patients had progressive disease, and the median duration of response for the other 8/10 patients was only 105 days (range 58–593). This may have been due to an insufficient dose of alloreactive NK cells and early rejection. Furthermore, appropriate donors were identified for only 30% of otherwise eligible patients. We therefore investigated whether NK cells from MM patients could be expanded and activated to kill autologous MM. We then examined whether pre-treatment of MM cell targets with elotuzumab, a humanized antibody to the MM tumor antigen CS1, could further enhance NK cell-mediated lysis. PBMC from 5 MM patients were co-cultured for 14 days with irradiated K562 cells transfected with 4-1BBL and membrane bound IL15 in the presence of IL2 (300U/ml) as previously described (Imai et al, Blood2005;106:376–383). The degree of NK cell expansion, NK immunophenotype, and ability to kill MM (4 hour 51Cr release assays) were assessed. To determine the ability of ex vivo expanded NK cells to traffic to bone marrow, activated NK cells were injected into the tail vein of NK cell depleted NOD-SCID mice, which were then sacrificed after 48 hours. Flow cytometry for human CD45, CD3, and CD56 was performed on cells from blood, marrow and spleen. There was an average 64-fold expansion of NK cells (range: 8–200) after 2 weeks of co-culture with K562 transfectants. Expansion of T cells was not observed. The NK cell activating receptor NKG2D, and natural cytotoxicity receptors NKp30, NKp44, and NKp46 were up-regulated following the expansion. Expanded NK cells were able to kill autologous MM (E:T ratio 10:1, average 31%, range 22–41%), whereas resting NK cells did not. Pretreatment of autologous MM cells with elotuzumab increased the activated NK cell-mediated killing by 1.7-fold over target cells pretreated with an isotype control antibody. This level of killing was similar to that of the highly NK kill-sensitive cell line K562 (Figure). Autologous PHA blasts and CD34+ stem cells were not killed. Activated human NK cells were detectable in the bone marrow of NOD-SCID mice 48 hours after injection. Ex vivo activation of NK cells from MM patients with K562 transfectants can induce killing of autologous MM and produce large numbers of NK cells for potential therapy. The addition of elotuzumab to activated NK cell therapy enhances anti-MM effects by ADCC thus invoking an additional NK cell-mediated mechanism of MM killing. Importantly, ex vivo activated NK cells traffic to the bone marrow in mice. Autologous NK cell therapy eliminates the issues related to allo-donor availability and early NK cell rejection, and could provide an option for patients refractory to chemotherapy agents. Figure Figure

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Adaptive NK Cell Therapy Modulated by Anti-PD-1 Antibody in Gastric Cancer Model

Frontiers in Pharmacology ◽

10.3389/fphar.2021.733075 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shahrokh Abdolahi ◽

Zeinab Ghazvinian ◽

Samad Muhammadnejad ◽

Mohammad Ahmadvand ◽

Hamid Asadzadeh Aghdaei ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Therapy ◽

Tumor Growth ◽

Growth Inhibition ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Tumor Growth Inhibition ◽

Cancer Model ◽

Nk Cell Therapy

Recently, adaptive NK cell therapy has become a promising treatment but has limited efficacy as a monotherapy. The identification of immune checkpoint inhibitor (ICI) molecules has opened a new horizon of immunotherapy. Herein, we aimed to demonstrate the cytotoxic effects of a polytherapy consisting of ex vivo expanded IL-2-activated NK cells combined with human anti-PD-1 antibody as an important checkpoint molecule in a xenograft gastric cancer mouse model. EBV-LCL cell is used as a feeder to promote NK cell proliferation with a purity of 93.4%. Mice (NOG, female, 6–8 weeks old) with xenograft gastric tumors were treated with PBS, ex vivo IL-2-activated NK cells, IL-2-activated NK cell along with human anti-PD-1 (Nivolumab), and IL-2-activated pretreated NK cells with anti-PD-1 antibody. The cytotoxicity of ex vivo expanded NK cells against MKN-45 cells was assessed by a lactate dehydrogenase (LDH) assay. Tumor volume was evaluated for morphometric properties, and tumor-infiltrating NK cells were assessed by immunohistochemistry (IHC) and quantified by flow cytometry. Pathologic responses were considered by H and E staining. Ex vivo LDH evaluation showed the cytotoxic potential of treated NK cells against gastric cancer cell line. We indicated that the adoptive transfer of ex vivo IL-2-activated NK cells combined with anti-PD-1 resulted in tumor growth inhibition in a xenograft gastric cancer model. Mitotic count was significantly decreased (*p < 0.05), and the tumor was associated with improved infiltration of NK cells in the NK-anti-PD-1 pretreated group (*p < 0.05). In conclusion, the combination approach of activated NK cells and anti-PD-1 therapy results in tumor growth inhibition, accompanied by tumor immune cell infiltration in the gastric tumor model.

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Adoptive transfer of NK cells in combination with chemotherapy to improve outcomes of patients with locally advanced colon carcinoma.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e15038 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e15038-e15038 ◽

Cited By ~ 1

Author(s):

Lingyu Li ◽

Jiuwei Cui ◽

Chang Wang ◽

Yizhuo Wang ◽

Chao Niu ◽

...

Keyword(s):

Cohort Study ◽

Cell Therapy ◽

Nk Cells ◽

Nk Cell ◽

Locally Advanced ◽

Cell Group ◽

Control Group ◽

Poorly Differentiated ◽

Local Advanced ◽

Nk Cell Therapy

e15038 Background: The prognosis of advanced colon cancer (CC) patients remains disappointing, partly due to their greater proportion of CC-initiating cells (CICs), which is responsible for cancer drug-resistance and immune escape. Immunotherapies by harnessing the immune system to eliminate tumors have attracted broad attention. This study was to detect whether chemotherapy could enhance cytotoxicity of natural killer (NK) cells to CC cells (CCs), especially for CICs in vitro, and further evaluate the efficacy and safety of NK-cell therapy combined with chemotherapy in patients with local advanced CC. Methods: We observed that cytotoxicity of NK cells to CCs and CICs pretreated with 5-Fu or oxaliplatin. Then, an open-label pilot cohort study was conducted with local advanced CC patients who had received surgical excision. 60 patients elected to receive either NK-cell therapy combined with chemotherapy (NK-cell group, 27 patients) or pure chemotherapy (control group, 33 patients). Progression-free survival (PFS), overall survival (OS) and adverse effects were investigated. Results: Chemotherapy sensitized CCs and CICs to NK cell lysis through upregulation of their NK cell activating ligands and reducing inhibitory ligands. Poorly differentiated CCs were more susceptible to NK-cell than well-differentiated CCs, and CICs were more easily to be killed by NK cell than their differentiated CCs. In the cohort study, the 5-year PFS and OS rates in the NK-cell group were significantly higher than those in the control group (51.1% vs. 34.9%, p= 0.043; 73% vs. 51.3%, p= 0.038, respectively).Among patients with poorly differentiated carcinomas or low expression of HLA-1, median PFS in the NK-cell group vs. the control group was 23.5 vs. 11.5 months ( p= 0.047), and median OS was 30 vs. 15 months ( p= 0.043), respectively. No significant adverse reaction was found during NK-cell therapy. Conclusions: NK-cell therapy in combination with chemotherapy in locally advanced CC prevented recurrence and prolonged survival with acceptable adverse effects, especially for poorly differentiated carcinomas.

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The Interplay of Exosomes and NK Cells in Cancer Biology

Cancers ◽

10.3390/cancers13030473 ◽

2021 ◽

Vol 13 (3) ◽

pp. 473

Author(s):

Inês A. Batista ◽

Sofia T. Quintas ◽

Sónia A. Melo

Keyword(s):

Nk Cells ◽

Cancer Progression ◽

Cell Biology ◽

Cancer Biology ◽

Immune Escape ◽

Nk Cell ◽

Lymphoid Cells ◽

Tumor Immune Escape ◽

Immunotherapeutic Strategy ◽

Open Question

Natural killer (NK) cells are innate lymphoid cells involved in tumor surveillance. These immune cells have the potential to fight cancer growth and metastasis, as such, their deregulation can result in tumor immune escape. Recently exosomes were described as mediators of intercellular communication between cancer and NK cells. The exact role of this subclass of extracellular vesicles (EVs), which transport genetic and molecular material to recipient cells, in NK cell biology in the context of cancer, is still an open question. Several reports have demonstrated that tumor-derived exosomes (TDEs) can exert immunomodulatory activities, including immunosuppression, thus promoting cancer progression. Some reports demonstrate that the interplay between cancer exosomes and NK cells allows tumors to escape immune regulation. On the other hand, tumor exosomes were also described to activate NK cells. Additionally, studies show that NK cell exosomes can modulate the immune system, opening up their potential as an immunotherapeutic strategy for cancer treatment. Our review will focus on the reprogramming effect of cancer exosomes on NK cells, and the immunotherapeutic potential of NK cells-derived exosomes.

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NK Cell Therapy: A Rising Star in Cancer Treatment

Cancers ◽

10.3390/cancers13164129 ◽

2021 ◽

Vol 13 (16) ◽

pp. 4129

Author(s):

Nawen Du ◽

Feifei Guo ◽

Yufeng Wang ◽

Jiuwei Cui

Keyword(s):

Cell Therapy ◽

Cancer Treatment ◽

Nk Cells ◽

Nk Cell ◽

Current Status ◽

Safety Issues ◽

Patients With Cancer ◽

Histocompatibility Complex ◽

Promising Solution ◽

Nk Cell Therapy

Immunotherapy has become a robust and routine treatment strategy for patients with cancer; however, there are efficacy and safety issues that should be resolved. Natural killer (NK) cells are important innate immune cells that have attracted increasing attention owing to their major histocompatibility complex-independent immunosurveillance ability. These cells provide the first-line defense against carcinogenesis and are closely related to cancer development. However, NK cells are functionally suppressed owing to multiple immunosuppressive factors in the tumor microenvironment; thus, releasing the suppressed state of NK cells is an emergent project and a promising solution for immunotherapy. As a result, many clinical trials of NK cell therapy alone or in combination with other agents are currently underway. This review describes the current status of NK cell therapy for cancer treatment based on the effector function and releasing the inhibited state of NK cells in the cancer microenvironment.

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CD38 knockout natural killer cells expressing an affinity optimized CD38 chimeric antigen receptor successfully target acute myeloid leukemia with reduced effector cell fratricide.

Haematologica ◽

10.3324/haematol.2020.271908 ◽

2020 ◽

Author(s):

Mark Gurney ◽

Arwen Stikvoort ◽

Emma Nolan ◽

Lucy Kirkham-McCarthy ◽

Stanislav Khoruzhenko ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Therapy ◽

Nk Cells ◽

Natural Killer ◽

Chimeric Antigen Receptor ◽

Myeloid Leukemia ◽

Nk Cell ◽

Cd38 Expression ◽

Nk Cell Therapy ◽

Acute Myeloid

There is a strong biological rationale for the augmentation of allogeneic natural killer (NK) cell therapies with a chimeric antigen receptor (CAR) to enhance acute myeloid leukemia (AML) targeting. CD38 is an established immunotherapeutic target in multiple myeloma and under investigation as a target antigen in AML. CD38 expression on NK cells and its further induction during ex vivo NK cell expansion represents a barrier to the development of a CD38 CAR-NK cell therapy. We set out to develop a CD38 CAR-NK cell therapy for AML, first by using an NK cell line which has low baseline CD38 expression and subsequently healthy donor expanded NK cells. To overcome anticipated fratricide due to NK cell CD38 expression when using primary expanded NK cells, we applied CRISPR/Cas9 genome editing to disrupt the CD38 gene during expansion achieving a mean knockdown efficiency of 84%. The resulting CD38 KD expanded NK cells, after expression of an affinity optimized CD38 CAR, showed reduced NK cell fratricide and an enhanced ability to target primary AML blasts. Furthermore, the cytotoxic potential of CD38 CAR-NK cells was augmented by pre-treatment of the AML cells with all-trans retinoic acid which drove enhanced CD38 expression offering a rational combination therapy. These findings support the further investigation of CD38 KD - CD38 CAR-NK cells as a viable immunotherapeutic approach to the treatment of AML.

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