Chimeric Antigen Receptor Natural Killer Cells Research Articles

Imaging CAR-NK cells targeted to HER2 ovarian cancer with human sodium-iodide symporter-based positron emission tomography.

Chimeric antigen receptor (CAR) cell therapies utilize CARs to redirect immune cells towards cancer cells expressing specific antigens like human epidermal growth factor receptor 2 (HER2). Despite their potential, CAR T cell therapies exhibit variable response rates and adverse effects in some patients. Non-invasive molecular imaging can aid in predicting patient outcomes by tracking infused cells post-administration. CAR-T cells are typically autologous, increasing manufacturing complexity and costs. An alternative approach involves developing CAR natural killer (CAR-NK) cells as an off-the-shelf allogeneic product. In this study, we engineered HER2-targeted CAR-NK cells co-expressing the positron emission tomography (PET) reporter gene human sodium-iodide symporter (NIS) and assessed their therapeutic efficacy and PET imaging capability in a HER2 ovarian cancer mouse model.NK-92 cells were genetically modified to express a HER2-targeted CAR, the bioluminescence imaging reporter Antares, and NIS. HER2-expressing ovarian cancer cells were engineered to express the bioluminescence reporter Firefly luciferase (Fluc). Co-culture experiments demonstrated significantly enhanced cytotoxicity of CAR-NK cells compared to naive NK cells. In vivo studies involving mice with Fluc-expressing tumors revealed that those treated with CAR-NK cells exhibited reduced tumor burden and prolonged survival compared to controls. Longitudinal bioluminescence imaging demonstrated stable signals from CAR-NK cells over time. PET imaging using the NIS-targeted tracer 18F-tetrafluoroborate ([18F]TFB) showed significantly higher PET signals in mice treated with NIS-expressing CAR-NK cells.Overall, our study showcases the therapeutic potential of HER2-targeted CAR-NK cells in an aggressive ovarian cancer model and underscores the feasibility of using human-derived PET reporter gene imaging to monitor these cells non-invasively in patients.

Chimeric antigen receptor-natural killer cell therapy: current advancements and strategies to overcome challenges.

Chimeric antigen receptor-natural killer (CAR-NK) cell therapy is a novel immunotherapy targeting cancer cells via the generation of chimeric antigen receptors on NK cells which recognize specific cancer antigens. CAR-NK cell therapy is gaining attention nowadays owing to the ability of CAR-NK cells to release potent cytotoxicity against cancer cells without side effects such as cytokine release syndrome (CRS), neurotoxicity and graft-versus-host disease (GvHD). CAR-NK cells do not require antigen priming, thus enabling them to be used as "off-the-shelf" therapy. Nonetheless, CAR-NK cell therapy still possesses several challenges in eliminating cancer cells which reside in hypoxic and immunosuppressive tumor microenvironment. Therefore, this review is envisioned to explore the current advancements and limitations of CAR-NK cell therapy as well as discuss strategies to overcome the challenges faced by CAR-NK cell therapy. This review also aims to dissect the current status of clinical trials on CAR-NK cells and future recommendations for improving the effectiveness and safety of CAR-NK cell therapy.

Informed by Cancer Stem Cells of Solid Tumors: Advances in Treatments Targeting Tumor-Promoting Factors and Pathways.

Cancer stem cells (CSCs) represent a subpopulation within tumors that promote cancer progression, metastasis, and recurrence due to their self-renewal capacity and resistance to conventional therapies. CSC-specific markers and signaling pathways highly active in CSCs have emerged as a promising strategy for improving patient outcomes. This review provides a comprehensive overview of the therapeutic targets associated with CSCs of solid tumors across various cancer types, including key molecular markers aldehyde dehydrogenases, CD44, epithelial cellular adhesion molecule, and CD133 and signaling pathways such as Wnt/β-catenin, Notch, and Sonic Hedgehog. We discuss a wide array of therapeutic modalities ranging from targeted antibodies, small molecule inhibitors, and near-infrared photoimmunotherapy to advanced genetic approaches like RNA interference, CRISPR/Cas9 technology, aptamers, antisense oligonucleotides, chimeric antigen receptor (CAR) T cells, CAR natural killer cells, bispecific T cell engagers, immunotoxins, drug-antibody conjugates, therapeutic peptides, and dendritic cell vaccines. This review spans developments from preclinical investigations to ongoing clinical trials, highlighting the innovative targeting strategies that have been informed by CSC-associated pathways and molecules to overcome therapeutic resistance. We aim to provide insights into the potential of these therapies to revolutionize cancer treatment, underscoring the critical need for a multi-faceted approach in the battle against cancer. This comprehensive analysis demonstrates how advances made in the CSC field have informed significant developments in novel targeted therapeutic approaches, with the ultimate goal of achieving more effective and durable responses in cancer patients.

Abstract 1323: Anti-B7-H3 chimeric antigen receptor NK cells suppress the growth of atypical teratoid/rhabdoid tumor orthotopic xenografts

Abstract Background: Atypical teratoid/rhabdoid tumors (AT/RTs) are the most common malignant CNS tumor in infants. AT/RT patients have a 5-year overall survival rate of ~35% and high rates of relapse, emphasizing a dire need for new safe and effective therapies. These therapy-resistant tumors frequently overexpress the immune checkpoint cell surface molecule B7-H3 (CD276). CAR-T cell studies have an established safety profile in pediatric patients. We developed B7-H3 targeted chimeric antigen receptor (CAR) natural killer (NK) cells as a tumor-specific cell therapy for aggressive pediatric brain tumors. CAR-NK cells have several advantages over CAR-T cells. NK cells can be obtained from healthy donors and produced as an “off-the-shelf” product. NK cells also have a lower risk of inflammatory toxicity and graft-versus-host disease compared to T-cells when transferred across the HLA barrier from healthy donors to patients. Methods: We have designed a library of variable affinity B7-H3-targeted CARs, produced replication incompetent γ-retroviral vector, and used this for generation of B7-H3 CAR-NK cells. We verified B7-H3 expression in a panel of AT/RT cell lines, and further engineered firefly luciferase expressing AT/RT (CHLA06.ffLuc, BT12.ffLuc, BT37.ffLuc) as well as a CHLA06-derived B7-H3 knockout. We developed an orthotopic CHLA06.ffLuc xenograft model. We tested CAR-NK cell functionality using in vitro co-culture and cytotoxicity assays. In vivo study was performed in our xenograft with intratumoral delivery of CAR-NK cells. Results: B7-H3 targeted CAR-NK cells demonstrate target-specific cytotoxicity when compared to untransduced NK cells (36.8±12.5% vs. 72.4±24.2% at effector:target ratio of 1:1, n=3). CRISPR/Cas9 mediated knockout of B7-H3 in target cells abolished the difference in CAR vs. no-CAR NK-mediated target killing. When delivered intracranially to CHLA-06 orthotopic xenograft bearing mice, B7-H3 CAR NK cells eliminate tumor cells and prolong survival, whereas unmodified NK cells did not (log-ranked median survival = 20 vs. 84 days, p<0.0001). We are currently testing intracranial delivery of our B7-H3 CAR NK against other AT/RT orthotopic xenografts. Conclusions: Targeting pediatric brain tumors with an anti-B7-H3 CAR-NK cell therapy may provide a safe and effective treatment for patients who have extremely limited therapeutic options. Direct intracranial injections of cell therapies into pediatric patients is currently being explored in clinical trials. As B7-H3 is a tumor associated antigen found to be overexpressed in a large variety of tumor types, this strategy may also be useful for treatment of other B7-H3 expressing cancers. Citation Format: Jun Choe, Sachiv Chakravarti, Natalie Holl, Ruyan Rahnama, Megan Zinsky, Danielle G. Jones, Stamatia Vorri, Arjun Modi, Eric H. Raabe, Challice L. Bonifant. Anti-B7-H3 chimeric antigen receptor NK cells suppress the growth of atypical teratoid/rhabdoid tumor orthotopic xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1323.

Abstract 1333: Preclinical data supporting the Phase 1 trial design of SENTI-202, a next generation allogeneic logic-gated selective CAR NK cell therapy, engineered to overcome key limitations of first generation cell therapies in AML

Abstract First generation natural killer (NK) and chimeric antigen receptor (CAR) NK cell products are well-tolerated and have clinical activity (~20-60% CR) in patients with AML. CRs generally increase with the inclusion of CARs or cytokines, increased cell doses, and when Flu/Ara-C lymphodepletion (LD) is used for conditioning. Key clinical limitations have been low PK of the infused NK cells, short durability of the observed responses potentially due to immune evasion of leukemic stem cells (LSCs), and manufacturing challenges precluding higher or multiple doses. SENTI-202 is a next generation allogeneic logic-gated selective CAR NK cell therapy specifically designed to address these limitations and to augment endogenous NK anti-AML activity. SENTI-202 expresses a bivalent activating CAR (aCAR) that targets CD33 and FLT3 on bulk AML blasts and LSCs. As FLT3 is also expressed on healthy hematopoietic stem cells (HSCs), SENTI-202 is further engineered to express an inhibitory CAR that recognizes endomucin expressed on HSCs, thereby conferring protection from aCAR-mediated cytotoxicity even in the presence of FLT3/CD33. SENTI-202 expresses a calibrated-release IL15 that provides both auto- and paracrine cytokine support, mediating expansion, activation, and persistence of SENTI-202 and host immune cells. SENTI-202 demonstrated robust and specific killing of primary AML blasts, LSCs, and AML cell lines in vitro. SENTI-202 protected HSCs from CAR-mediated cytotoxicity while preserving their function. In vivo, SENTI-202 revealed robust efficacy that increased with higher E:T ratio and with 3 weekly vs 1 dose. Preclinically, 3 doses of SENTI-202 that were > 60-fold the planned starting clinical trial dose were well tolerated in acute and chronic toxicology studies with no CAR NK-related body weight, laboratory, or histopathology findings. SENTI-202 non-clinical PK revealed greater than dose proportional exposure, which was ~2-fold greater compared to non-engineered NK cells. Pretreatment of CD33/FLT3 negative AML cell lines with Ara-C resulted in upregulation of CD33 and FLT3 expression, sensitizing cells to robust SENTI-202-mediated killing, providing additional rationale for the use of Ara-C-based LD. In the presence of exogenous IL2, persistence, cytotoxicity, and serial killing activity of SENTI-202 were increased, supporting the use of low dose IL2 to further augment SENTI-202 clinical activity. Taken together, these results support the Phase 1 trial design of SENTI-202-101 in patients with R/R CD33 and/or FLT3 positive malignancies including AML, which uses Flu/Ara-C as LD followed by 3 weekly doses of SENTI-202 and includes the option of enrolling patients into cohorts that additionally receive low dose IL2 following SENTI-202 administration. Citation Format: Deepika Kaveri, Pearley Chinta, Elizabeth Leitner, Lawrence Naitmazi, Gozde Yucel, Mengxi Tian, Niran Almudhfar, Han Deng, Yongshuai Li, Alice Lam, Chen-Ting Lee, Enping Hong, Rochelle Emery, Brian Garrison, Aaron N. Nguyen, Kanya Rajangam. Preclinical data supporting the Phase 1 trial design of SENTI-202, a next generation allogeneic logic-gated selective CAR NK cell therapy, engineered to overcome key limitations of first generation cell therapies in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1333.

Abstract 3603: 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

Abstract 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 transduced with 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 IC50 and IC90 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 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, further validating NKG2D-Ls as therapeutic targets. 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 in a synergistic manner, supporting continued investigation of NKX101 CAR NK cell therapy after lymphodepletion incorporating Ara-C for the treatment of r/r AML. Citation Format: Cynthia Cho, Nikki Kimura, Jessica Sood, Damie Juat, Max Zhang, Carmel Chan, Tao Geng, Kyle Hansen, James Trager. 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 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3603.

Abstract 1327: Affinity tuning anti-CD123 chimeric antigen receptor natural killer cell therapy to treat acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a highly prevalent blood and bone marrow cancer characterized by the uncontrolled growth of abnormal myeloblasts. Current treatments for AML often result in systemic toxicities for patients, many of whom will still experience relapse. There is thus an unmet need to develop safer and more effective therapies targeting AML. Chimeric antigen receptor (CAR) T cells, which are engineered to express a cancer-targeting extracellular antibody single-chain variable fragment (scFv) linked to an intracellular T cell-activating domain, have shown promise in treating B cell leukemias, but their success has been limited in myeloid leukemias. This is due in part to the lack of AML-specific target antigens. Moreover, impaired T cell function, a hallmark of AML, is also problematic when relying on autologous T cell activation upon reinfusion. We overcame the limitations of CAR T therapy in AML by developing CAR-expressing natural killer (NK) cells targeting CD123. While present on both healthy and AML cells, CD123 is expressed at >10-fold higher levels on malignant cells. Furthermore, use of NK allows use of fully functional allogeneic cells from healthy donors, since NK cells do not induce graft-versus-host-disease. To optimize the tumor cell selectivity of CAR NK cells, we employed the yeast display directed evolution platform to isolate anti-CD123 scFvs with varying affinities and are identifying and characterizing clones that maximize the interaction with CD123high AML cells over healthy CD123low cells, as determined by binding, cytotoxicity, cytokine secretion, and intracellular signaling studies. Collectively, our efforts introduce a new paradigm for engineered cell therapeutics with significant promise for AML treatment. Citation Format: Monika Kizerwetter, Huilin Yang, Ruyan Rahnama, Challice Bonifant, Jamie Spangler. Affinity tuning anti-CD123 chimeric antigen receptor natural killer cell therapy to treat acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1327.

IL-15-secreting CAR natural killer cells directed toward the pan-cancer target CD70 eliminate both cancer cells and cancer-associated fibroblasts

BackgroundIt remains challenging to obtain positive outcomes with chimeric antigen receptor (CAR)-engineered cell therapies in solid malignancies, like colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC). A major obstacle is the lack of targetable surface antigens that are not shared by healthy tissues. CD70 emerges as interesting target, due to its stringent expression pattern in healthy tissue and its apparent role in tumor progression in a considerable amount of malignancies. Moreover, CD70 is also expressed on cancer-associated fibroblasts (CAFs), another roadblock for treatment efficacy in CRC and PDAC. We explored the therapeutic potential of CD70 as target for CAR natural killer (NK) cell therapy in CRC, PDAC, focusing on tumor cells and CAFs, and lymphoma.MethodsRNA-seq data and immunohistochemical analysis of patient samples were used to explore CD70 expression in CRC and PDAC patients. In addition, CD70-targeting CAR NK cells were developed to assess cytotoxic activity against CD70+ tumor cells and CAFs, and the effect of cytokine stimulation on their efficacy was evaluated. The in vitro functionality of CD70-CAR NK cells was investigated against a panel of tumor and CAF cell lines with varying CD70 expression. Lymphoma-bearing mice were used to validate in vivo potency of CD70-CAR NK cells. Lastly, to consider patient variability, CD70-CAR NK cells were tested on patient-derived organoids containing CAFs.ResultsIn this study, we identified CD70 as a target for tumor cells and CAFs in CRC and PDAC patients. Functional evaluation of CD70-directed CAR NK cells indicated that IL-15 stimulation is essential to obtain effective elimination of CD70+ tumor cells and CAFs, and to improve tumor burden and survival of mice bearing CD70+ tumors. Mechanistically, IL-15 stimulation resulted in improved potency of CD70-CAR NK cells by upregulating CAR expression and increasing secretion of pro-inflammatory cytokines, in a mainly autocrine or intracellular manner.ConclusionsWe disclose CD70 as an attractive target both in hematological and solid tumors. IL-15 armored CAR NK cells act as potent effectors to eliminate these CD70+ cells. They can target both tumor cells and CAFs in patients with CRC and PDAC, and potentially other desmoplastic solid tumors.

An Injectable Puerarin Depot Can Potentiate Chimeric Antigen Receptor Natural Killer Cell Immunotherapy Against Targeted Solid Tumors by Reversing Tumor Immunosuppression.

Chimeric antigen receptor natural killer (CAR-NK) cell therapy represents a potent approach to suppressing tumor growth because it has simultaneously inherited the specificity of CAR and the intrinsic generality of NK cells in recognizing cancer cells. However, its therapeutic potency against solid tumors is still restricted by insufficient tumor infiltration, immunosuppressive tumor microenvironments, and many other biological barriers. Motivated by the high potency of puerarin, a traditional Chinese medicine extract, in dilating tumor blood vessels, an injectable puerarin depot based on a hydrogen peroxide-responsive hydrogel comprising poly(ethylene glycol) dimethacrylate and ferrous chloride is concisely developed. Upon intratumoral fixation, the as-prepared puerarin depot (abbreviated as puerarin@PEGel) can activate nitrogen oxide production inside endothelial cells and thus dilate tumor blood vessels to relieve tumor hypoxia and reverse tumor immunosuppression. Such treatment can thus promote tumor infiltration, survival, and effector functions of customized epidermal growth factor receptor (HER1)-targeted HER1-CAR-NK cells after intravenous administration. Consequently, such puerarin@PEGel-assisted HER1-CAR-NK cell treatment exhibits superior tumor suppression efficacy toward both HER1-overexpressing MDA-MB-468 and NCI-H23 human tumor xenografts in mice without inducing obvious side effects. This study highlights a potent strategy to activate CAR-NK cells for augmented treatment of targeted solid tumors through reprogramming tumor immunosuppression.

Manufacturing CAR-NK against tumors: Who is the ideal supplier?

Chimeric antigen receptor-natural killer (CAR-NK) cells have emerged as another prominent player in the realm of tumor immunotherapy following CAR-T cells. The unique features of CAR-NK cells make it possible to compensate for deficiencies in CAR-T therapy, such as the complexity of the manufacturing process, clinical adverse events, and solid tumor challenges. To date, CAR-NK products from different allogeneic sources have exhibited remarkable anti-tumor effects on preclinical studies and have gradually been applied in clinical practice. However, each source has advantages and disadvantages. Selecting a suitable source may help maximize CAR-NK cell efficacy and increase the feasibility of clinical transformation. Therefore, this review discusses the development and challenges of CAR-NK cells from different sources to provide a reference for future exploration.

Harnessing natural killer cell effector function against cancer.

Natural killer (NK) cells are cytotoxic innate lymphoid cells that participate in anti-tumour and anti-viral immune responses. Their ability to rapidly destroy abnormal cells and to enhance the anti-cancer function of dendritic cells, CD8+ T cells, and macrophages makes them an attractive target for immunotherapeutic strategies. The development of approaches that augment NK-cell activation against cancer is currently under intense preclinical and clinical research and strategies include chimeric antigen receptor NK cells, NK-cell engagers, cytokines, and immune checkpoint inhibitors. In this review, we highlight recent advances in NK-cell therapeutic development and discuss their potential to add to our armamentarium against cancer.

Nicotinamide-Expanded Allogeneic Natural Killer Cells with CD38 Deletion, Expressing an Enhanced CD38 Chimeric Antigen Receptor, Target Multiple Myeloma Cells.

Natural killer (NK) cells are a vital component of cancer immune surveillance. They provide a rapid and potent immune response, including direct cytotoxicity and mobilization of the immune system, without the need for antigen processing and presentation. NK cells may also be better tolerated than T cell therapy approaches and are susceptible to various gene manipulations. Therefore, NK cells have become the focus of extensive translational research. Gamida Cell's nicotinamide (NAM) platform for cultured NK cells provides an opportunity to enhance the therapeutic potential of NK cells. CD38 is an ectoenzyme ubiquitously expressed on the surface of various hematologic cells, including multiple myeloma (MM). It has been selected as a lead target for numerous monoclonal therapeutic antibodies against MM. Monoclonal antibodies target CD38, resulting in the lysis of MM plasma cells through various antibody-mediated mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis, significantly improving the outcomes of patients with relapsed or refractory MM. However, this therapeutic strategy has inherent limitations, such as the anti-CD38-induced depletion of CD38-expressing NK cells, thus hindering ADCC. We have developed genetically engineered NK cells tailored to treat MM, in which CD38 was knocked-out using CRISPR-Cas9 technology and an enhanced chimeric antigen receptor (CAR) targeting CD38 was introduced using mRNA electroporation. This combined genetic approach allows for an improved cytotoxic activity directed against CD38-expressing MM cells without self-inflicted NK-cell-mediated fratricide. Preliminary results show near-complete abolition of fratricide with a 24-fold reduction in self-lysis from 19% in mock-transfected and untreated NK cells to 0.8% of self-lysis in CD38 knock-out CAR NK cells. Furthermore, we have observed significant enhancements in CD38-mediated activity in vitro, resulting in increased lysis of MM target cell lines. CD38 knock-out CAR NK cells also demonstrated significantly higher levels of NK activation markers in co-cultures with both untreated and αCD38-treated MM cell lines. These NAM-cultured NK cells with the combined genetic approach of CD38 knockout and addition of CD38 CAR represent a promising immunotherapeutic tool to target MM.

Chimeric antigen receptor natural killer cells: a promising antitumor immunotherapy.

Chimeric antigen receptor (CAR) T cells have been successfully used in adoptive cell therapy for malignancies. However, some obstacles, including side effects such as graft-versus-host disease and cytokine release syndrome, therapy resistance, limited sources, as well as high cost, limited the application of CAR T cells. Recently, CAR natural killer (NK) cells have been pursued as the effector cells for adoptive immunotherapy for their attractive merits of strong intrinsic antitumor activity and relatively mild side effects. Additionally, CAR NK cells can be available from various sources and do not require strict human leukocyte antigen matching, which suggests them as promising "off-the-shelf" products for clinical application. Although the use of CAR NK cells is restrained by the limited proliferation and impaired efficiency within the immunosuppressive tumor microenvironment, further investigation in optimizing CAR structure and combination therapies will overcome these challenges. This review will summarize the advancement of CAR NK cells, CAR NK cell manufacture, the clinical outcomes of CAR NK therapy, the challenges in the field, and prospective solutions. Besides, we will discuss the emerging application of other immune cells for CAR engineering. Collectively, this comprehensive review will provide a valuable and informative summary of current progress and evaluate challenges and future opportunities of CAR NK cells in tumor treatment.

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.

Chimeric Antigen Receptor-Engineered NK Cells Overcome CD226 Downregulation As an Immune Escape Mechanism in Acute Myeloid Leukemia

Background: Natural killer (NK) cells are part of the innate immune system, and their killing capacity depends on the inputs from activating and inhibitory receptors. CD226 is an activating receptor that plays a role in the immune surveillance of acute myeloid leukemia (AML). AML cells have developed mechanisms to escape NK cell cytotoxicity including downregulating activating receptors such as CD226 on the surface of NK cells. Chimeric antigen receptors (CARs) are synthetic receptors integrated into NK cells to direct their anti-leukemia activity, and CD38 is a potential target for NK CAR-based cellular therapy against AML. Understanding the dynamics between the endogenous activating and inhibitory NK cell receptors (such as CD226) and the introduced CAR can determine the development of more effective NK CAR-based therapy for AML. We hypothesize that CD38-directed CAR NK cells may overcome CD226 downregulation as a mechanism of immune escape in AML. Purpose: we aimed to investigate (1) the mechanisms developed by AML cells to escape recognition by the activating receptor CD226, (2) whether and how anti-CD38 CAR-armed NK cells can overcome AML immune escape mechanisms. Methods: we performed multiparametric mass spectrometry to characterize changes in CD226 expression in NK cells of individuals with AML. Then, we investigated CD226 expression in NK cell-AML cocultures to characterize CD226-related mechanisms by imaging flow cytometry. Finally, to explore the impact of CD226 knockout (KO) together with the role of CD38 CAR in NK cell functionality, we retrovirally transduced CD38 CAR combined with CRISPR-Cas9 CD226 KO followed by short- and long-term cytotoxicity assays, cytokine production techniques and immune synapse assays by imaging flow cytometry. Results: NK cells from AML patients expressed low levels of CD226 with impaired cytotoxicity compared to heathy donor NK cells. Co-culture of healthy donor NK cells with AML blasts resulted in CD226 downregulation secondary to its internalization and was dependent on expression of the CD226 ligand CD155 in AML cells. CD226 KO diminished NK cell cytokine production and decreased their killing capacity against AML cells. In contrast, CD38-targeting NK cells displayed enhanced and specific cytotoxic activity against primary AML cells. When we combined the CD38 CAR with a CD226 KO in NK cells, these cells maintained their activating potential, cytokine secretion levels, and anti-leukemic cytotoxicity (Figure 1). CD38 CAR / CD226 KO NK cells could build strong immune synapses (Figure 2) even in the absence of LFA-1, an integrin activated by CD226 signaling and essential for NK cell immune synapse formation, suggesting that the CAR molecule compensates for the absence of CD266-mediated LFA signaling. Furthermore, CD38 CAR NK cells were capable of forming immune synapses, that were at least partially independent from LFA-1. Conclusion: CAR-directed NK cells can surpass resistance mechanisms in AML, such as CD226 downregulation and dampening of LFA-1 recruitment into the immunological synapse. Thus, NK cell activation by artificially introduced and germline-encoded receptors can overcome distinct tumor-resistance mechanisms, CAR-driven NK cells being a potent option for the treatment of AML.

CD70 CAR NK Cells in the Treatment of Multiple Myeloma

Despite the high response rates of chimeric antigen receptor (CAR) - T cells targeting the B-cell maturation antigen (BCMA) for multiple myeloma (MM), patients eventually still relapse. At this time there is no clear subsequent therapy. Furthermore, CAR T-cell therapy is associated with significant toxicities such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. CD70 is a normally transiently expressed on activated T-cells, B-cells, or dendritic cells, but have been found to be upregulated on many hematologic and solid cancer cells such as acute myeloid leukemia, triple negative breast cancer, and renal cell carcinoma. However, CD70 as a potential target for treatment of multiple myeloma, including patients who have progressed on BCMA targeted therapies, has not been fully investigated. We assessed bone marrow aspirates from MM patients by flow cytometry and demonstrated that CD70 was expressed on the majority of myeloma cells, and on average, relapsed myeloma patients had higher expression of CD70. In addition, using immunohistological staining, we found that CD70 was expressed on 10 of 10 patients who had progressed on BCMA targeted therapies (seven who had progressed on belantamab mafodotin, an antibody drug conjugate, three who had progressed on idecabtagene vicleucel, a BCMA CAR-T), suggesting that CD70 could be a viable target in patients who have failed BCMA targeted therapy. As natural killer (NK) cells are a promising off-the-shelf treatment with minimal toxicity as compared to CAR-T cells, we generated CD70-targeting CAR NK cells for the treatment of MM. We found in the chromium cytotoxicity assay that CD70 CAR NK cells had higher killing as compared to non-transduced (NT) NK cells at multiple effector-to-target ratios against MM1s, a myeloma cell line confirmed to express CD70. There was no difference in killing when co-cultured with H929, a myeloma cell line lacking CD70 expression, demonstrating the specificity of the CD70 CAR NK cell. The enhanced killing was also confirmed in an incucyte assay in which the CD70 CAR NK cells cleared tumor faster than NT NK, even in MM1s in which BCMA was knocked out using CRISPR (simulating BCMA antigen loss). When tested in a myeloma mouse model, the CD70 CAR NK exhibited greater tumor control as shown in Figure 1, when compared to NT NK, which lead to increased survival of the mice. Similar results were seen in a mouse model using BCMA KO myeloma cells. In summary, we were able to demonstrate that CD70 can be a feasible target for the treatment of multiple myeloma, including in patients who have failed BCMA targeted therapy. Based on these results, we have initiated a Phase I/II clinical trial (NCT05092451) that is currently recruiting.

Generation and Screening of Various CD70 CAR NK Cells Identify the Most Effective Construct Against Hematologic Malignancies

The ability to re-direct the intrinsic power of the immune system against cancer has resulted in unprecedented outcomes in certain patients with otherwise incurable diseases. Natural killer (NK) cells, similar to T cells, are powerful immune effectors that possess intrinsic anti-tumor properties and are capable of generating a strong cytotoxic response upon engaging tumor cells. However, unlike T cells, NK cells do not identify specific antigens and do so in a way that is HLA-independent, posing minimal risk of GvHD. Thus, NK cells have are being explored as a possible allogeneic source for over-the-counter cellular immunotherapy. CD70, the ligand for the CD27 receptor, is an attractive “pan-cancer antigen”, since in addition to being expressed in hematologic malignancies such as acute myeloid leukemia (AML) and lymphoma, it is also expressed on many solid tumors, including bladder, lung, triple negative breast cancer, renal cell carcinoma, pancreatic cancer, and melanoma. Moreover, CD70 is only transiently found on activated T and B lymphocytes and on dendritic cells. To target CD70-expressing cancers, we designed and tested a number of different chimeric antigen receptors (CAR). Briefly, we used the natural interaction between CD70 and CD27 to make a CAR construct that incorporated the extracellular domain of human CD27, which is the natural receptor for CD70. We generated various second-generation CAR constructs that differed in their transmembrane domains (CD27 vs. CD28) and costimulatory domains. We included co-stimulatory domains that are NK-centric (DAP10 and DAP12), T-cell-centric (CD28), or bi-centric (4-1BB). The CD3ζ chain was used as a signaling endodomain, and all the CAR constructs also included IL-15 to enhance in vivo proliferation and persistence. Cord blood (CB)-derived NK cells were efficiently retrovirally transduced with the different CAR constructs and subjected to an in vitro functional screen against various cancer cells to evaluate the best constructs for in vivo validation (Figure 1). The top constructs from our in vitro screen were further evaluated in multiple in vivo models, including a Raji lymphoma model and a THP-1 AML model. The best CAR construct in terms of superior tumor control and prolongation of survival was selected for GMP manufacturing. Based on these preclinical findings, a Phase I/II clinical study is now being conducted at our center to assess the safety and effectiveness of CD70 CAR NK cells against CD70-expressing hematologic malignancies (NCT05092451). Figure 1: Flow chart showing the screening process to identify the most effective CAR construct.

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.

Anti-TIM3 chimeric antigen receptor-natural killer cells from engineered induced pluripotent stem cells effectively target acute myeloid leukemia cells

BackgroundAcute myeloid leukemia (AML) is a clonal malignant disorder which originates from a small number of leukemia-initiating cells or leukemic stem cells (LSCs)—the subpopulation that is also the root cause of relapsed/refractory AML. Chimeric antigen receptor (CAR)-T cell therapy has proved successful at combating certain hematologic malignancies, but has several hurdles that limit its widespread applications. CAR-natural killer (NK) cells do not carry the risk of inducing graft-versus-host disease (GvHD) frequently associated with allogeneic T cells, thereby overcoming time-consuming, autologous cell manufacturing, and have relatively safer clinical profiles than CAR-T cells. The present study aimed to generate anti-TIM3 CAR-NK cells targeting LSCs from a clonal master induced pluripotent stem cells engineered with the third-generation anti-TIM3 CAR.MethodsA clonal master umbilical cord blood NK-derived induced pluripotent stem cell (iPSC) line, MUSIi013-A, was used as a starting cells for engineering of an anti-TIM3 CAR harboring TIM3 scFv fragment (clone TSR-022), CD28, 4-1BB, and CD3ζ signaling (CAR-TIM3). The established CAR-TIM3 iPSCs were further differentiated under serum- and feeder-free conditions into functional CAR-TIM3 NK cells and tested for its anti-tumor activity against various TIM3-positive AML cells.ResultsWe successfully established a single-cell clone of CAR-TIM3 iPSCs, as validated by genomic DNA sequencing as well as antibody and antigen-specific detection. We performed thorough iPSC characterization to confirm its retained pluripotency and differentiation capacity. The established CAR-TIM3 iPSCs can be differentiated into CAR-TIM3 NK-like cells, which were further proven to have enhanced anti-tumor activity against TIM3-positive AML cells with minimal effect on TIM3-negative cells when compared with wild-type (WT) NK-like cells from parental iPSCs.ConclusionsiPSCs engineered with CARs, including the established single-cell clone CAR-TIM3 iPSCs herein, are potential alternative cell source for generating off-the-shelf CAR-NK cells as well as other CAR-immune cells. The feasibility of differentiation of functional CAR-TIM3 NK cells under serum- and feeder-free conditions support that Good Manufacturing Practice (GMP)-compliant protocols can be further established for future clinical applications.

Application of chimeric antigen receptor-natural killer cells for the treatment of type 1 diabetes

For the past 100 years, insulin supplementation has been the mainstay of treatment for type 1 diabetes (T1D), which is characterized by progressive autoimmune-mediated loss of insulin-producing β cells in the islets of Langerhans over the last decades, technological advances in glucose monitoring and therapeutics have greatly improved the care and management of these patients. However, morbidity, mortality, and quality of life remain challenges for patients with T1D. Islet transplantation has been successfully performed, but there are several limiting factors, such as the lack of cadaveric donors and the need for lifelong immunosuppressive therapy. Therefore, there is a great medical need for alternative therapeutic approaches. In the current review, the current knowledge on novel approaches for the treatment of T1D with a focus on the potential of using chimeric antigen receptor (CAR)-T cells and natural killer (NK) cells is summarized.