CAR-NK cell therapy for hematological malignancies.

Introduction

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the management of hematologic malignancies, providing durable responses even in patients with relapsed or refractory disease. 1 , 2 , 3 However, significant challenges with CAR-T-cell therapy include the risk of life-threatening toxicities, including cytokine release syndrome (CRS) and neurotoxicity, the requirement for individualized, cumbersome cell manufacturing, and substantial costs with autologous generation, limiting accessibility and the feasibility of this therapeutic strategy. 4 , 5 Additionally, the autologous derivation of CAR-T cells from heavily pretreated patients may compromise product quality and quantity. 6 , 7 , 8 , 9 Ultimately, the complex logistics of generating autologous CAR-T cells impede timely deployment for many patients in need. 10

In contrast, natural killer (NK) cells, which are innate immune effector cells, offer specific immunotherapeutic advantages. 11 NK cells rapidly identify and kill malignant or virally infected cells without previous sensitization through a diverse array of activating and inhibitory receptors on target cells. 12 They recognize missing-self signals on major histocompatibility complex (MHC) class I–negative targets, release cytotoxic granules containing perforin and granzyme B (GZMB) to induce apoptosis, and mediate both direct cytolytic and antibody-dependent cellular cytotoxicity (ADCC). 13 Their inherent antitumor functionality, suitability for allogeneic use without risk of graft-versus-host disease (GVHD), and lower risk of cytokine storm position them as a viable platform for cell therapy. 11 CAR-NK cells combine innate cytotoxic functions of NK cells with CAR-driven specificity, addressing several limitations of CAR T-cell therapy. 14 CAR-NK cells can be generated from diverse sources, enabling “off-the-shelf” allogeneic therapies. 5 Although investigation is underway to generate allogeneic, off-the-shelf CAR-T cells with genetic modifications to reduce GVHD risk, this often disrupts the T-cell receptor, compromising CAR-T-cell quality and their intrinsic T-cell functionality. 15 , 16 , 17 , 18 This is avoided with CAR-NK cells, which do not require such genetic engineering for allogeneic implementation. Early trials, including our group’s phase 1/2 study of umbilical cord blood (CB)-derived CD19-targeting CAR-NK cells for relapsed/refractory (R/R) hematologic malignancies, have shown promising efficacy and safety with NK cell persistence for >1 year. 19 , 20 , 21

Despite their potential, CAR-NK cell therapy is challenged by limited persistence, immunosuppression by tumor cells, and metabolic constraints. 4 , 5 , 11 , 22 This review examines engineering and manufacturing strategies, addresses biological and tumor-specific barriers, and highlights emerging preclinical and clinical advances optimizing the therapeutic potential of CAR-NK cells.

CAR-NK cell manufacturing

CAR-NK cells can be generated from various sources, including CB, peripheral blood (PB), NK cell lines, and pluripotent stem cells (PSCs; Figure 1 ). Each source has distinct benefits and limitations, influencing the efficacy and phenotype of derived CAR-NK cells ( Table 1 ). Expansion and cryopreservation considerations are key for scalable CAR-NK cell therapeutic application. Finally, the design and delivery of CAR constructs for NK cells involve tailoring intracellular signaling motifs, vector elements, or selecting gene delivery approaches, which all affect efficiency, safety, and therapeutic durability. Figure 1. Derivation and application of CAR-NK cell therapy. CAR-NK cells can be produced from multiple sources, such as PBMCs and CB from which NK cells are isolated, PSCs differentiated into NK cells, or the NK-92 cell line established from a patients with NHL and requiring irradiation before clinical application. Introduction of the CAR transgene into NK cells can be achieved through retroviral or lentiviral transduction, mRNA electroporation, or transposon systems, with additional genetic modifications, such as CRISPR/Cas9-mediated editing, possible during this process. After expansion, CAR-NK cells are cryopreserved to create good manufacturing practice–compliant, off-the-shelf cell therapy banks for clinical use. PBMCs, PB mononuclear cells; QC, quality control. Figure created with biorender.com . Manzar GS. (2026) https://BioRender.com/ao35jjm . Table 1. Comparison of cell origins that can be utilized to derive CAR-NK cells PBMCs CB NK-92 cells PSCs Availability Readily available, autologous or allogeneic donors Banked units widely available, 1 unit can treat multiple patients Established master cell banks; unlimited supply Potentially unlimited supply Variability High donor-to-donor variability Moderate donor variability mitigated with CB unit selection Minimal variability Low variability with standardized cell lines Risk of tumorigenicity Very low Very low Requires irradiation to prevent malignant proliferation Theoretical risk from reprogramming/genetic instability and undifferentiated cells Expandability Low NK frequency; requires robust ex vivo expansion High proliferative capacity supporting large-scale expansion Rapid and efficient clinical expansion to clinical scale High expansion potential Maturity or phenotype Mature NK phenotype Relatively immature but highly proliferative with good persistence Activated phenotype, potent cytotoxicity Often immature phenotype, low CD16 and KIR expression Transduction efficiency Generally low, requires optimization Moderate to high genetic engineering efficiency High genetic engineering efficiency Highly amenable to precise genetic engineering In vivo persistence Variable, may be limited by exhaustion and host rejection Favorable persistence seen clinically Limited persistence due to mandatory irradiation Limited persistence seen clinically, under investigation Off-the-shelf feasibility Limited, autologous manufacturing frequent Strong Strong Strong Clinical experience Early-to-intermediate clinical evaluation; fewer trials compared with CB-based products Most advanced clinical experience with demonstrated safety and efficacy in phase 1 trials Multiple phase 1 trials showing safety and transient efficacy with limited persistence Early clinical translation; limited but emerging clinical experience Notable issues Self-MHC inhibition (autologous); donor variability; expansion challenges Donor unit selection critical Requires repeated dosing; irradiation limits persistence Time- and labor-intensive derivation; regulatory and safety considerations PBMCs, CB, the NK-92 cell line, and PSCs are all potential sources for CAR-NK cell therapy. Each has unique advantages and challenges, which are highlighted in the table. KIR, killer cell immunoglobulin-like receptors; PBMCs, PB mononuclear cells.

NK cell sources

PB is a readily accessible source of mature NK cells with a short differentiation period. Autologous PB-derived NK cells carry minimal immune rejection risk but may have reduced efficacy because of self-MHC–based inhibition or suboptimal functionality due to previous treatments or health issues. 23 Allogeneic PB-derived NK cells are thus preferred and can be isolated from healthy donors to treat multiple patients but face the issue of donor-dependent variability. PB-derived NK cells require expansion due to the naturally low NK cell frequency, 24 and are challenged by lower transduction efficiency and exhaustion from prolonged activation. 25 Cytokine cocktails, feeder cells, and metabolic supplements are being optimized to enhance PB NK cell expansion and function. 26 , 27 , 28

CB provides NK cells with high proliferative capacity, supporting large-scale expansion for off-the-shelf CAR-NK therapies to treat many patients from 1 cord. 29 Current expansion methods enable the production of therapeutic-grade CAR-NK cells from CB at clinical scales, yielding robust antitumor function and durable persistence. 30 , 31 We reported the first-in-human phase 1/2 trial using CD19-CAR-NK cells derived from CB to treat R/R hematological malignancies, demonstrating strong clinical potency and an excellent safety profile, highlighting CB as a source for effective CAR-NK cell therapies. 20 As with allogeneic PB, donor variability affects CB-derived NK cells. Correlative analysis of our clinical trial identified that CB units with ≤8 × 10 7 nucleated red blood cells and a collection-to-cryopreservation time of ≤24 hour represented the strongest predictors of superior outcomes and CAR-NK cell functionality. 20 These observations emphasize the critical role of donor selection in allogeneic cell therapy development.

Derived in 1992 from the PB of a 50-year-old man with non-Hodgkin lymphoma (NHL), NK-92 is an NK cell line notable for ease of expansion to therapeutic quantities with minimal variability and can be readily engineered to express CARs. However, NK-92 cells must be irradiated before infusion to prevent uncontrolled in vivo proliferation and mitigate tumorigenic risk. Irradiated NK-92 cells were safe and showed encouraging short-term antitumor activity in clinical trials, but their limited persistence in patients may reduce long-term efficacy and necessitates multiple infusions for sustained benefit. 32 , 33 , 34

Human embryonic stem cells or induced PSCs (iPSCs) can produce large batches of CAR-NK cells with consistent properties, offering potentially unlimited expansion potential for off-the-shelf therapeutic application and reducing donor variability. Limitations include cumbersome, time-intensive, and inefficient derivation of NK cells with potential immature phenotypes evidenced by low killer cell immunoglobulin-like receptors and CD16 expression. 35 The reprogramming process also introduces the risk of genetic aberrations with tumorigenic potential. 36 Nevertheless, protocol improvements continue to reveal substantial potential for stem cell–derived CAR-NK cell therapies that have translated into clinical application. 37 , 38

NK cell expansion and cryopreservation

Ex vivo expansion is essential for therapeutic application of NK cells due to their low frequency and the large quantities needed for clinical use. Various expansion strategies are used, such as cytokine supplementation; coculture with feeder cells such as modified K562 expressing membrane-bound cytokines; and feeder-free, particle-based methods. 31 These techniques support robust NK cell proliferation but require several weeks to achieve clinically relevant cell numbers. Additional challenges include optimizing gene transduction or gene editing efficiency, while sustaining proliferative capacity and NK cell viability. 39 , 40 , 41 The K562 cell line, derived from a patient with chronic myelogenous leukemia in blast crisis, is the most commonly used feeder for NK cell expansion. Because K562 cells retain tumorigenic properties, irradiation is required before coculture, but incomplete removal can result in residual K562 cells in the final NK cell product. This necessitates stringent US Food and Drug Administration regulations to prevent feeder cell contamination in clinical-grade NK cell preparations. 42 Alternative expansion platforms, such as artificial antigen-presenting cells or feeder-free, particle-based techniques, are under investigation to enhance safety and regulatory compliance for clinical use. 39

Cryopreservation is crucial for large-scale, off-the-shelf clinical application of NK cell therapies. However, unlike T cells, which generally maintain cytotoxic function after freeze-thaw cycles, NK cells experience markedly reduced viability, recovery, and functionality after thawing, impairing their expansion, persistence, and cytotoxicity in vivo. 43 , 44 Several biological variables underlie these disparities. NK cells possess greater granularity than T cells due to abundant preformed cytotoxic granules. 45 They also express innate, germ line–encoded activating receptors that detect stress ligands (eg, MHC class I chain-related protein A/B and UL16-binding proteins) on damaged cells. 46 These features make NK cells more susceptible to apoptosis after cryopreservation, likely from cryoinjury-induced GZMB leakage or fratricide triggered by stress-induced NK cell receptor activation. 47 Overcoming cryopreservation-associated challenges are essential for effective deployment of off-the-shelf NK cell therapies.

CAR construction and expression

CARs enable immune cells to recognize and target cancer cells in an antigen-specific manner. Structurally, CARs consist of an ectodomain, which is often a single-chain variable fragment conferring antigen specificity, a hinge region providing flexibility and stability, a transmembrane domain to link extracellular and intracellular components, and intracellular signaling plus costimulatory domains that activate immune responses. 48 , 49 , 50 Once a CAR extracellular domain binds to the target antigen, the intracellular signaling domain spurs signaling cascades that stimulate NK cell cytotoxicity. The choice of domain sources affects stability, function, and persistence, and includes CD8α, CD28, or immunoglobulin for hinge and transmembrane elements, or CD3ζ, 4-1BB, or CD28 for intracellular signaling components. 50 , 51 The vector backbone that delivers CAR transgenes to NK cells contains promoters that regulate CAR expression and influence activity. Polyadenylation signals on the vector stabilize CAR mRNA and support efficient translation of the CAR protein. 52 , 53

To exploit innate NK cell signaling, CAR-NK designs may incorporate NK-specific motifs, such as DNAX-activating protein 10 (DAP10), DNAX-activating protein 12 (DAP12), 2B4/CD244, or DNAX-acessory molecule 1 (DNAM1), alongside traditional CD3ζ-based signaling, aiming to exploit NK-specific natural costimulatory mechanisms and maximize activation. 54 , 55 , 56 Studies comparing different endodomains indicate that CD28-based costimulation with CD3ζ enhances CAR-NK cytotoxicity and persistence in hematologic and solid tumor models, as opposed to NK-specific DAP10 and DAP12, or bicentric 4-1BB endodomains. 57 However, optimal costimulatory domains can vary by disease contexts and therapeutic indications.

Strategies for gene delivery in CAR-NK cell engineering

Various delivery methods can be used in CAR-NK cell production, including viral or nonviral approaches. Retroviruses integrate efficiently into dividing NK cells and have shown success in phase 1/2 clinical trials, achieving 68% progression-free survival and a favorable safety profile. 19 , 20 Of note, RD114-pseudotyped retroviruses outperform gamma retroviruses and lentiviruses in NK cell transduction. 58 , 59 Lentiviruses, which transduce both dividing and resting NK cells, often have lower transduction efficiency, limiting widespread application. 52 When pseudotyped with the baboon envelope, lentiviruses have improved transduction efficiency and promising preclinical efficacy. 60 , 61

The Sleeping Beauty transposon system involves a “cut-and-paste” approach to integrate CAR transgenes into NK cell genomes. However, because these integrations are semirandom, transposase-based methods may carry a risk of oncogenic transformation. Indeed, piggyBac transposase-engineered CAR-T cells led to malignancy in treated patients with cancer. 62 Newer methods in CAR-NK cell generation combined minicircle DNA and a Sleeping Beauty transposase system, demonstrating preferential integration into safer genomic harbors, with robust antitumor activity. 63

Finally, mRNA electroporation uses electrical pulses to transiently permeabilize NK cell membranes, introducing short-lived CAR-encoding mRNA that does not integrate in host genomes. This nonviral method enables temporary CAR expression, reducing long-term toxicity, insertional mutagenesis risk, off-tumor side effects, and regulatory burden. 64 It is especially useful for early-phase studies or when safety of a new CAR target is uncertain.

Advantages of CAR-NK cell–based therapy

CAR-NK cells present notable advantages over CAR-T cells, particularly as allogeneic products, due to their favorable safety and lower manufacturing costs, described hereafter and in Figure 2 . Figure 2. Summary of the major strengths and drawbacks of CAR-T cell therapy and CAR-NK cell therapy. Favorable features or benefits are highlighted in green checkmarks, and limitations or challenges are shaded in red crosses. KIR, killer cell immunoglobulin-like receptors; LDC, lymphodepleting chemotherapy; PBMCs, PB mononuclear cells; TCR, T-cell receptor; TME, tumor microenvironment. Figure created with biorender.com . Manzar GS. (2026) https://BioRender.com/nd03tji .

Scalability, allogeneic use, and cost reduction

There are active efforts looking to derive allogeneic, “off-the-shelf” CAR-T cells with deletion of the native T-cell receptor (T-cell receptor alpha constant, or TRAC, knockout) to reduce the risk of GVHD, but this further reduces CAR-T-cell quantity and quality, with compromised innate functionality due to disruption of the T-cell receptor. 15 , 16 , 17 , 18 Unlike CAR-T cells, which carry the risk of GVHD even with HLA matching, 65 NK cells do not induce GVHD, enabling safe allogeneic use even in HLA-mismatched settings. 66 If suitably cryopreserved, CAR-NK cells become a rapid, scalable, and cost-effective off-the-shelf therapy. This eliminates lengthy patient-specific manufacturing from potentially heavily pretreated patients, reduces costs, and accelerates treatment initiation in the setting of rapidly progressing disease. 67 Of note, as discussed above, cryopreservation adversely affects CAR-NK cells both in quality and quantity, which is a challenge in allogeneic, scalable, off-the-shelf use that merits continued investigation for improvement.

Favorable safety profile

CAR-T-cell therapy can cause severe toxicities such as CRS and immune effector cell–associated neurotoxicity syndrome (ICANS), leading to prolonged hospitalization and high costs. The massive release of proinflammatory “cytokine storm” by activated effector cells results in systemic inflammation that drive high fever, hypotension, hypoxia, and multiorgan dysfunction. Severe CRS (grade ≥3) occurs in up to 30% of patients receiving CAR-T-cell therapy, with rates varying by CAR design and disease setting. 68 , 69 , 70 , 71 ICANS manifests in up to 50% of patients after CAR-T-cell therapy. 68 , 70 Recent findings also describe atypical neurotoxicities, such as parkinsonism and movement disorders, in patients with multiple myeloma after CAR-T-cell therapy, implicating possible off-tumor effects on the basal ganglia or alternative neuroinflammatory pathways beyond classical ICANS. 72 , 73 In contrast, CAR-NK cell therapy in early trials have not shown grade ≥3 CRS or neurotoxicity, with most side effects limited to manageable cytopenias, grade ≤2 CRS, or mild infusion reactions. 19 , 20 , 34 , 38 The favorable side effect profile of CAR-NK cells may be due to their lower persistence and cytokine release but, for now, still underscores the superior safety profile of CAR-NK cells.

Dual innate and CAR-mediated antitumor function

NK cells possess innate germ line–encoded activating receptors such as NKG2D, NKp46, NKp30, NKp44, NKG2C, DNAM-1, and CD16, allowing them to kill malignant cells by both natural cytotoxicity and ADCC. CAR-NK cells combine engineered and innate mechanisms for cytotoxicity, without a need for previous sensitization, potentially limiting antigen escape in heterogenous tumors. 74

Challenges with CAR-NK cell therapy

Despite showing promise against hematologic malignancies, 19 , 38 CAR-NK cells face several important limitations. Opportunities for progress include boosting expansion, enhancing persistence, and improving resistance to exhaustion. 75

Limited persistence

Unmodified NK cells have a natural life span of ∼2 weeks and generally fail to persist or expand in vivo unless sustained by endogenous cytokines. 75 Genetic engineering to induce cytokine armoring such as with interleukin-15 (IL-15) 75 or IL-21 76 in NK cells improves persistence and long-term function and has been successful in clinical trials. 19 Still, tumor-derived immunosuppressive factors can accelerate NK apoptosis, and chronic activation by tumor exposure can drive exhaustion and activation-induced cell death.

Metabolic and nutrient constraints

Tumors rapidly consume glucose, amino acids, and lipids, depriving NK cells of critical nutrients needed for their metabolism, survival, and cytotoxicity. 77 Immunosuppressive byproducts prostaglandin E2 and lactic acid disrupt key NK signaling pathways, including in distant metastases from patients. 78 , 79 In tissue biopsies containing human colorectal cancer metastasis–resident NK cells, lactate induces apoptosis through acidification and mitochondrial dysfunction. 80 Overcoming these constraints is a focus of research aiming to enhance NK cell fitness and resilience.

Immunosuppressive cells and biochemical factors

Regulatory T cells, myeloid-derived suppressor cells (MDSCs), and M2 tumor-associated macrophages secrete immunosuppressive cytokines including prostaglandin E2, transforming growth factor β (TGF-β), and IL-10, which inhibit NK function. 81 , 82 , 83 , 84 TGF-β blocks mammalian target of rapamycin (mTOR) signaling, and dampens NK cell glycolysis and oxidative phosphorylation, inducing NK cell metabolic dysfunction in metastatic breast cancer and acute myeloid leukemia (AML). 85 , 86 , 87 , 88 Both TGF-β and IL-10 reduce expression of key activation receptors such as CD16 and NKG2D. 89 Our group described how AML blasts exhaust NK cells in a cell-cell contact–dependent manner through the TGF-β/SMAD/basic leucine zipper ATF-like transcription factor axis, driving epigenetic scaring of NK cells. 88 MDSCs inhibit NK cell function by downregulating NKG2D, and CD3ζ, a key subunit of the NK-activating receptors NKp46, NKp30, and CD16. 90 , 91 Regulatory T cells, M2 tumor-associated macrophages, and MDSCs also impair NK cell degranulation and IFN-γ secretion in a contact-dependent manner. 81 , 82 , 84

NK cell memory

Unlike T cells, the memory state in NK cells is poorly defined, particularly in the context of cancer. Although NK cell memory subsets have been described during viral infections or toxic exposures, cancer-provoked memory is limited and often manifests as transient hyperactivation rather than long-lived antigen-specific persistence capable of surveillance and clonal expansion that occurs in T cells. 92 Protocols using cytokines such as IL-12, IL-15, and IL-18 can create memory-like NK cells and improve long-term cytotoxicity, but their efficacy varies by tumor type, and recurrence remains a problem. 92 , 93

Challenges in target selection

Selecting tumor-specific antigens is a major challenge because evolving tumors can mutate, downregulate, or lose antigens, causing immune escape and heterogeneity. CAR-NK cells, with both direct CAR-targeted killing and intrinsic cytotoxicity, may partially overcome this, but antigen escape remains a hurdle.

CAR-NK cells also conversely risk attacking normal tissues expressing the target antigen, termed “on-target, off-tumor” effects. Tumor-specific antigens that are expressed only on cancer cells are preferred, but tumor-associated antigens are common and may be expressed on normal cells at lower levels. Even with tumor-specific antigens, fratricide can occur via trogocytosis, which is when NK cells acquire antigen from target cells, leading to self-recognition and NK cell death. 94 Our group described the prevention of trogocytosis-mediated fratricide by engineering NK cells with a logic-gated dual-CAR system, in which 1 CAR activates upon binding a tumor-specific antigen, and a second CAR delivers an inhibitory signal upon recognizing an NK self-antigen. 94

Preclinical approaches to improve CAR-NK cell function

Modern preclinical research on CAR-NK cells focuses on enhancing persistence, potency, and safety by reprogramming cell metabolism, engineering cytokine support, suppressing inhibitory signals, exploring combinatorial strategies, creating multispecific or tandem CARs, and incorporating safety switches.

Targeting metabolic checkpoints

NK cell metabolism is pivotal for their antitumor activity. mTOR complex 1 (mTORC1) is a crucial metabolic regulator, necessary for producing effector molecules such as IFN-γ and GZMB, and supporting NK cell maturation. 95 , 96 Amino acid transporters such as SLC1A5 and SLC7A5 activate metabolic regulators such as mTORC1 and c-Myc, which drive NK cell growth and function. 97 CISH encodes cytokine-inducible Src homology 2, a protein that inhibits IL-15 and IL-2 signaling in NK cells. 67 , 98 , 99 Our group demonstrated that CRISPR–associated protein 9–mediated deletion of CISH removes cytokine signaling brakes, boosts protein kinase B/mTORC1 and c-MYC signaling, enhances metabolic fitness, and increases CAR-NK cytotoxicity, especially in IL-15–secreting CAR-NK cells. 100 Recently, we identified that the transcription factor cyclic AMP response element modulator restricts key effector genes in NK cells and serves as a transcriptional checkpoint in NK cell stimulation. 101 Deleting the cyclic AMP response element modulator improves chromatin accessibility, proliferation, glycolytic capacity, and antitumor activity. Additionally, the transcription factor sterol regulatory element-binding protein supports cytokine-induced metabolic reprogramming to enhance NK function. 102 Thus, metabolic bottlenecks can be targeted to enhance the potency and resilience of CAR-NK cells.

Boosting persistence and expansion with embedded cytokine support

A key challenge is CAR-NK cell persistence in vivo, because NK cells are naturally short-lived and require cytokines for survival after infusion. Initially, systemic infusions of exogenous cytokines, such as IL-2 and IL-15, were used to enhance NK cell expansion and survival, but adverse effects such as capillary leak syndrome have limited this strategy. 22 , 103 An approach that has shown feasibility in the clinic is implementing CAR constructs with embedded proinflammatory cytokine support to improve their persistence and antitumor function. IL-15 armoring produces NK cells with notable efficacy and prolonged persistence of up to a year in responder recipients, even in HLA-mismatched settings. 19 , 75 IL-15 armored CAR-NK cell function is further enhanced with deletion of the checkpoint CISH , as described earlier. 100 CAR-NK cells engineered with expression of IL-12 show improved metabolic fitness and antitumor activity. 104 We also reported that NK cells armored with IL-21 demonstrate superior persistence and durable in vivo efficacy against human glioblastoma xenografts in mice. 76

Memory-like NK cells can be induced by exposure to IL-12, IL-15, and IL-18, yielding NK cells that persist longer and have greater antitumor potency. 92 , 105 Synergy between the memory-like phenotype and CARs has been demonstrated, with CD19-CAR memory-like NK cells exhibiting superior survival, proliferation, and cytotoxicity in vivo against NK-resistant lymphoma in NSG murine models, compared with unmodified memory-like NK cells. 93

Gene editing approaches to boost potency and curb inhibition

Tumors may use suppressive elements, such as TGF-β, which curtails cytokine secretion and NK metabolic fitness by inhibiting mTOR pathways, leading to reduced efficacy. 67 , 86 , 87 Knockout of TGFBR2 in CAR-NK cells, prevents TGF-β1–mediated NK cell dysfunction in B acute lymphoblastic leukemia and glioblastoma models. 106 , 107 Tumors also upregulate ligands for inhibitory NK cell receptors, such as NKG2A, T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT), and T-cell immunoglobulin and mucin-domain containing-3 (TIM3), curbing NK cell activation. Disrupting these inhibitory signals by knockdown or pharmacologic blockade prevents NK cell dysfunction. 108 , 109 , 110 , 111 , 112 NKG2A knockout enhanced the potency of anti-CD33 CAR-NK cells.

Combination therapies

CAR-NK cells can be combined with checkpoint inhibitors, chemotherapy, and targeted therapies to improve efficacy.

Checkpoint inhibition

Antibodies that block NK inhibitory receptors can synergize with CAR-NK treatment by preventing dysfunction and boosting antitumor immunity in both NK cells and T cells. 113 , 114 Pharmacological checkpoint inhibitors allow greater flexibility than genetic alterations, permitting titratable and temporally controlled immune enhancement.

Chemotherapy

Besides their own cytotoxicity, standard chemotherapies can sensitize tumor cells by upregulating activating ligands and increasing susceptibility to CAR-NK cell killing. 115 Cisplatin did not impair the viability and function of anti-CD133 CAR-NK-92 cells but increased their antitumor potency. 116

Targeted therapy

Preclinical research in lung and colon cancer demonstrated that agents such as kinase inhibitors (eg, regorafenib), proteasome inhibitors, and stimulator of interferon genes agonists (eg, rocaglamide) can synergize with CAR-NK therapy by increasing NK cell infiltration, activation, and tumor control. 117 , 118

Multispecific and tandem CARs to target multiple tumor antigens

A hurdle is the identification of tumor antigens with high specificity and stable expression, because heterogeneity or antigen loss enable immune escape and off-target effects introduce toxicity. Dual or multispecific CARs, designed to target >1 antigen simultaneously, address antigen escape and can help minimize off-target effects. For example, CAR-NK cells expressing both anti-CD19 CAR and high-affinity CD16–mediated ADCC when combined with monoclonal antibodies, preferentially targeting tumor cells, and surmounting antigen loss and tumor heterogeneity in B-cell malignancies, mediating superior preclinical outcomes. 74 Clinically, the ability to leverage ADCC to boost adoptive NK cell therapy was recently validated in our phase 1 trial, in which CB-derived NK cells were administered alongside a bispecific innate cell engager targeting CD16 on NK cells and CD30 on tumor cells. The combination yielded a 92.9% overall response rate (ORR) and a 66.7% complete response (CR) rate in patients with R/R CD30 + lymphoma. 119

Given the risk for on-target, off-tumor toxicity, CAR constructs are now being designed with suicide switches such as inducible caspase 9 used by our group to allow selective elimination of CAR-NK cells with AP1903 (rimiducid) in cases of severe toxicity. 30

Clinical experience of CAR-NK cells in hematological malignancies

Worldwide, the number of CAR-NK cell clinical trials is growing. A search on clinicaltrials.gov for “CAR-NK cells” identifies 123 trials, most of which are phase 1 or early phase 1, with select phase 1/2 trials. Fifteen trials focus on autoimmune diseases, 1 targets COVID-19, and 38 trials focus on solid tumors, but most anticancer CAR-NK trials address hematological malignancies, with 69 total. Four of these have completed accrual, of which 3 have published outcomes on CD19-targeted CAR-NK cells, cited in various earlier sections. 19 , 21 , 38

Our initial experience reported outcomes in 11 heavily pretreated patients with heterogenous R/R hematological malignancies treated with CB-derived IL-15 armored CD19-CAR-NK cells. We demonstrated 7 CRs and 1 partial response within 30 days, with persistence of CAR-NK cells for up to 1 year, even with HLA mismatch. 19 Our follow-up study in 37 heavily pretreated patients revealed an ORR of 48.6% at both day 30 and day 100, with CR rates of 27% (day 30) and 29.7% (day 100), and a 1-year CR rate of 37.8%. 20 The 1-year CR rate was highest in low-grade NHL at 83% and lower for chronic lymphocytic leukemia (CLL) with or without Richter transformation (20% and 50%, respectively) and diffuse large B-cell lymphoma (DLBCL; 29%). Responses were generally rapid and durable, with most early CRs persisting at 6 months. Patients achieving day-30 CR had a 70% chance of maintaining CR at 1 year. Correlative analysis showed that CB units with ≤8 × 10 7 nucleated red blood cells and a collection-to-cryopreservation interval of ≤24 hours strongly associated with favorable outcomes, underscoring the importance of donor selection. 20

Another clinical trial of FT596, an off-the-shelf iPSC-derived CD19-CAR-NK cell product engineered with IL-15, given without (n = 18) or with (n = 68) rituximab showed similarly high response rates and safety but lower persistence of the NK cells. 38 Among 86 heavily pretreated patients, the CR rate and ORR were 85% and 100%, respectively, for follicular lymphoma, and 25% and 38%, respectively, for LBCLs. Patients with de novo DLBCL had a low CR rate of 5%, but exclusion of these patients from the LBCL group improved the CR rate and ORR to 64% and 82%, respectively. Of note, 20 patients in this trial received previous CAR-T-cell therapy, and the ORR was 45%, with 30% CR among these patients. Persistence of CAR-NK cells was limited to 15 days, but response once achieved was durable. Of note, cryopreservation of CAR-NK cells was implemented in this trial, unlike our trial, which we deployed before optimizing patented cryopreservation techniques.

A third phase 1 trial from China was recently published in which 8 patients with R/R LBCL received a 3-dose regimen of freshly generated CB-derived CD19-CAR-NK cells equipped with 4-1BB and CD3ζ signaling endodomains. 21 The ORR was 62.5%, with a CR rate of 50%. Three patients had durable CR past 1 year, and 1 patient had durable CR beyond 2 years. CAR-NK cell persistence correlated with response, with 1 patient with CR showing CAR copies detectable by quantitative polymerase chain reaction at 15 months after infusion, and another patient with partial response harboring CAR copies at 3 months. The lack of CAR copies 2 weeks after infusion correlated with the absence of response. In addition, CAR-NK cells of patients with progressive disease showed elevated TGF-β signaling in relation to increased CBLB-mediated ubiquitination, reduced cytotoxicity, and lower antigen presentation activity.

Emerging work that is only published in abstract form includes SENTI-202-101, a PB-derived CAR-NK cell therapy with bivalent CD33 OR FLT3 NOT EMCN logic-gated system to target CD33 + or Flt3 + AML cells, while protecting normal hematopoietic progenitor cells expressing endomucin to circumvent on-target off-tumor toxicity. 120 Applied to 6 patients with AML, the authors reported no dose-limiting toxicities and transient cytopenias attributed to lymphodepleting chemotherapy, with 3 patients achieving measurable residual disease–negative CRs, and 1 patient showing complete remission with partial hematologic recovery. 120 Announced only in abstract form as well, FT522 is an iPSC-derived CD19-CAR-NK cell product genetically edited to express (1) IL-15/IL-15 receptor fusion for persistence, (2) high-affinity noncleavable CD16 Fc receptor to enhance ADCC, (3) CD38 deletion to augment mitochondrial respiratory capacity and resilience against oxidative stress, and (4) a novel alloimmune defense receptor to target 4-1BB–expressing host cells that may harm CAR-NK cells, enabling NK persistence in the allogeneic host immune system. 121 Tested in 3 patients with R/R B-cell malignancies, CRs were seen in 2 patients with low-grade follicular and marginal zone lymphomas; the third patient with DLBCL had progressive disease.

Thus, CAR-NK cell therapy holds tremendous promise against hematological malignancies, with highest efficacy seen in low-grade NHL. As the widening portfolio of trials increases, further insights into CAR-NK cell therapy are anticipated. Most of the other currently accruing trials originate in China and prioritize safety as the main end point, with secondary measures including efficacy, quality of life, and immune markers such as NK cell persistence and cytokine levels. Some studies, including with FT596, reported outpatient administration, underscoring the favorable safety profile. 38

Side effects of CAR-NK cell therapy

Thus far, clinical trials of CAR-NK cells in hematological malignancies have reported manageable side effects, such as transient cytopenias, mild infusion reactions, or grade ≤2 CRS. 19 , 21 , 34 , 38 There have been no documented cases of grade ≥3 CRS, ICANS, tumor lysis syndrome, or GVHD, complications commonly seen with CAR-T cells. Safety was the primary outcome of our trial. Lymphodepleting chemotherapy caused reversible hematological toxicity, and only 1 patient developed grade 1 CRS (2.7%). The FT596 trial described grade 1 CRS in 6% of patients receiving FT596 alone, or grade ≤2 CRS in 13% of patients receiving FT596 with rituximab. In all 3 trials, no patient developed neurotoxicity or GVHD.

In comparison, among CAR-T-cell recipients, 57% to 93% experienced grade ≥1 CRS, and 13% to 32% grade ≥3 CRS. 122 Thus, both the frequency and severity of CRS are lower in CAR-NK cell therapy. Neurotoxicity such as ICANS occurs in up to 50% of patients after CAR-T-cell therapy. 68 , 70 The favorable side effect profile of NK-based therapies is attributed to their lower persistence and cytokine release.

To further enhance safety, some CAR-NK cell products include inducible suicide genes to enable their rapid, controlled elimination if necessary. 30 Although CAR-NK cell therapy demonstrates favorable safety, ongoing patient monitoring and systematic evaluation in clinical trials remain essential.

Conclusions and future directions

CAR-NK cell therapy delivers promising safety and early efficacy in hematologic malignancies. Although still in its infancy, the future of CAR-NK cell therapy is focused on unlocking the full potential of scalable, accessible, and effective immunotherapies for solid tumors and autoimmune diseases through advancements in construct engineering and manufacturing strategies. Optimizing cryopreservation methods and logistics will ensure rapid availability, viability, and functionality of NK cells after thawing, minimizing vein-to-vein time for patients across global settings. Although manufacturing costs and affordability with cell therapies remain a major concern, in our practice, the high scalability of CB-derived NK cells, optimal expansion and cryopreservation methods, high-fidelity gene editing approaches, and streamlined enterprise-scale good manufacturing practice processes have contributed to an affordable product with which broad accessibility is feasible and financial toxicity is orders of magnitude lower than for CAR-T cells. Concomitantly, rapid progress in precise gene editing technologies, including CRISPR screening approaches, enables the generation of next-generation CAR-NK cells with enhanced cytotoxicity, metabolic resilience, and resistance to immunosuppressive tumor microenvironments. 109 The integration of CAR-NK cell therapy with other anticancer modalities is conceivable in clinical settings, and is being intensively explored in preclinical settings and some early phase clinical trials. Additionally, strategies involving multispecific, tandem, or logic gated CAR constructs capable of integrating dual-antigen sensing, combinatorial signaling, or Boolean “AND/OR” logic gating, may circumvent tumor heterogeneity and minimize off-tumor effects through precise cellular activation. Ultimately, future clinical success of CAR-NK cell therapies depends on improving persistence and potency broadly across cancers, combining scalable manufacturing, robust cryopreservation, advanced gene editing, rational combination therapies, and innovative CAR designs, together catalyzing a new era of safer, more effective, and universally accessible cellular immunotherapy.

Conflict-of-interest disclosure: M.D. and The University of Texas MD Anderson Cancer Center have an institutional financial conflict of interest with Takeda Pharmaceutical for the licensing of the chimeric antigen receptor natural killer cell technology. M.D. participates on the scientific advisory board of Cellsbin, Aurigene, and Bruker Cellular Analysis. The remaining authors declare no competing financial interests.

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Acknowledgments

The authors are grateful to the contributions of all patients and families who have participated in studies advancing the field of chimeric antigen receptor natural killer (CAR-NK) cell therapy. They thank their research collaborators and clinical partners for their dedicated efforts and technical expertise; and they also appreciate the generous support from funding agencies and philanthropic organizations, which has enabled numerous preclinical and clinical investigations in CAR-NK cell immunotherapy. Finally, they are grateful to the institutions, staff, and study coordinators that have facilitated the conduct of clinical trials and fostered an environment of innovation and excellence in cellular therapy research.

The development of this manuscript did not specifically require any funding. G.S.M. cites funding from the 10.13039/100006293 American Society of Clinical Oncology Conquer Cancer Young Investigator Award, and is supported, in part, by the K12 Calabresi Scholars Program at MD Anderson Cancer Center (5K12CA088084). M.D. is supported by an RO1 grant (National Cancer Institute R01-CA280827-01), a Leukemia Specialized Program of Research Excellence (SPORE) grant (P50CA100632), and by grants from the 10.13039/100011619 Society for Immunotherapy of Cancer , Andrew Sabin Family, and from the SPORE developmental research program in leukemia.

Authorship

Contribution: M.D. and G.S.M. supervised the creation of the review; G.S.M. rendered tables and figures; G.S.M., M.F.H., M.M., S.T., R.B., and M.D. wrote the manuscript; and all authors edited the manuscript and agree with the contents enclosed.