Induced pluripotent stem cell-derived chimeric antigen receptor natural killer cells: Engineering innovations, translational hurdles and clinical prospects in immune therapy

Table 1 Comparison of chimeric antigen receptor T lymphocytes and chimeric antigen receptor natural killer cells

Characteristic	CAR-T cells	CAR-NK cells
Source	Usually derived from patients’ autologous T cells, but they may also be derived from T cells from healthy donors	Can be derived from peripheral blood, cord blood, iPSCs, the NK-92 cell line, bone marrow, placenta, and tumor-infiltrating NK cells
Cell type	T cells	NK cells
Mechanism of action	CAR-mediated antigen recognition activates T-cell cytotoxic effector function, thereby killing target cells	CAR-NK cells mediate CAR-directed killing while retaining the endogenous cytotoxic activity of NK cells; when CD16 is expressed, they can also mediate ADCC
Target specificity	Can be engineered to target various tumor-associated surface antigens	Can be engineered to target various tumor-associated surface antigens and may supplement antigen recognition through endogenous NK-cell receptors
Product manufacturing and application model	Autologous CAR-T cells usually require individualized manufacturing; allogeneic CAR-T cells require additional immune-safety engineering	Theoretically more amenable to standardized allogeneic manufacturing and “off-the-shelf” production, and may reduce variability caused by interindividual differences
Safety	May be associated with CRS, ICANS, GvHD, and other treatment-related toxicities	Existing studies suggest that the risks of CRS and neurotoxicity may be lower; however, systematic clinical monitoring and long-term safety validation are still required
Cost	The manufacturing process is complex, and individualized production usually results in relatively high costs	More compatible with scalable manufacturing models and may theoretically reduce costs in allogeneic application settings; however, this still requires validation in terms of GMP manufacturing data, quality-control costs, and real-world evidence
Immune escape and in vivo limitations	Susceptible to antigen loss, T-cell exhaustion, and the immunosuppressive tumor microenvironment	Endogenous NK-cell recognition and ADCC may partially complement CAR-mediated recognition; these cells do not carry the risk of αβ TCR-mediated GvHD, but may still face host immune clearance, insufficient in vivo persistence, limited tumor infiltration, and suppression by the tumor microenvironment

CAR: Chimeric antigen receptor; NK: Natural killer; iPSCs: Induced pluripotent stem cells; ADCC: Antibody-dependent cellular cytotoxicity; CRS: Cytokine release syndrome; ICANS: Immune effector cell-associated neurotoxicity syndrome; GvHD: Graft-vs-host disease; GMP: Good Manufacturing Practice; TCR: T cell receptor.

Table 2 Comparison of induced pluripotent stem cell-derived chimeric antigen receptor natural killer cells with major competing off-the-shelf cellular immunotherapy platforms

Platform	Main source/product form	Key features/potential advantages	Major limitations	Key differences compared with iPSC-derived CAR-NK cells	Maturity of clinical translation
Peripheral blood-derived CAR-NK cells	Peripheral blood NK cells from healthy donors or patients, followed by ex vivo expansion and CAR engineering	Relatively strong natural cytotoxic activity; mature cell source; relatively preserved primary NK-cell effector functions; an existing clinical research basis	Limited frequency of NK cells in the starting material; expansion efficiency and functional status are affected by donor variability; donor-to-donor differences may lead to insufficient batch-to-batch consistency; large-scale standardized manufacturing remains challenging	Cell function is closer to that of primary NK cells, but standardization, scalability, and batch-to-batch consistency are generally weaker than those of the iPSC-derived platform	Early clinical exploration has been reported
Cord blood-derived CAR-NK cells	Cord blood NK cells or cord blood-derived NK precursor cells, followed by expansion and CAR engineering	Relatively low immunogenicity; suitable for allogeneic application; relatively standardized sample source; potential for off-the-shelf development	Limited cell numbers from a single cord blood unit; ex vivo expansion and maturation still require optimization; inter-sample variability may affect manufacturing consistency; limited in vivo persistence	Existing clinical development basis, but difficult to achieve long-term, stable, large-scale production comparable to that enabled by a single-clone-derived iPSC	Early clinical exploration
NK-92 cell line-derived CAR-NK cells	NK-92 cell line engineered with a CAR	Easy to expand; relatively stable manufacturing workflow; suitable for early process development and mechanistic validation	Cell line origin raises safety concerns; pre-infusion irradiation is usually required, limiting in vivo proliferation and persistence; intrinsic lack of CD16 results in insufficient ADCC capacity	Standardized expansion is relatively straightforward, but in vivo persistence and clinical applicability are limited by pre-infusion irradiation and cell line-related properties	Preclinical research and early clinical exploration
iPSC-derived CAR-NK cells	Single-clone-derived iPSCs subjected to CAR introduction and multiplex engineering, followed by differentiation into NK cells	Master cell banks can be established; theoretically favorable for scalable and standardized manufacturing with improved batch-to-batch consistency; amenable to multiplex gene editing; suitable for modular engineering	Complex differentiation workflow; high manufacturing cost and quality-control requirements; genetic stability, residual undifferentiated cells, and risks associated with multiplex editing must be controlled; long-term in vivo persistence and real-world cost advantages still require validation	Compared with primary NK and NK-92 platforms, this platform has development potential in platform-based manufacturing and multiplex engineering, but its comparative clinical advantages still require further validation	Early-stage clinical validation
Allogeneic CAR-T cells	Healthy donor-derived T cells engineered with a CAR, often requiring TCR- and HLA-related gene editing	Strong antigen-specific cytotoxic capacity of T cells; relatively mature CAR-T development experience; potential for off-the-shelf development	Need to address TCR-mediated GvHD, host anti-graft responses, immune rejection, and gene-editing safety; CRS/ICANS may still occur; multiplex editing increases regulatory and quality-control complexity	Compared with iPSC-derived CAR-NK cells, CAR-T cell therapy has more extensive clinical development experience, but allogeneic T cell-related immune safety concerns and editing requirements are greater	Multiple early clinical studies are ongoing
γδ T-cell and CAR-γδ T-cell therapies	Peripheral blood- or tissue-derived γδ T cells, followed by ex vivo expansion or CAR engineering	MHC-unrestricted recognition; both innate-like and adaptive immune features; theoretically lower risk of GvHD; ability to recognize stress-associated antigens	Marked subset heterogeneity of γδ T cells; expansion stability and engineering efficiency require optimization; functional differences among subsets are substantial; consistency of clinical efficacy still requires validation	Compared with iPSC-derived CAR-NK cells, γδ T cells have T cell-like cytotoxicity and tissue homing- or tissue residency-related features, but product standardization and batch-to-batch consistency remain challenging	Preclinical to early clinical exploration

The “key features/potential advantages” listed in this table include theoretical platform advantages and technical features reported in early studies; they should not be equated with clinical superiority demonstrated by prospective randomized clinical trials. Some platforms should be understood as off-the-shelf or potentially off-the-shelf cellular immunotherapy strategies. The information in this table was synthesized from studies and reviews related to peripheral blood- and cord blood-derived chimeric antigen receptor (CAR)-natural killer (NK) cells, NK-92 cell line-derived CAR-NK cells, induced pluripotent stem cell (iPSC)-derived CAR-NK cells, allogeneic CAR-T cells, and γδ T-cell and CAR-γδ T-cell therapies[8-10,18,22-24,40,42,43,45,47,48,50-52]. The potential advantages of iPSC-derived CAR-NK cells in scalable manufacturing, batch-to-batch consistency, and cost control still require further validation through Good Manufacturing Practice manufacturing data, clinical studies, and real-world evidence. iPSCs: Induced pluripotent stem cells; CAR: Chimeric antigen receptor; NK: Natural killer; ADCC: Antibody-dependent cellular cytotoxicity; CRS: Cytokine release syndrome; ICANS: Immune effector cell-associated neurotoxicity syndrome; TCR: T cell receptor; HLA: Human leukocyte antigen; GvHD: Graft-vs-host disease.

Table 3 Evidence levels and translational maturity of induced pluripotent stem cell-derived chimeric antigen receptor natural killer cells across different disease contexts

Disease/application context	Representative product or strategy	Ref.	Current primary evidence type	Conclusions supported by current evidence	Major limitations	Translational maturity
Relapsed/refractory B-cell lymphoma	FT596; FT516 may serve as a reference for a non-CAR-engineered iPSC-derived NK-cell platform	[23,92]	Early phase I clinical studies	The FT596 study suggests early safety and preliminary antitumor activity signals of iPSC-derived CAR-NK cells in B-cell lymphoma. FT516 may serve as a clinical safety and feasibility reference for a non-CAR-engineered iPSC-NK platform	The sample size and follow-up duration are limited. Randomized controlled studies and direct comparisons with CAR-T cells, bispecific antibodies, or antibody-based combination therapies are lacking. FT516 is not a CAR-engineered product and therefore cannot serve as direct evidence for the efficacy of iPSC-CAR-NK cells	Early clinical stage
Multiple myeloma	FT576; multi-module iPSC-derived NK/CAR-NK strategies involving BCMA-CAR, hnCD16, IL-15-related support modules, CD38-related engineering, and other modifications	[50,91,114]	Early clinical data, interim disclosures, and related mechanistic studies	FT576-related data suggest that BCMA-targeted, multi-module engineered iPSC-derived NK/CAR-NK strategies have a degree of translational potential. Antigen-escape studies may help explain insufficient durability of response and relapse risk	Full peer-reviewed clinical publications and long-term follow-up remain limited. The duration of response, patterns of treatment failure, antigen escape involving BCMA, GPRC5D, and other targets, and comparative effectiveness against existing BCMA-targeted therapies remain unclear	Preliminary clinical evidence stage
AML	FT538; anti-TIM3 iPSC-CAR-NK cells; NKG2C-KE; a registered clinical study of CLL1/CD33-targeted iPSC-derived NK cells	[93-95]; ClinicalTrials.gov: NCT06367673	Preclinical studies, early clinical recruitment, or preliminary translational data	Current evidence supports the feasibility of target identification, engineering design, and functional validation for iPSC-derived NK/CAR-NK strategies in AML	Most evidence remains preclinical. AML targets are often shared with normal hematopoietic cells, creating a risk of on-target, off-tumor toxicity. In vivo safety, the therapeutic dose window, and the GMP scale-up pathway still require validation	Preclinical to early clinical exploratory stage
Solid tumors	CD276-, MSLN-, and GPC3-targeted iPSC-derived CAR-NK strategies, as well as iPSC-derived NK/CAR-NK strategies incorporating CCL19, CCR2B, IL-15, NKG2D, or other functional-enhancement modules	[21,35,98-100,106,107]	In vitro experiments, patient-derived organoid studies, animal models, and preclinical mechanistic studies	Current evidence suggests the preclinical feasibility, antigen-dependent killing, and some in vivo antitumor activity signals of iPSC-derived CAR-NK cells in solid tumors	Immunodeficient animal models have limited clinical predictive value. Stromal barriers, abnormal vasculature, immunosuppressive networks, metabolic stress, and antigen heterogeneity in human solid tumors have not been sufficiently modeled. Current evidence cannot be directly extrapolated to clinical efficacy	Mainly preclinical stage
Single-case compassionate-use exploration in systemic sclerosis; autoimmune-disease applications have not yet been established	CD19/BCMA dual-targeting iPSC-derived CAR-NK cells	[22,103]	Single compassionate-use case report and related commentary articles	The original case study suggests that B-cell depletion, clinical improvement, and manageable early safety signals were observed in this individual patient. This result should be regarded only as an early feasibility signal in the context of single-patient compassionate use	This is a single, uncontrolled case and cannot demonstrate reproducibility of efficacy. It should not be equated with disease-level proof of concept in systemic sclerosis, nor does it support broad extrapolation to other autoimmune diseases. Long-term safety, B-cell reconstitution, infection risk, host immune responses, and the respective contributions of CD19 and BCMA dual targeting still require validation in larger cohorts with longer follow-up	Single-case early translational signal

CAR: Chimeric antigen receptor; iPSCs: Induced pluripotent stem cells; NK: Natural killer; BCMA: B-cell maturation antigen; GPRC5D: G protein-coupled receptor class C group 5 member D; AML: Acute myeloid leukemia; GMP: Good Manufacturing Practice; IL: Interleukin; NKG2D: Natural killer group 2 member D.

Table 4 Bottleneck layers, engineering and mitigation strategies, and key validation metrics in the clinical translation of induced pluripotent stem cell-derived chimeric antigen receptor natural killer cells

Bottleneck layer	Core bottlenecks/key translational constraints	Representative engineering/mitigation strategies	Key validation metrics	Key considerations for translational interpretation
Product definition layer	Insufficient differentiation consistency, batch-to-batch variability, risk of genetic instability, and risk of residual undifferentiated iPSCs	Clonal screening, establishment of master cell banks, standardized differentiation workflows, control of residual iPSCs, and optimization of cryopreservation and thawing processes	NK-cell purity, CAR positivity rate, cell viability, differentiation yield, post-thaw function, karyotype, CNV, genomic stability, residual pluripotency markers, and sterility, mycoplasma, and endotoxin testing	Determines whether the product can be manufactured consistently, assessed for quality, and compared across batches
Effector execution layer	Insufficient target-cell recognition, inadequate cytotoxic function, target-antigen heterogeneity or antigen downregulation, and insufficient serial killing capacity	NK-adapted CAR structural optimization, optimization of the antigen-recognition domain and affinity, hnCD16-mediated ADCC enhancement, multi-target design, logic gating, and cytokine support	Antigen-dependent killing, cytotoxicity at different E:T ratios, CD107a degranulation, granzyme B and perforin expression, cytokine release, serial killing capacity, antigen-negative escape, and killing of non-target cells	Enhanced in vitro effector function alone cannot be equated with in vivo efficacy or clinical benefit
Tissue delivery layer, including TME adaptation	Insufficient homing to solid tumors, extravasation, and infiltration into the tumor parenchyma; local TME-mediated suppression; hypoxia/metabolic stress; and remodeling of the antigenic landscape	Chemokine receptor engineering, adaptation to stromal barriers, TGF-β resistance, adenosine/A2A-axis adaptation, hypoxia-resistant or metabolically adaptive designs, and local TME-responsive modules	Chemotactic migration ability, tumor-tissue infiltration ratio, tumor-to-peripheral-blood cell ratio, spatial distribution, local persistence duration, markers related to TGF-β, adenosine, and hypoxia, and in vivo tumor-growth inhibitory activity	Requires determination of whether the main limitation arises from tissue delivery, local suppressive pathways, or antigen escape
Host interaction layer	Allogeneic immune-mediated clearance, insufficient in vivo persistence, accelerated clearance after repeat dosing, immunogenicity, and risk of “missing-self” recognition	HLA-related engineering, B2M/CIITA editing, HLA-E/HLA-G expression, evasion of host NK-cell-mediated clearance, and in vivo persistence-enhancing modules	In vivo expansion and persistence, cellular exposure levels in peripheral blood and tissues, host T/NK-cell clearance responses, anti-product immune responses, changes in cellular exposure after repeat dosing, and long-term safety	Determines the effective exposure duration and feasibility of repeat dosing; immune-evasion benefits need to be balanced against safety risks
Translational implementation layer	Safety risks associated with multiplex engineering, inadequate potency assays, non-uniform release criteria, insufficient comparability after process changes, and uncertainties in regulatory pathways and costs	Potency assay systems, release criteria, comparability studies, safety switches, long-term safety monitoring, manufacturing cost control, and optimization of cold-chain and supply-chain logistics	Batch production success rate, release pass rate, potency consistency, vector copy number, gene-editing off-target risk, replication-competent virus detection, manufacturing cycle, cost per dose, and long-term AE/SAE monitoring	Determines whether an engineering strategy can become a cell product that is amenable to regulatory evaluation, manufacturable, and clinically deployable

iPSCs: Induced pluripotent stem cells; NK: Natural killer; CAR: Chimeric antigen receptor; CNV: Copy number variation; ADCC: Antibody-dependent cellular cytotoxicity; TME: Tumor microenvironment; TGF-β: Transforming growth factor-β; B2M: Beta-2 microglobulin; HLA: Human leukocyte antigen; AE: Adverse event; SAE: Serious adverse event.