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

Abstract

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.

Introduction

Despite the remarkable effectiveness of chimeric antigen receptor-T (CAR-T) cell therapy in human malignancies, it is associated with a plethora of challenges (1). In addition to optimizing CAR-T cell efficacy, the alternative role of natural killer (NK) cells has garnered significant interest. The distinct characteristics of chimeric antigen receptor-natural killer (CAR-NK) cells highlight their superior potential for anti-tumor efficacy compared to CAR-T cells (2). In addition to CAR-mediated cytotoxicity, NK cells can lyse cancer cells with downregulated or deficient target antigens via NK-specific cytotoxicity, indicating that CAR-NK cells are excellent candidates for eliminating heterogeneous solid tumors (3). CAR-NK therapy is also believed to be safer than CAR-T cell therapy, given the short-term survival of CAR-NK cells in vivo and the distinctive cytokine spectrum compared to CAR-T cells (4). “Multiple sources” is another significant advantage of CAR-NK therapy. In addition to autologous peripheral blood-derived NK cells, multiple allogeneic NK cells further enhance the possibility of fabricating off-the-shelf CAR-NK products to help reduce patient waiting times and treatment costs (5). However, each source has its drawbacks. Therefore, in this review, different CAR-NK cell sources are discussed to assist in making an informed choice when applying CAR-NK cells.

Comparison of CAR-NK cells derived from different sources

Peripheral blood (PB), cord blood (CB), NK cell lines, and iPSC are the main sources of NK cells for CAR modification (5). NK cells from four promising cell types possess their advantages but also exhibit different problems upon application (Figure 1).

Figure 1.

Different sources for manufacturing CAR-NK cells. (A) PB-derived CAR-NK cells; (B) CB-derived CAR-NK cells; (C) NK cell line (NK-92)-derived CAR-NK cells; (D) iPSC-derived CAR-NK cells. A sufficient number of CAR-NK cells derived from the four allogeneic sources can be delivered to cancer through systemic or regional administration (e.g., pleural, peritoneal, and direct intra-tumoral injection). CAR-NK, chimeric antigen receptor-natural killer; PB, peripheral blood; PBMC, peripheral blood mononuclear cells; CB, cord blood; CBMC, cord blood mononuclear cells; aAPC, artificial antigen-presenting cells. The figure is created with BioRender.com.

PB-derived CAR-NK cells

PB-NK cells

Human PB mononuclear cells (PBMC) are conventionally used as NK cell sources in the early adoptive immunotherapy (6,7). PB-NK cells have two subsets (8): CD56brightCD16−/dim PB-NK cells that are the mostly immature and CD56dimCD16+ that are more mature. CD56brightCD16−/dim PB-NK has longer telomeres (9) and will gradually acquire CD56dim phenotypes (obtaining CD16 and KIRs and losing NKG2A) as it matures (10). PB-NK cells used for immunotherapy can be divided into autologous and allogeneic sources. Autologous NK cells usually exhibit more limited anti-tumor effects than allogeneic NK cells (11), since autologous KIR/HLA matching between NK and tumor cells and immunosuppressive conditions are critical contributors to NK cell inactivation and dysfunction (12). In contrast, mismatched KIR/HLA between allogeneic NK and tumor cells excludes interference of inhibitory signals and ensures sufficient NK activity (13). However, it should be noted that T cells in allogeneic peripheral blood must be removed before NK cell application to avoid graft versus host disease (GVHD) (14). As a popular choice for gene modification, universal accessibility, low cost, and high safety are the main advantages of PB-NK. The overexpression of killer cell-activating receptors in PB-NK, such as CD16, NKP44, and NKP46, also plays an essential role in enhancing anti-tumor activity (15).

Advances in research on PB-CAR-NK cells

PB-CAR-NK cells have demonstrated significant efficacy in treating both hematological and solid malignancies. The 2G CAR structure is the most popular. Kruschinski et al. constructed human epidermal growth factor receptor 2 (HER2)-specific CAR-NK cells with CD28-CD3ζ structure. This could override inhibitory signals in primary NK cells and exhibit high-level cytokine release and degranulation to eradicate tumor cells in HER2-positive carcinoma models (16). Similarly, Imai et al. demonstrated that anti-CD19 CAR-NK cells with 4-1BB-CD3ζ structure were capable of surmounting NK resistance and producing incremental amounts of INF-γ and GM-CSF to promote the killing of leukemic cells (17). Recently, additional NK cell-specific CAR structures have been devised to optimize CAR-NK cell functions. For example, a unique CAR-NK cell line with NKG2D-DAP10-CD3ζ structure designed to exploit NKG2D-MICA/B interactions has exhibited remarkable cytotoxicity in acute lymphoblastic leukemia (ALL), osteosarcoma, prostate carcinoma, and rhabdomyosarcoma models (18).

To date, three clinical trials have been conducted to examine PB-CAR-NK cells. Regarding hematological malignancies, one was conducted to explore the efficacy of haploidentical PB-derived anti-CD19-CAR-NK (4-1BB-CD3ζ) in B-lineage ALL patients (NCT00995137), and another was carried out to evaluate the anti-tumor effect of a similar anti-CD19-CAR-NK in patients with refractory ALL (NCT01974479). For solid tumors, a phase I clinical trial is being planned by Guangzhou Medical University in China to explore both the efficacy and safety of autologous/allogeneic broad-spectrum NKG2D-ligand-targeting CAR-NK in multiple metastatic solid tumors (NCT03415100). Considering the exceptional outcomes observed in the corresponding pre-clinical studies, there is anticipation for noteworthy results from these clinical trials (Table 1).

Table 1. Clinical trials and products of CAR-NK cell therapy.

Sources of NK cells	Trial identifier	Product name	Tumor type	Target	Stage	Status
CAR-NK, chimeric antigen receptor-natural killer; PB, peripheral blood; CB, cord blood; NHL, non-Hodgkin lymphoma; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; MDS, myelodysplastic neoplasms; MM, multiple myeloma; BCL, B-cell lymphoma; CLL, chronic lymphocytic leukemia; HER2, human epidermal growth factor receptor 2. *, In this trail, CAR structure refered to novel chimeric costimulatory converting receptor, comprising mainly extracellular domain of PD1, transmembrane and cytoplasmic domains of NKG2D, and cytoplasmic domain of 41BB.
PB-NK	NCT05645601	JD010	B-cell malignancies	CD19	Phase I	Recruiting
	NCT05410717	−	Ovarian cancer, testis cancer and other endometrial cancers	Claudin6	Phase I/IIa
	NCT05487651	KUR-502	B-cell NHL or leukemia	CD19	Phase I	Recruiting
	NCT03692637	−	Epithelial ovarian cancer	Mesothelin	Early phase I	Unknown status
	NCT00995137	−	B-lineage ALL	CD19	Phase I	Completed
	NCT01974479	−	B-lineage ALL	CD19	Phase I	Suspended
	NCT03415100	−	Metastatic solid tumors	NKG2D ligands	Phase I	Unknown status
	NCT05020678	NKX019	B-cell malignancies	CD19	Phase I	Recruiting
	NCT04623944	NKX101	AML or MDS	NKG2D ligands	Phase I	Recruiting
CB-NK	NCT05247957	−	AML	NKG2D ligands	Phase I	Terminated
	NCT05472558	−	B-cell NHL	CD19	Phase I	Recruiting
	NCT04796675	−	B-cell malignancies	CD19	Phase I	Recruiting
	NCT05922930	−	Ovarian cancer, mesonephric-like adenocarcinoma, and pancreatic cancer	TROP2	Phase I/II	Not yet recruiting
	NCT05110742	−	T-cell malignances	CD5	Phase I/II	Not yet recruiting
	NCT05008536	−	MM	BCMA	Early phase I	Recruiting
	NCT03056339	−	B-cell malignancies	CD19	Phase I/II	Completed
	NCT05092451	−	Leukemia, lymphoma, or multiple myeloma	CD70	Phase I/II	Recruiting
	NCT05667155	−	B-cell NHL	CD19, CD70	Phase I	Recruiting
	NCT05020015	−	B-cell NHL or indolent NHL	CD19	Phase II	Recruiting
	NCT05703854	−	Renal cell carcinoma, mesothelioma and osteosarcoma	CD70	Phase I/II	Recruiting
	NCT05842707	−	B-cell NHL	CD19, CD70	Phase I/II	Recruiting
	NCT05008575	−	AML	CD33	Phase I	Recruiting
NK-92	NCT02944162	−	AML	CD33	Phase I/II	Unknown status
	NCT03940833	−	MM	BCMA	Phase I/II	Unknown status
	NCT02892695	PCAR-119	Leukemia, lymphoma	CD19	Phase I/II	Unknown status
	NCT02742727	−	Leukemia, lymphoma	CD7	Phase I/II	Unknown status
	NCT02839954	−	Hepatocellular carcinoma, non-small cell lung cancer, pancreatic carcinoma, triple-negative invasive breast carcinoma, malignant glioma of brain, colorectal carcinoma, gastric carcinoma	MUC1	Phase I/II	Unknown status
	NCT03383978	−	Glioblastoma	HER2	Phase I	Recruiting
	NCT03656705	−	Non-small cell lung cancer	−*	Phase I	Completed
iPSC-NK	NCT05182073	FT576	MM	BCMA	Phase I	Recruiting
	NCT04245722	FT596	BCL, CLL	CD19	Phase I	Active, not recruiting
	NCT04555811	FT596	NHL	CD19	Phase I	Active, not recruiting
	NCT05395052	FT536	Non-small cell lung cancer, colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, head and neck cancer, gastroesophageal cancer	MICA/ MICBα3	Phase I	Active, not recruiting
	NCT05336409	CNTY-101	B-cell malignancies	CD19	Phase I	Recruiting

CB-derived CAR-NK cells

CB-NK cells

CB-NK cells are NK cells that differentiate from hematopoietic stem cells in umbilical CB. CB is an off-the-shelf source of allogeneic NK cells for several reasons. First, the convenient collection and cryopreservation of CB makes it easy to store (19). Second, compared to other sources of grafts, CB has fewer T cells, thereby reducing the risk of GVHD (20). Moreover, CB provides a more stable and higher number of NK cells than PB (21). CB also comprises more NK cell progenitors (22).

Advances in research on CB-CAR-NK

Examination of CB-NK cells has entered clinical trials. A phase I/II clinical trial (NCT03056339) was carried out to test the efficacy and safety of anti-CD19 CAR-CB-NK cells in 11 patients with relapsed or refractory CD19-positive cancers. CB-NK cells were designed to co-express anti-CD19, interleukin (IL)-15, and inducible caspase-9 (iCasp9). IL-15 is a crucial cytokine involved in the proliferation and persistence of NK cells. Suicide genes are safety switches that prevent toxicity. A total of eight (73%) patients presented a response, of which seven had complete remission (CR), and one had remission with respect to Richter’s transformation with persistent CLL. The most striking finding of this study was the complete irrelevance of cytokine release syndrome (CRS)/neurotoxicity/GVHD and CAR-NK therapy (23).

Other phase I clinical studies using CB-CAR-NK cells with a similar CAR structure (anti-CD19) in patients with lymphoma and leukemia have also been launched and are currently underway. Furthermore, the effects and safety of CB-CAR-NK cells in solid cancers are also being studied (NCT05703854 and NCT05922930) (Table 1).

NK cell line-derived CAR-NK cells

NK cell lines

In addition to PB/CB-derived NK cells, there are numerous mature NK cell lines, such as NK-92, NKL, HANK1, IMC-1, SNK-6, and SNT-8, all of which can be cultured on a large scale in vitro to provide sufficient cells for application. Among them, NK-92 is the only NK cell line approved for clinical trials and is an important supplier for the manufacture of CAR-NK (24).

The NK-92 cell line has unique molecular characteristics, such as the expression of CD2+, CD56+, CD3−, and CD16− (25). NK-92 cells are functionally characterized by their high activation and dependence on IL-2 for their cytotoxic killing capabilities. These cells have an exceptional ability to recognize tumor cells and exert potent cytotoxic effects (26). NK-92 cells are equipped with activating receptors and sufficient cytolytic molecules (27,28). NK-92 cells express low levels of inhibitory receptors such as NKG2A to avoid cancer cell-mediated inhibition (25). Antibody-dependent cellular cytotoxicity (ADCC) is another pathway used by NK cells to kill cancer cells. Despite the low expression of CD16 reported in NK-92 cells, NantKwest successfully engineered an NK cell product with a high affinity for CD16 (29). This modification aimed to address this limitation and enhance the cytotoxicity of NK-92 cells. Clinical trials registered under the identifiers NCT03027128 and NCT03853317 are currently being conducted to investigate the effectiveness of this approach. For clinical use, the immortalized NK-92 cell line can be easily cryopreserved in vitro and expanded to large numbers via good manufacturing practices (GMP) (30). According to GMP standards, Tam et al. successfully developed a highly efficient protocol that allowed the proliferation of NK-92 cells more than 200-fold within a 2−2.5-week period. This protocol not only meets clinical immunotherapeutic requirements but also ensures the quality and safety of proliferated cells (31).

Advances in research on CAR-NK-92 therapy

CAR-NK-92 therapy has achieved outstanding results for hematological malignancies and solid tumors. Oelsner et al. constructed a CAR-NK-92 cell line that targeted FMS-like tyrosine kinase 3 (FLT3), which is overexpressed in B-ALL, and confirmed its notable anti-tumor cytotoxicity in SEM B-ALL xenograft models (32). Romanski et al. found that engineered NK-92 cells with an anti-CD19 CAR structure could recover their significant cytotoxicity against CD19-expressing B-precursor leukemia cell lines and lymphocytes from patients with leukemia (33). A phase I/II clinical trial (NCT02944162) reported the efficacy and safety of CAR-NK-92 cells targeting CD33 in three patients with relapsed and refractory acute myeloid leukemia (AML) (34). Clinical trials NCT02892695 exploring the efficacy of 3G CD19-CAR-NK-92 cells and NCT02742727 exploring the safety of CD7-CAR-NK-92 cells in lymphoma and leukemia are still underway.

As for solid tumors, CAR-NK-92 also presented outstanding efficacy. To overcome ovarian cancer, one of the most lethal gynecological cancers, Ao et al. designed anti-folate receptor α (αFR) CAR-NK-92 cells and confirmed their effective killing effect on ovarian cancer cells in vitro and ovarian cancer mouse models (35). Klapdor et al. constructed a novel dual-CAR structure in NK-92 cells (anti-CD24 and anti-mesothelin) that showed high cytotoxicity in both ovarian cancer cell lines and primary ovarian cancer cells (36). Han et al. found that intracranial administration of anti-EGFR CAR-NK-92 cells inhibited tumor growth and prolonged tumor-bearing mouse survival in glioblastoma mouse models (37). Esser et al. showed that CAR-NK-92 cells targeting GD2 showed enhanced cytotoxicity in neuroblastoma pre-clinical models (38). In addition, anti-MUC1 CAR-NK-92 cells were also utilized to treat relapsed or refractory solid tumors (NCT02839954).

In addition to single CAR modifications, promising combination strategies have been used to boost CAR-NK efficacy in solid tumors. Zhang et al. showed the synergistic effects of regorafenib and CAR-NK-92 cells targeting epithelial cell adhesion molecules (EpCAM) in colorectal cancer xenograft mouse models (39). A phase I clinical trial (NCT03383978) targeting glioblastoma with anti-HER2 CAR-NK-92 and another phase I clinical trial (NCT03656705) designed for non-small cell lung cancer were initiated to further explore the efficacy and safety of CAR-NK-92 cells in the treatment of solid clinical tumors (Table 1).

iPSC-derived CAR-NK cells

iPSC-derived NK cells

The development of iPSCs was first initiated by Takahashi using Oct3/4, Sox2, c-Myc, and Klf4 (40). Subsequently, human iPSCs were successfully generated and used as renewable cell sources (41). Similar to embryonic stem cells (ESCs), iPSCs exhibit indefinite proliferation and pluripotency. Successful induction of iPSCs allows for the convenient acquisition of pluripotent stem cells without embryos. Blood cells derived from iPSCs have characteristics similar to those derived from ESCs (42).

A robust system for the production of clinical grade iPSC has been developed (43). For iPSC-CAR-NK products, CAR structural modifications were executed in the undifferentiated state of iPSCs. Subsequently, modified iPSCs are differentiated in a feeder-dependent or feeder-free manner in two steps: First, iPSCs are stimulated by stem cell factor (SCF), vascular endothelial growth factor, and bone morphogenic protein 4 to differentiate into hematopoietic stem cells. Subsequently, CD34+ hematopoietic stem cells are selected to further differentiate into NK cells under stimulation by IL-3, IL-7, IL-15, SCF, and FLT3L (44). Finally, differentiated iPSC-CAR-NK cells were co-cultured with artificial antigen-presenting cells (aAPC and K562 cells expressing mbIL15/mbIL21 + 41BBL) for clinical-scale expansion (45). Zeng et al. established a practical strategy to expand the number of NK cells without KIRs, thereby broadening the range of eligible patients (46). iPSC-derived NK cells display many of the same characteristics as primary NK cells, including the presence of specific markers, such as CD56, NKG2D, and CD16 (47). Recent evidence suggests that iPSC-derived NK cells have comparable or more potent anti-tumor effects than PB/CB NK cells (48). Moreover, iPSC-derived NK cells are more suitable for expressing CAR structures than primary NK cells (44).

Advances in research on iPSC-CAR-NK cells

Pre-clinical and clinical studies of iPSC-derived NK cells have been conducted in recent years (49). A high-affinity, non-cleavable variant of CD16a was introduced and engineered in human iPSCs, resulting in the creation of potent high-affinity noncleavable variant of CD16a (hnCD16)-iPSC-NK cells with improved ADCC properties (50). Furthermore, iPSC-NK cells armed with anti-EpCAM CAR have been developed that show potent cytotoxicity against EpCAM-positive cancer cells that are resistant to NK cell attack (51).

Clinical studies have investigated the effectiveness of iPSC-CAR NK cells against various malignancies. The ELiPSE-1 study (NCT05336409) is currently recruiting participants to evaluate the safety, pharmacokinetics, and preliminary efficacy of CNTY-101 in CD19-positive B cell malignancies. Furthermore, clinical trials using iPSC-CAR-NK cells such as FT576 and FT596 targeting BCMA (NCT05182073) and CD19 (NCT04245722 and NCT04555811), respectively, are underway for examining their therapeutic potential for hematological malignancies. In particular, FT536 is specifically designed to target MICA/MICBα3, which are NKG2D ligands for the treatment of solid cancers (NCT05395052). Due to the novelty of iPSC-CAR NK cells, more clinical experiments are required to explore their safety and efficacy (Table 1).

Challenges and potential solutions for CAR-NK cells derived from different sources

In vitro expansion and cryopreservation of PB-NK cells

Due to the low frequency of NK cells in PB, in vitro expansion is necessary to obtain clinical-scale quantities (52). Feeder-dependent and feeder-free approaches are the two main expansion approaches.

Co-stimulatory signaling is essential for NK cell expansion. Feeder cells can provide different co-stimulatory signals to NK cells via cell-to-cell contact, allowing clinical-scale expansion of NK cells (53). K562, an erythroleukemia cell line lacking HLA antigen expression, was initially found to be co-stimulatory. Irradiated K562 cells have been widely used to enhance NK cell expansion (54). K562, which was modified to express specific co-stimulatory molecules, exhibited better NK-expansion efficacy in clinical trials. For example, K562-mb15-4-1BBL is capable of increasing NK expansion and upregulating NK activation markers such as CD69, CD25, NKG2D, NKp30, NKp44, and NKp46. No T cell expansion, genetic alterations, or potential oncogenic effects has been observed (17,55). MbIL-21 is an excellent substitute for mbIL-15. K562 cells expressing mbIL-21 showed enhanced expansion of NK cells (>47,000-folds in 21 d), and these proliferated cells always exhibited increased activation and purity without senescence, represented by telomere shortening (56). The irradiated EBV-transformed lymphoblastoid cell line has also been used to obtain clinical-scale NK cells, and these expanded NK cells exhibit superior activation and cytotoxicity compared to naïve NK cells (57,58).

To further strengthen the safety of feeder-dependent expansion systems, many non-tumor or non-genetically modified feeder cells have been explored. Two phase I clinical trials examining adoptive NK therapy used RetroNectin-stimulated T cells (RN-T) to expand autologous NK cells, and both obtained sufficient NK cells with high purity, functionality (high expression of NKG2D and CD16), and safety (59,60). Irradiated autologous PBMCs are alternate choices (61), and their combination with anti-CD16 monoclonal antibodies was confirmed to be an effective approach to expand highly purified cytotoxic NK cells with potent anti-tumor function both in vitro and in vivo (61,62). Compared to feeder cells, feeder-free approaches may facilitate easier licensing as a clinical-grade expansion platform, regardless of the infusion of viable feeder cells. Based on the existing application of different non-cell-based activating supplements (e.g., cytokines) for clinical-grade NK cell expansion, optimization of the combination, adjusting sequencing, and duration of exposure time will be more extensively examined in the future (63,64).

PB-NK cells exhibit reduced cytotoxicity after cryopreservation and long-distance transportation; however, this can be partially overcome by the expansion process, which largely limits the therapeutic effect of adoptive NK therapy (65). Therefore, the exploration of effective cryoprotective agents (CPAs) is a promising strategy. Dimethyl sulfoxide (DMSO), a widely used cryopreservation reagent, exhibits broad toxicity. It is capable of altering the expression of NK cell markers and impairing NK in vivo functions (66). In view of this, Yao et al. designed a biocompatible chitosan-based nanoparticle, which can mediate efficient DMSO-free NK-cell cryopreservation via intracellular delivery of non-DMSO CPAs. More importantly, NK cells cryopreserved in this manner maintain their strong cytotoxicity, degranulation, and cytokine production functions (67).

Simultaneous maturation and expansion of CB-NK cells

The use of CB-NK cells presents two significant challenges for investigators: the limited number of NK cells in a CB unit and their naïve phenotype. Although the percentage of NK cells was similar or even higher in CB than in PB, the total number of NK cells was limited by the small volume of a single CB unit (68). In addition, CB-NK cells are less mature than their PB counterparts (69). CB-NK cells exhibit a different phenotype characterized by decreased expression of molecules such as CD16 and granzyme B and increased expression of NKG2A and CXCR3 (68,70). The immature phenotype observed in umbilical CB-NK cells is indicative of reduced cytotoxicity and increased homing propensity. Consequently, CB-NK cells generally demonstrate lower anti-cancer activity than PB-NK cells. Therefore, any attempt to expand CB-NK cells must be accompanied by an effort to enhance their activation.

The simultaneous resolution of insufficient in vitro expansion and immature killing capacity may be achieved via the use of expansion techniques, which commonly exploit the benefits of interleukins, particularly IL-2 and IL-15 (71). Through ex vivo expansion technology, the number of NK cells can be substantially augmented, with an approximately (1,800−2,400)-fold increase observed in fresh or cryopreserved CB units (53). Furthermore, the activation and maturation of NK cells, contributing to their effective killing abilities, are achieved via the influence of cytokines (72). CB-NK cells can also be activated by feeder cells. Liu et al. successfully created universal antigen-presenting cells (uAPC) by engineering K562 cells with increased expression of CD48, 4-1BBL, and mbIL21 to promote the expansion and activation of CB-CAR-NK cells (73).

In vivo persistence of NK92 cells

Despite the advantages of NK-92 cells over PB/CB-NK cells in terms of expansion and modification, they still face challenges due to their limited persistence in vivo. There are two possible reasons for the limited persistence of NK-92 cells. First, for lymphoma, NK-92 cells must be irradiated before adoptive transfer to eliminate the risk of tumorigenicity and Epstein-Barr viral susceptibility (74). However, irradiation was detrimental to the persistence of NK-92 cells in vivo. Furthermore, the viability and proliferation of NK-92 cells are highly dependent on a continuous supply of low-dose IL-2. In the absence of IL-2, NK-92 cells exhibited a rapid decline in survival, leading to death in 72 h. Therefore, when introduced into the human body, the absence of IL-2 poses a significant threat to the longevity of NK-92 cells (25).

Enhancing the persistence of NK-92 cells can be achieved via gene modifications, particularly by targeting various elements, such as cytokines. For example, NK-92 cells can be modified by attaching IL-2 to their plasma membranes to stimulate nearby IL-2 receptors. This membrane-bound protein (MBP) NK-92 cells exhibited exogenous IL-2 independent proliferation and showed improved infiltration and persistent activity in an A549 spheroid solid tumor model (75). Similarly, mbIL-2-expressing NK-92 cells showed improved persistence and enhanced anti-tumor activity in a leukemia xenograft model (76). IL-15 is another cytokine commonly used in laboratory and clinical investigations to sustain survival and enhance the cytotoxicity of NK cells. NK-92 cells co-expressing IL-15 and EpCAM/breast cancer-specific CAR showed persistent and increased cytotoxicity against EpCAM-expressing breast carcinoma cells in vitro (77).

In addition to cytokines, other elements can be modified to optimize the persistence of NK-92 cells. For example, in vivo studies have shown that NK-92 cells expressing erythropoietin or thrombopoietin receptors have a longer lifespan (78). Similarly, NK-92 cells with truncated PD-1 (tPD-1) exhibit enhanced persistence compared to standard NK-92 cells, as tPD-1 blocks the PD-1/PD-L1 inhibition (79).

Common challenges associated with CAR-NK cells

Gene modification of CAR-NK products

Overcoming the resistance of NK cells to transduction is a significant hurdle in the successful and efficient gene modification of CAR-NK cells. Compared to T cells, NK cells show a higher sensitivity to foreign genetic materials and a lower transduction efficiency (80). Mechanically, pattern recognition receptors in NK cells recognize foreign genetic materials and trigger an innate defense against them (81). Therefore, the potential risks of insertional gene mutations should not be overlooked. In the presence of high titers of vital vectors, NK cells encounter the potential genotoxicity associated with insertional mutations (82).

Currently, transfection methods are divided into two types: viral and non-viral. Viral vectors have emerged as highly efficient and superior gene modification tools compared to non-viral alternatives (83). As an important component of viral transfection systems, lentiviral transfection has been used in phase I/II clinical research on CAR NK cell therapy for hematological (NCT05472558) and solid (NCT05410717) malignancies. Other retroviral vectors are also primary choices to modify NK cell genes. For example, Suerth et al. confirmed that alpha-retroviral vectors are useful tools for the application of NK cell-mediated cancer immunotherapy (84).

To date, many efforts have been made to enhance the transduction efficiency. These include the implementation of multiple rounds of transduction, co-culture techniques involving feeder cells, and the development and refinement of novel vector designs.

(1) Multiple rounds of transduction

Adopting multiple rounds of transduction represents a direct approach to improving the efficiency of gene expression. Guven et al. observed that the percentage of NK cells transduced on d 21 was higher in cells that underwent two rounds of transduction than in cells that underwent a single round of transduction (75.4% vs. 51.9%) (85). In addition, in the presence of the lentiviral transduction enhancer dextran, the transduction rate of NK cells reached 40% after one round of transduction and increased to 100% after two rounds (86). Therefore, repeated transduction may be a potential method to increase transduction efficiency.

(2) Co-culture with feeder cells

Feeder cells can be used to improve the production of CAR-NK cells. Despite their potential impact on viral transfection efficiency, feeder cells effectively enhance the recovery and expansion of transfected NK cells. Allan et al. investigated the delicate balance between gene editing efficiency and expansion potential and showed that the ideal pre-activation duration of feeder cells is 5−7 d prior to the lentiviral transduction (87). EBV-transformed lymphoblastoid cell lines and genetically modified K562 cells are feeder cells that are commonly used to achieve clinical-scale NK cell proliferation (23,88). Mechanically, the secretion of IL-2 by feeder cells serves as a highly effective enhancer of NK cell expansion and transduction (57). Furthermore, physical contact between feeder cells and NK cells, as well as the binding of ligands on the feeder cells (e.g., 4-1BBL (CD137L), mbIL-15, and mbIL-21) to their corresponding stimulatory receptors on NK cells, is crucial for successful expansion of NK cells (89,90).

(3) Vector and transduction enhancer

The design of new vectors appears to be another feasible method. One such example is the use of baboon envelope-pseudotyped lentiviral vectors, which have been developed to promote viral entry. Colamartino et al. demonstrated that these vectors achieved a transduction efficiency of 38.3%±23.8% in NK cells, which increased to 58.4%±7.8% after re-expansion (91).

The use of transduction enhancers, such as dextran and IL-2, is another option. Müller et al. conducted a comparative study of two different retroviral vector platforms with the addition of retronectin and vectofusin-1. They found that the combination of RD114-TR pseudotyped retroviral and vectofusin-1 was the optimal strategy to engineer PB-derived NK cells (92).

(4) Non-viral gene modification

Considering the restricted vector capacity and potential risk of tumorigenicity associated with viral transfection, various non-viral vectors have been extensively investigated in recent years to overcome these limitations. Electroporation, nucleofection, and lipofection are the most commonly used non-viral transduction methods (93). Compared to lipofection, electroporation has shown greater efficiency and suitability for clinical translation, although it may be less cost-effective (94). mRNAs are the main cargo involved in non-viral transduction. Carlsten et al. used electroporation to deliver the mRNA of high-affinity CD16 and the chemokine receptor C-C motif chemokine receptor 7 to primary NK cells. This resulted in 95% of NK cells successfully expressing the target mRNA. The engineered cells exhibit enhanced migration and cytotoxicity against antibody-coated lymphoma cells (95). However, while mRNA has a lower risk of insertional mutation as it cannot integrate into DNA, it poses a limitation for non-viral transduction methods in achieving permanent CAR structure expression. Additionally, the limited capacity of mRNA as a cargo hinders the development of non-viral transduction (96).

Various strategies have been explored to overcome the aforementioned hurdles, such as the utilization of DNA transposons, CRISPR/Cas9 technology, and nanoparticles. DNA transposons have become popular non-viral gene delivery tools for achieving stable gene expression (97). Transposon-mediated CAR transduction, especially with piggyBac and sleeping beauty transposon systems, has shown promising efficacy in achieving stable CAR expression in NK cells through a close-to-random profile of genomic integration (98). Another promising approach is the combination of non-viral gene delivery methods with CRISPR delivery systems (99). Specifically, Nguyen et al. electroporated Cas9 ribonucleoproteins in NK cells, which improved the transfection efficiency. Moreover, multifunctional nanoparticles (MF-NPs) can be used to genetically express anti-EGFR-CAR structures on NK cells and image MF-NP-loaded NK cells in vivo (100).

Challenges of CAR-NK cells in solid tumor treatment

CAR-NK cells from different sources have limited effectiveness in solid tumor treatment and yield unsatisfactory outcomes. This can be attributed to several factors. First, decreased expression of chemokine ligands, such as CXCL1 and CXCL9, impairs NK cell trafficking ability, restricting their infiltration into tumor sites (101). For example, in hepatocellular carcinoma, miR-561-5p overexpression downregulates CXCL1 expression and blocks CXCR1+NK cell infiltration (102). Furthermore, the immunosuppressive characteristics and the abnormal metabolic profile of tumor microenvironment (TME) hinder the survival and function of NK cells (103,104). Fortunately, various strategies have shown promise in addressing these challenges in pre-clinical tumor models.

(1) Enhancing infiltration of CAR-NK cells

One approach to enhance the infiltration of NK cells into tumor sites is to increase the expression of chemokines in NK cells and stimulate cancer cells to secrete the corresponding chemokine ligands. This can be achieved by various methods. First, NK cells can be designed to co-express a CAR structure and chemokine receptors, such as CXCR3 and CXCR4, which are key mediators of NK cell trafficking. Müller et al. successfully enhanced tumor infiltration by NK cells by designing novel NK cells with EGFRvIII-specific CAR and CXCR4 (105). Similarly, CXCR1-engineered NK cells exhibited increased infiltration of human tumors in a xenograft model (106). Second, certain anti-tumor agents, such as radiotherapy and autophagy inhibitors, can disrupt normal tumor processes, leading to increased secretion of chemokine ligands (107,108). For example, dipeptidyl peptidase inhibitors and curaxins have been shown to enhance the secretion of CXCL9 and CXCL10 in different solid tumors, promoting the infiltration of CXCR3+ NK cells into the TME (109). In addition, nanoparticles can facilitate NK cell infiltration. For instance, magnetic nanoparticles loaded with NK-92 cells showed a 17-fold increase in infiltration when a magnetic field was applied compared to normal NK-92 cells (110).

(2) Optimization of CAR-NK cells

To improve the function of NK cells, it is important to directly modify NK cells to overcome unfavorable conditions in the TME. One promising approach is to adapt and modulate CAR-NK cells to respond effectively to the components of TME. Therefore, reengineering the CAR structure has shown great potential. A unique CAR, namely the novel chimeric costimulatory converting receptor (CCCR), was designed to address this challenge. CCCR incorporates the domain of PD1 (extracellular), NKG2D (transmembrane and cytoplasmic), and 41BB (cytoplasmic). This design aimed to convert the inhibitory PD-1 signal into an activating one (111). When expressed in NK-92 cells, CCCR-NK-92 cells were able to overcome immunosuppressive TME and exhibited enhanced anti-tumor efficacy.

Memory-like NK cells have increased production of IFN-γ, which is related to the upregulation of killing-activating receptors and downregulation of killing inhibitory receptors. This ultimately leads to a superior killing effect compared to common NK cells (112). Inducing immunological memory of NK cells is an alternative method to enhance NK cell activity in solid tumors (113). For example, Chang et al. developed PD-L1-induced memory-like iPSC-NK cells that respond to PD-L1 signaling, resulting in enhanced proliferation and cytotoxicity. In a mouse xenograft model, these cells exhibited higher anti-tumor cytotoxicity than other CAR-NK cells (114).

Additionally, the effectiveness of NK cells with different CAR structures may vary depending on the type of cancer. For example, in prostate cancer mouse models, anti-PSCA-DAP12 CAR-NK cells showed greater cytotoxicity compared to anti-PSCA-CD3ζ CAR-NK cells (115). Similarly, Li et al. evaluated different CAR structures in iPSC-NK cells and found that NKG2D-2B4-CD3ζ CAR exhibited the strongest activating effect on NK cells in an ovarian cancer model (98). Therefore, it is crucial to compare and optimize various CARs for different types of cancer to maximize the cytotoxic effects of CAR-NK cells.

(3) TME modification

In addition, it is important to regulate the immune and metabolic conditions in the TME to enhance the effectiveness of CAR-NK cells. Various agents, such as chemotherapy and immune checkpoint inhibitors (ICIs), can modulate the immune response in the TME and improve the persistence and cytotoxicity of NK cells (116,117). Chemotherapy can directly activate NK cells and modify immune response in the TME to support NK cell functions. It achieves this by improving the immunogenicity of cancer cells and regulating the recruitment and function of immunosuppressive cells (118). For example, Klapdor et al. found that treating CD44-positive ovarian cancer cells with anti-CD44 NK cells and cisplatin simultaneously improved the anti-tumor capacity of NK cells (119).

ICIs are effective in regulating immune interactions between NK and tumor cells. In solid cancers, prominent inhibitory checkpoints, such as PD-1, NKG2A, and KIRs, can inhibit NK cell activation (120,121). Various antibodies have been developed to block these inhibitory checkpoints and restore CAR-NK cell function to overcome immune inhibition (122). Wang et al. showed that combining anti-PSMA CAR NK-92 cells with an anti-PD-L1 antibody improves anti-tumor efficacy in a castration-resistant prostate cancer mouse model (123). Furthermore, clinical trials have confirmed that monalizumab, an anti-NKG2A monoclonal antibody, improves NK cell activity in solid cancers (124). The combination of monalizumab with CAR-NK cell therapy has the potential to enhance its therapeutic effects.

Additionally, abnormal metabolic conditions in TME, such as hypoxia, nutrient deprivation, and accumulation of metabolites (e.g., reactive oxygen species and adenosine), have a significant effect on the metabolism and energy supply of NK cells, ultimately affecting their anti-tumor properties (125,126). Therefore, modulating metabolism can be a promising method to enhance the performance of NK cells in TME. For example, combining NKG2D-specific CAR-NK-92 cells with CD73 blockade improves therapeutic efficacy in CD73+ human lung cancer models (127). CD73 is associated with the accumulation of adenosine, a metabolite with immunosuppressive effects that leads to the exhaustion of NK cells (128) (Figure 2).

Figure 2.

Individual and common challenges faced by CAR-NK cells derived from different sources. Common challenges: Left, NK cells are initially resistant to transfection during gene modification; Right, the efficacy of CAR-NK therapy in solid tumors is still limited. Individual challenges: The efficiency of expansion needs to be improved to meet clinical requirements in both PB- and CB-derived CAR-NK cells. More advanced techniques are required to maintain the activity of PB-CAR-NK cells during cryopreservation. Compared to PB-NK cells, CB-NK cells are less mature. Preliminary irradiation and reliance on IL-2 leads to limited in vivo persistence of CAR-NK-92 cells. The clinical safety and efficacy of iPSC-CAR-NK cells require further verification. CAR-NK, chimeric antigen receptor-natural killer; PB, peripheral blood; CB, cord blood. The figure is created with BioRender.com.

Conclusions and perspective

CAR-NK cells derived from PB, CB, and NK-92 cell lines and iPSCs are effective in anti-tumor therapy. The abundant allogeneic suppliers of NK cells undoubtedly provide a promising opportunity for clinical-scale manufacturing and application of CAR-NK therapy. However, each source also has its problems that need to be solved and is associated with common challenges that impede effective CAR-NK production. Although CAR-NK therapy has shown outstanding anti-tumor effects on pre-clinical studies of both hematological and solid tumors, corresponding clinical trials with CAR-NK cells are just beginning, and many critical results have not yet been published. Therefore, to obtain reliable clinical-level off-the-shelf CAR-NK products, follow-up comparisons and verification of the clinical efficacy and safety of CAR-NK cells from different sources and optimization and standardization of CAR-NK manufacturing processes are required.