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

Feifei Guo, Yi Zhang, Jiuwei Cui

Citation: Feifei Guo, Yi Zhang, Jiuwei Cui. Manufacturing CAR-NK against tumors: Who is the ideal supplier?. Chin J Cancer
Res 2024; 36(1): 1-16. doi: 10.21147/j.issn.1000-9604.2024.01.01

View online: http://article.cjcrcn.org/article/doi/10.21147/j.issn.1000-9604.2024.01.01

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 Review Article

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

Feifei Guo*, Yi Zhang*, Jiuwei Cui

The First Hospital of Jilin University, Cancer Center, Changchun 133021, China
*These authors contributed equally to this work.
Correspondence to: Jiuwei Cui. The First Hospital of Jilin University, Cancer Center, Changchun 133021, China. Email: cuijw@jlu.edu.cn.

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.

Keywords: CAR-NK cells; cord blood; iPSC; NK-92 cells; peripheral blood

Submitted Nov 02, 2023. Accepted for publication Jan 12, 2024.
doi: 10.21147/j.issn.1000-9604.2024.01.01
View this article at: https://doi.org/10.21147/j.issn.1000-9604.2024.01.01

Introduction CAR-NK products to help reduce patient waiting times

and treatment costs (5). However, each source has its
Despite the remarkable effectiveness of chimeric antigen
drawbacks. Therefore, in this review, different CAR-NK
receptor-T (CAR-T) cell therapy in human malignancies, it
cell sources are discussed to assist in making an informed
is associated with a plethora of challenges (1). In addition to
choice when applying CAR-NK cells.
optimizing CAR-T cell efficacy, the alternative role of
natural killer (NK) cells has garnered significant interest.
Comparison of CAR-NK cells derived from
The distinct characteristics of chimeric antigen receptor-
different sources
natural killer (CAR-NK) cells highlight their superior
potential for anti-tumor efficacy compared to CAR-T cells Peripheral blood (PB), cord blood (CB), NK cell lines, and
(2). In addition to CAR-mediated cytotoxicity, NK cells
iPSC are the main sources of NK cells for CAR
can lyse cancer cells with downregulated or deficient target
modification (5). NK cells from four promising cell types
antigens via NK-specific cytotoxicity, indicating that CAR-
possess their advantages but also exhibit different problems
NK cells are excellent candidates for eliminating
upon application (Figure 1).
heterogeneous solid tumors (3). CAR-NK therapy is also
believed to be safer than CAR-T cell therapy, given the PB-derived CAR-NK cells
short-term survival of CAR-NK cells in vivo and the
distinctive cytokine spectrum compared to CAR-T cells (4). PB-NK cells
“Multiple sources” is another significant advantage of Human PB mononuclear cells (PBMC) are conventionally
CAR-NK therapy. In addition to autologous peripheral used as NK cell sources in the early adoptive
blood-derived NK cells, multiple allogeneic NK cells immunotherapy (6,7). PB-NK cells have two subsets (8):
further enhance the possibility of fabricating off-the-shelf CD56brightCD16−/dim PB-NK cells that are the mostly

© Chinese Journal of Cancer Research. All rights reserved. www.cjcrcn.org Chin J Cancer Res 2024;36(1):1-16
2 Guo et al. CAR-NK cells: Characteristics and barriers

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.

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

© Chinese Journal of Cancer Research. All rights reserved. www.cjcrcn.org Chin J Cancer Res 2024;36(1):1-16
Chinese Journal of Cancer Research, Vol 36, No 1 February 2024
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).
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
© Chinese Journal of Cancer Research. All rights reserved.
3
(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
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4 Guo et al. CAR-NK cells: Characteristics and barriers

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

Sources of Product
Trial identifier Tumor type Target Stage Status
NK cells name
PB-NK NCT05645601 JD010 B-cell malignancies CD19 Phase I Recruiting
NCT05410717 − Ovarian cancer, testis cancer Claudin6 Phase I/IIa
and other endometrial cancers
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 Phase I Unknown status
ligands
NCT05020678 NKX019 B-cell malignancies CD19 Phase I Recruiting
NCT04623944 NKX101 AML or MDS NKG2D Phase I Recruiting
ligands
CB-NK NCT05247957 − AML NKG2D Phase I Terminated
ligands
NCT05472558 − B-cell NHL CD19 Phase I Recruiting
NCT04796675 − B-cell malignancies CD19 Phase I Recruiting
NCT05922930 − Ovarian cancer, mesonephric-like TROP2 Phase I/II Not yet recruiting
adenocarcinoma, and pancreatic
cancer
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 CD70 Phase I/II Recruiting
myeloma
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 CD70 Phase I/II Recruiting
and osteosarcoma
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 MUC1 Phase I/II Unknown status
cell lung cancer, pancreatic
carcinoma, triple-negative invasive
breast carcinoma, malignant glioma
of brain, colorectal carcinoma,
gastric carcinoma
NCT03383978 − Glioblastoma HER2 Phase I Recruiting
NCT03656705 − Non-small cell lung cancer −* Phase I Completed
Table 1 (continued)

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Chinese Journal of Cancer Research, Vol 36, No 1 February 2024 5

Table 1 (continued)

Sources of Product
Trial identifier Tumor type Target Stage Status
NK cells name
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 MICA/ Phase I Active, not
cancer, breast cancer, ovarian MICBα3 recruiting
cancer, pancreatic cancer, head and
neck cancer, gastroesophageal
cancer
NCT05336409 CNTY-101 B-cell malignancies CD19 Phase I Recruiting
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.

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-

© Chinese Journal of Cancer Research. All rights reserved. www.cjcrcn.org Chin J Cancer Res 2024;36(1):1-16
6 Guo et al. CAR-NK cells: Characteristics and barriers
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).
© Chinese Journal of Cancer Research. All rights reserved.
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
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Chinese Journal of Cancer Research, Vol 36, No 1 February 2024
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
© Chinese Journal of Cancer Research. All rights reserved.
7
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).
www.cjcrcn.org Chin J Cancer Res 2024;36(1):1-16
8 Guo et al. CAR-NK cells: Characteristics and barriers
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.
© Chinese Journal of Cancer Research. All rights reserved.
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
www.cjcrcn.org Chin J Cancer Res 2024;36(1):1-16
Chinese Journal of Cancer Research, Vol 36, No 1 February 2024
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).
© Chinese Journal of Cancer Research. All rights reserved.
9
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
www.cjcrcn.org Chin J Cancer Res 2024;36(1):1-16
10 Guo et al. CAR-NK cells: Characteristics and barriers

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.

© Chinese Journal of Cancer Research. All rights reserved. www.cjcrcn.org Chin J Cancer Res 2024;36(1):1-16
Chinese Journal of Cancer Research, Vol 36, No 1 February 2024
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.
Acknowledgements
None.
Footnote
Conflicts of Interest: These authors have no conflicts of
interest to declare.
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