Generating universal chimeric antigen receptor expressing cell products from induced pluripotent stem cells: beyond the autologous CAR-T cells

Abstract

Adoptive therapeutic immune cells, such as chimeric antigen receptor (CAR)-T cells and natural killer cells, have established a new generation of precision medicine based on which dramatic breakthroughs have been achieved in intractable lymphoma treatments. Currently, well-explored approaches focus on autologous cells due to their low immunogenicity, but they are highly restricted by the high costs, time consumption of processing, and the insufficiency of primary cells in some patients. Induced pluripotent stem cells (iPSCs) are cell sources that can theoretically produce indefinite well-differentiated immune cells. Based on the above facts, it may be reasonable to combine the iPSC technology and the CAR design to produce a series of highly controllable and economical “live” drugs. Manufacturing hypoimmunogenic iPSCs by inactivation or over-expression at the genetic level and then arming the derived cells with CAR have emerged as a form of “off-the-shelf” strategy to eliminate tumor cells efficiently and safely in a broader range of patients. This review describes the reasonability, feasibility, superiority, and drawbacks of such approaches, summarizes the current practices and relevant research progress, and provides insights into the possible new paths for personalized cell-based therapies.

Introduction

Regenerative therapies based on stem cells have evolved quickly and have been applied widely since induced pluripotent stem cells (iPSCs) were first reported by Takahashi and Yamanaka^[1] in 2006. By simultaneously introducing four genes (Oct3/4, Sox2, Klf4, and cMyc), iPSCs were successfully reprogrammed from somatic cells. iPSCs exhibit almost identical characteristics and properties to embryonic stem cells (ESCs), which proliferate indefinitely and are able to differentiate into any type of cells.^[1,2] In addition to solid organ transplantation, immune cells differentiated from iPSCs have also been used in the design of cell-based therapies for hematological malignancies in recent studies. iPSC-derived cells outperform conventional, patient-derived autologous cells when utilized in adoptive immunotherapy due to their easy editing,^[3–5] availability to mass production,^[3,4,6] free from viral and cancer cells,^[7] relatively mature protocols,^[5,7,8] standardized quality control,^[4,7,8] and avoidance of ethical restrictions.^[5]

The chimeric antigen receptor (CAR)-T cell immunotherapy has promoted the treatment of multiple types of cancer, especially CD19-expressing B-cell malignancies. CAR is designed based on the natural T cell receptor (TCR) and consists of three domains: 1) a single-chain variable fragment (scFv) derived from a monoclonal antibody for targeted recognition; 2) a transmembrane domain; and 3) the intracellular domain, which incorporates a co-stimulatory domain, such as 4-1BB or CD28,^[9] and a CD3ζ signaling domain (4-1BB/CD28-CD3ζ, designated T-CAR). Most recent studies have reported the fourth generation of CARs, which additionally contain an inducible module for the expression of transgenic proteins, such as cytokines, to enhance T-cell-mediated killing.^[10] Other immune cells equipped with CAR have arisen along with more cell-specific CAR constructs. Researchers have screened possible alternatives for each part of the CAR molecule and tested different combinations, ultimately optimizing CAR for their adaptation to natural killer (NK) cells (NKG2D-2B4ζ, designated NK-CAR)^[4] and macrophages (multiple epidermal growth factor [EGF]-like domains protein 10 [Megf10]/FcRγ-CD3ζ, designated P-CAR).^[11]

In light of various gene-editing operations generating both hypoimmunogenic iPSC-derived and CAR-based cells,^[3] it provides an attractive and feasible way to combine iPSCs and CAR using new multiplex editing technologies^[12] to produce robust, safe, economical, and universal cell products for the treatment of clinically troublesome and refractory hematological malignancies.

Immune Cells Armed with CARs Utilized in Leukemia and Lymphoma

The CAR-T-cell marks a technical milestone in the field of hematological malignancies over the last decade. Targeted CAR endows effector immune cells with the ability to directly recognize and eliminate the selected tumor cells independently of the major histocompatibility complex (MHC). The specificity of CAR lies in the design of scFv, which extends the application of adoptive cell therapy beyond traditional tumor chemical therapy.

The CAR technology most maturely developed in T cells. CD19 CAR T therapies against B-cell malignancies were recently approved by the United States Food and Drug Administration (FDA), and are now commercially available for relapsed or refractory (R/R) acute lymphoblastic leukemia (ALL) and some specific types of B-cell lymphoma.^[13] Anti-B cell maturation antigen (BCMA) CAR T therapies have been identified as powerful approaches against multiple myeloma (MM), characterized by high response rates and long-term duration in clinical practice.^[14,15] Potential CAR target antigens against acute myeloid leukemia (AML) are now being studied in laboratories and preclinical tests, including the folate receptor-β (FR-β),^[16] feline McDonough sarcoma (FMS)-like tyrosine kinase-3 (FLT-3),^[17] CD33,^[18] and CD123.^[19] The efficiency and safety of CD5^[20,21] and CD7^[22] CAR T-cell therapies have been verified primarily to treat other intractable hematological malignancies, such as T-cell acute lymphoblastic leukemia (T-ALL). Optimization strategies, involving bispecific designs,^[23] safety switches,^[18] and the bi-epitope recognition,^[21] aiming to enhance specificity have also been emphasized by researchers.

CAR NK-cell therapies have emerged over the last 5 years. Compared with T cells, NK cells function without human leukocyte antigen (HLA)-matching, and they exhibit safer properties, as indicated by less evidence for graft-versus-host disease (GvHD) and cytokine release syndrome (CRS) in adoptive therapies.^[24] The CAR reconstructed for NK cells has evolved into the second generation, and its basic components are chosen from the signaling domains DNAX-activation protein (DAP) 10,^[25] DAP12,^[26] natural killer cell receptor 2B4 (NKR2B4),^[4] or CD3ζ. Present programs on CAR-NK cells are mostly at the stage of clinical phase I, which still requires strict and long-term inspection. Hematological malignancies, including B-cell lymphoma, AML and MM, and solid tumors such as colorectal cancer and breast cancer, were tested for the antitumor activities of NK cells equipped with anti-CD19,^[27] CD33,^[28] BCMA (Number of National Clinical Trial: NCT03940833), natural killer group 2, member D (NKG2D) ligand,^[29] and human epidermal growth factor receptor-2 (HER2)^[30] CAR. Although there exist challenges for CAR-NK therapies, such as limited in vivo persistence without the support of cytokines and inefficient trafficking to tumor beds,^[24] CAR-NK therapy remains a prospective approach to developing “living drugs”.

To circumvent the limitations of CAR-T therapies, for example, T cells cannot easily penetrate into the tumor microenvironment (TME) of solid tumors, CAR macrophages have been developed over the last two years.^[31] Compared with T cells and NK cells, macrophages are abundant in the TME, giving rise to the idea that macrophage phagocytosis may be enhanced by CAR to stimulate the immune response in the immunosuppressive TME. However, until now, the CAR constructs for macrophages were less studied, using either the traditional T-CAR targeting CD19 or HER2,^[32,33] or the new P-CAR. The cytoplasmic domain of P-CAR was redesigned to trigger phagocytosis, such as Megf10 or Fc-receptor γ chain (FcRγ).^[11] It is worth emphasizing that the transition between the pro-inflammatory M1 phenotype and the pro-tumorigenic M2 phenotype of macrophages could be influenced by transduction vectors.^[32] The relevant clinical trials have focused mainly on breast cancer,^[34] and some epithelial malignancies.^[31] Due to the unique feature of macrophages to convert a “cold” tumor into a “warm” tumor, well-engineered CAR-macrophage therapies might benefit patients with various tumors.^[35]

To summarize, CAR-based therapy applying various immune cells has been generally acknowledged as one of the most effective approaches against fatal malignancies [Figure 1]. Comparisons among CAR-expressing T cells, NK cells, and macrophages were listed in Table 1.

Figure 1: The CAR construct designed for T cell, NK cell, and macrophages. The part above these small blue molecules (phospholipid, the cell membrane) is the extracellular domain of CAR, including the hinge and scFv. The part embedded in the membrane is the transmembrane region. The part below the membrane is the intracellular domain, including co-stimulatory domain and signaling domain. CAR: Chimeric antigen receptor; DAP10/12: DNAX-activation protein 10/12; FcRγ: Fc-receptor γ chain; Megf10: Multiple epidermal growth factor (EGF)-like domains protein 10; NK: Natural killer; NKG2D: Natural killer group 2, member D; P-CAR: Chimeric antigen receptors that trigger phagocytosis; PI3K: Phosphoinositide 3-kinase; scFv: Single-chain variable fragment; TM: Transmembrane domain; VH: Variable region of heavy chain; VL: Variable region of light chain.

Table 1 - Comparisons among CAR-T cells, CAR-NK cells, and CAR-macrophages.

Items	CAR-T cells	CAR-NK cells	CAR-macrophages	Citations
Stages	Commercialized	In clinical trials	In laboratory tests	[3,4,33]
Antitumor mechanism	Perforin, granzymes, Fas-FasL, and cytokines	Perforin, granzymes, ADCC, and cytokines	Phagocytosis, cytokines	[3,4,11]
Immunogenicity	Relatively high	Relatively low	Unknown	[3,4,42]
Infiltration	Relatively low	Relatively high	High	[11,24,31–33]
Killing capacity	High	Relatively high	Relatively low	[24,28,29]
Safety	CRS, GvHD, HvGD	Relatively safe	Toxicity is not statistically significant	[3,8]
Persistence	Long-time	Short-time	Unknown	[4,8]
Application	Hematological malignancies	Hematological malignancies, solid tumors	Solid tumors	[4,24]

ADCC: Antibody-dependent cell-mediated cytotoxicity; CAR: Chimeric antigen receptor; CRS: Cytokine release syndrome; Fas-L: Fas-ligand; GvHD: Graft-versus-host disease; HvGD: Host-versus-graft disease; NK: Natural killer.

Cell Sources of CAR-Equipped Cell Products

Several different cell sources have been chosen to generate CAR-expressing immune cells, including peripheral blood (PB), umbilical cord blood (UCB), stem cells (hematopoietic stem cells [HSCs], and iPSCs), and corresponding cell lines.^[24] PB and iPSCs are subdivided into two classes, autologous cells, and allogeneic cells, each with unique strength and weakness [Table 2].

Table 2 - Comparisons between PB and iPSCs in CAR-based immunotherapies.

Cell sources	Strength	Weakness	Citations
PB
Autologous	1. Low immunogenicity 2. Heterogenous components (CD4+/CD8+ T cells) 3. Highly active and cytotoxic 4. Mature phenotype 5. Well-established protocols	1. Large time consumption (due to the complicated production processes for each patient) 2. Long-distance transportation 3. Possible reduced number, viability, and functionality (due to the poor condition of patients) 4. Propensity for exhaustion after multiplex editing	[13,24,36]
Allogeneic	1. Preparation in advance 2. Time saving 3. Relatively low-cost 4. Sufficient quantity 5. Mature phenotype	1. Immune rejection 2. Reduced therapeutic effects 3. Limited in vivo persistence 4. Higher rates and severity of CRS 5. Propensity for exhaustion after multiplex editing	[13,24,69]
iPSC
Autologous	1. Infinite multiplication 2. Pluripotency 3. Low immunogenicity 4. Suitable for gene manipulations 5. Similar functionality to the primary cells	1. Formation of teratoma 2. Prolonged culture cycle 3. Accumulated gene mutations during the differentiation 4. Long-distance transportation 5. Optimization of production processes might be needed	[4,5,7,36,39,40]
Allogeneic	1. Infinite multiplication 2. Pluripotency 3. Homogenous components (for standardized production) 4. Similar functionality to the primary cells 5. Readily available iPSC banks 6. Suitable for gene manipulations 7. Adaption to strict quality control 8. Reduced costs compared with autologous iPSCs	1. Formation of teratoma 2. Prolonged culture cycle 3. Accumulated gene mutations during the differentiation 4. Immune rejection 5. Heterogeneity across different iPS cell lines 6. General protocols need to be designed 7. Difficulty in resource sharing between iPSC banks	[3,6–8,36,42]

CAR: Chimeric antigen receptor; CRS: Cytokine release syndrome; iPSC: Induced pluripotent stem cell; PB: Peripheral blood.

Autologous PB is the most widely used cell source in CAR-T therapies. The advantage of autologous cells is that they reduce the incidence of the immunological rejection and GvHD. The protocols for generating CAR-T cells have been well established and are being continuously improved. The relatively mature phenotype, stable cytotoxicity, and heterogeneous components which include both CD4⁺ and CD8⁺ T cells, ensure the effectiveness of the products. However, the complicated process from cell mobilization to the final reinfusion is labor-intensive and time-consuming since these T cells are highly personalized, and these processes must be executed separately for every single patient. Viral or cancerous contamination can be a constant risk. Additionally, primary T cells have been reported to be inclined to exhaust phenotypes after multiple strikes brought by gene editing.^[3] Cryopreservation and shipment can also be challenging. Additionally, the leukocytes extracted in the process of leukapheresis might not be sufficient for engineering and quality tests due to the poor condition of patients. The use of allogeneic PB inevitably involves even more gene manipulation, such as knockout of the HLA-I or HLA-II genes (genes encoding human leukocyte antigens, the MHC molecules of human), to avoid lethal immune rejection. These facts raise the requirements for cell sources that are more suitable to largely produce CAR-T cells in a clinical scale.

Stem cells then come into researchers’ sight for their inexhaustible self-renewal and differentiation activity.^[2] By changing cultural environment during certain periods of differentiation, immune cells can be abundantly derived from stem cells for immunotherapies [Figure 2].^{[8,33,36–38]} Contamination-free conditions can be implemented due to the standardized procedure. UCB, which contains ESCs, are now available as off-the-shelf frozen units stored in large global cord blood banks,^[39] and they were preferred by some researchers because of their fewer genomic changes, persistent epigenetic memory, and high-proliferative transcriptomic profile.^[40] However, extensive ex vivo expansion is needed since ESCs are few in UCB. Another challenge for its application is the competition between public banks for donated UCB and private banks for certain child or family.^[41] Instead, iPSCs could be generated simply from somatic cells in PB and bone marrow, leading to better accessibility than UCB. Recently, several articles have proposed that iPSCs could also be derived from UCB and filtered for homozygous HLA units, converting UCB banks into iPSC banks for wider utilization.^[40,41] iPSCs exhibit comparable functionality and properties to ESCs, which were validated by the karyotype analysis, genetic testing, and the formation of teratoma.^[1,2] Furthermore, the homogeneity of iPSCs enables stringent quality control. The feeder-free differentiation culture systems also confirmed the possibility of regenerating immune cells in a large-scale and a more economical way. Iriguchi et al^[42] demonstrated that mature T cells can be derived from iPSCs in quantity and further expanded by >200 folds, indicating the sufficiency for current CAR-T therapy. In conclusion, iPSCs could provide a better platform to generate a large number of immune cells for CAR-based clinical application and bioresearch compared with primary cells and ESCs.

Figure 2: The process of generating iPSC-derived CAR-expressing immune cell products. The first step is genetic editing in iPSC cells, including knockout of HLA-A and -B, forced expression of HLA-E and B2M fusion protein, and the replacement of TCR with CAR. The second step is generating HSPCs from the edited iPSCs. The following steps are separately performed according to the different demands for the products. bFGF: Basic fibroblast growth factor; BMP4: Bone morphogenetic protein 4; CAR: Chimeric antigen receptor; CAR iMacrophage: CAR-expressing iPSC-derived macrophages; CAR iNK: CAR-expressing iPSC-derived natural killer cells; CAR iT: CAR-expressing iPSC-derived T cells; FcDLL4: Fc- delta-like ligand 4 (DLL4) fusion protein; Flt-3L: Feline McDonough sarcoma (FMS) like tyrosine kinase 3 ligand; HLA: Human leukocyte antigen; IL: Interleukin; iPSCs: Induced pluripotent stem cells; M-CSF: Macrophage colony-stimulating factor; NK: natural killer; PBMC: Peripheral blood mononuclear cell; PHA-P: Phytohemagglutinin-P; SCF: Stem cell factor; TCR: T cell receptor; TPO: Thrombopoietin; VEGF: Vascular endothelial growth factor.

However, to produce “off-the-shelf” CAR-equipped immune cells, the quantity was not sufficient since allogeneic iPSCs will bring up issues about immune rejection. Constructing a system for the generation of hypoimmunogenic iPSCs could overcome these limitations. There are several manipulations at the genetic level that aim to reduce the immunogenicity of allogeneic iPSCs or iPSC-derived cells. Strategies for circumventing the recognition of host CD8⁺ and CD4⁺ T cells are usually involved in the silencing or deletion of MHC molecules, such as HLA-I or HLA-II genes and the genes essential for HLA expression.^[3] However, the absence of HLA molecules will sufficiently activate host NK cells, leading to the rapid consumption of products. Disruptions of B2M gene (gene encoding the β2-microimmunoglobulin) fully eliminated the expression of all classes of HLA molecules but did bring a new danger. Host NK cells were sensitive to the absence of HLA molecule, which left the edited cells vulnerable to the lysis, also named “missing-self” response. Two universally accepted methods are knocking out B2M with forced HLA-E expression (the HLA-E/B2M fusion transgene) and knocking out HLA-A and HLA-B alleles while remaining the intact HLA-C.^[2] Knockout of B2M leads to the deficiency of β2-microglobulin, resulting in complete HLA-I deletion and the loss of recognition of host CD8⁺ T cells, while the introduced HLA-E/B2M fusion gene enables the cell to evade the “missing self” of host NK cells.^[43,44] Homozygous iPSC cell lines and the HLA-C-only approach operate similarly: HLA-A and HLA-B are critical for immune rejection, while HLA-C binds to killing inhibiting receptors (KIRs), then suppresses NK cells.^[45,46] Some studies have additionally knocked out CIITA, a transcription factor required for the expression of HLA-II genes, to avoid the stimulation of host helper CD4⁺ T cells.^[47] The knockout of PVR (poliovirus receptor, encoding a ligand of killing activating receptor [KAR]) was also proved effective in eluding NK cells.^[3] Several studies have also reported that the upregulation of CD47, which interacts with cell surface receptors to inhibit phagocytosis, can evade host macrophages.^[46,48] Other strategies for the generation of universal immune cell products focus either on the microenvironment of adoptive cells, such as alloimmune defense receptor (ADR)-expressed immune cells,^[49] or on the alleviation of GvHD, such as UCAR T cells in which the TCR is deleted by inactivation of TRAC (the gene encoding constant region of T-cell receptor α chain).^[50]

The cell line used to generate CAR products is NK92, an NK-cell line with high proliferative capacity, homogeneity, and suitability for engineering.^[24] Nevertheless, the need for irradiation and the subsequent impairment of in vivo dynamics have limited its application. Studies of CAR-based therapies applying NK92 are also relatively few. CAR-NK cells can also be generated from PB of healthy donors with a more mature phenotype, but with relatively low efficiency (5–10% of lymphocytes are NK cells) which limits its utilization. Cord blood- and iPSC-derived NK cells were proven to be high-quality and readily available (banks) cell sources for CAR engineering. However, just like PB, after the extensive ex vivo expansion, the cord blood-derived NK cells ultimately generate heterogeneous cells, which made it difficult for the quality control. Despite the long culture condition, homogeneous NK cell products can be massively generated by using iPSC technology.

In summary, iPSC might be a feasible and superior cell source to engineer CAR cell products, and together with gene manipulations for reducing immunogenicity, iPSCs will potentially pave the way for universal CAR products, benefiting a broader range of patients.

Methods and Quality Controls for Generating iPSC-Derived Immune Cells

To increase the efficiency and yields as much as possible, researchers have devoted efforts to optimizing each stage of the production procedure. CAR engineering for different types of immune cells has been discussed before. The integration methods of transgenes have been reported to possibly influence the results of CAR expression on immune cells and even cause tumorigenesis. The two most frequently used gene integration methods are retroviruses, such as lentiviruses, and designer nucleases which contain three main subtypes: zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) nucleases.^[7] Integration can occur randomly in euchromatin when using retroviruses, and the most severely affected regions are oncogenes or tumor suppressors. Compared with retroviruses, designer nucleases can mediate locus-specific integration with predefined gene copy numbers, stabilizing and standardizing the output. CRISPR technologies outperform the other two nucleases in terms of on-target specificity, fleetness, and simplicity.^[7,51,52]

To ensure that these iPSC-derived cells meet a series of clinical-grade standards, such as safety and persistence, scientists have developed generally accepted manufacturing criteria for the quality control. Mandatory tests for the characterization of iPSC cell lines, iPSC-derived immune cells, and ultimately the CAR-equipped universal products contain examinations of differentiation, genetic stability, pluripotency (for iPSCs), morphology, viability,^[8] and tumorigenicity,^[53,54] while tests for only collecting information are alternatives, such as HLA typing.^[54] For CAR-expressing products, there are also necessary tests including maintenance, CAR expression, expansion capability, cytotoxicity, in vivo dynamics, systemic toxicity, and immunogenicity, especially for off-the-shelf products.^[4,8,33] The laboratory techniques required are listed in Table 3, including karyotype analysis, photography, flow cytometry, polymerase chain reaction (PCR), whole-genome sequencing (WGS), immunochemistry, and teratoma formation.^[52,53]

Table 3 - Methods and laboratory techniques used for quality control of iPSC-derived products.

Characteristics	Methods for quality control	Techniques	Citations
Morphology	Photography	Light microscope	[3,53,54]
Differentiation	Directed differentiation	Immunohistochemical analysis and FC	[4,8]
Pluripotency	Surface and intracellular markers	FC and PCR	[3,53,54]
Cell number	Total live cells	Trypan blue stain and cell counter
Cell viability	Viability	Trypan blue stain and cell viability analyzer, or FC
Purity	Homogeneity	FC
Genetic testing	Genetic stability	Karyotype analysis, SNP, array CGH, FISH assay
	Genetic mutations	Whole-genome sequencing, whole-exome sequencing
	Cancer predisposition testing	Sequencing for tumor suppressor genes and proto-oncogenes
Immunogenicity	Alloreactivity	Long-term culture with alloreactive immune cells	[4,8]
Sterility	Bacterial	Bacterial contamination	[3,53,54]
	Mycoplasma	Mycoplasma testing
	Endotoxin	Endotoxin testing
CAR expression	CAR expression and density	PCR, FC, immunohistochemical staining, and mass spectrometry	[4,8,33]
Cytotoxicity	Killing capacity and cytokine production	Killing assay, CD107 assay, 51Cr releasing test, and cytokine secretion assay
System toxicity	Adverse effects	Detection of CRS, histologic analysis for infiltrated lymphocytes in organs, etc.

CAR: Chimeric antigen receptor; CGH: Comparative genome hybridization; CRS: Cytokine release syndrome; FC: Flow cytometry; FISH: Fluorescence in situ hybridization; iPSC: Induced pluripotent stem cell; PCR: Polymerase chain reaction; SNP: Single nucleotide polymorphism.

Engineer iPSC-Derived Cells to Express CAR

Several groups have established projects using CAR-iPSC-derived cells. Wang et al^[3] successfully designed efficient procedures for generating hypoimmunogenic CAR-T cells for off-the-shelf utilization. They in turn validated the hypoimmunogenicity of B2M-knockout iPS-T cells, B2M and CIITA double-knockout (dKO) iPS-T cells, dKO-iPS-T cells with forced expression of HLA-E (dKO/E), and dKO/E-iPS-T cells with deletion of PVR (triple-knockout [tKO]/E), which were exhibited through the ability of immune escape from host CD8⁺ T cells, host CD4⁺ T cells, host natural killer group 2, member A (NKG2A)+NK cells, and host DNAM-1+NK cells, respectively. The resulting tKO/E-iPS-T cells showed simultaneously reduced immunogenicity to allogeneic T, B, and NK cells. Subsequent CAR engineering endowed the tKO/E T cells with similar target-specific suppression of tumors in vitro and prolonged overall survival in vivo when compared with primary CAR-T cells, together with longer T-cell graft survival. Iriguchi et al^[42] demonstrated another clinically applicable method for generating off-the-shelf iPSCs. They chose a strategy similar to iPSC banks that preserve HLA-homozygous iPSCs, then CAR was transduced into HLA-homo iPSC-T cells. Compared with the placebo and iPSC-T cells, iCAR-T cells exhibited better in vivo dynamics and exerted more powerful antitumor activity. The authors confirmed the feasibility of cell production by generating 1 × 10⁹ iPSC-T cells from 3 × 10⁵ iPSCs and further expanding them by >200 folds, which was sufficient for safety tests and CAR-T therapies. Additionally, the successful results could inspire other approaches to realize the commercialization of iPSC-derived immune cells, such as the improvement of automated bioreactor systems and TCR truncation for escaping GvHD. The latest findings published by Shen et al^[55] offered an optimized proposal for the in vitro generation of iPSC-derived T cells. Decreased oxygen metabolism was identified as a molecular driver of endothelial-to-hematopoietic transition (EHT) in vitro, and based on this finding, hypoxia conditions or pretreatment of arterial endothelium (AE) clusters involved in the hypoxia-associated mechanism during the onset of EHT of iPSCs were recommended. Intriguingly, AE-primed iPSC-derived T cells transduced with CD19-CAR exhibited comparable cytotoxicity against CD19⁺ target cells and robust tumor-killing capacity both in vitro and in vivo. iPSC-derived CAR-NK cells are also becoming a popular trend in this new field. A large number of studies have shown that iPSC-NK cells possess the same phenotypes and antitumor activities as PB NK cells and similar homogeneous properties to the NK92 cell line. An important issue the iPSC-NK cells might overcome is that clinical-scale NK cells will be sufficiently produced for multiple doses, alleviating the toxicity brought by high level of cytokines in the single-dose treatment. Li et al^[4] designed and tested a specific CAR construct for NK cells in iPSC-derived NK cells. Compared with PB-NK and T cells, NK-CAR iPSC-NK cells demonstrated superior antitumor activity in vitro and mediated improved survival in vivo. Interestingly, NK-CAR-expressing iPSC-NK cells performed better than T-CAR PB-T cells in reducing tumor burden, improving overall survival, and reducing toxicity. Another study by Ueda et al^[8] offered important information about the preservation and shipping of iPSC-derived CAR-NK cells. After 3 h of cryopreservation and subsequent thawing and long-distance transportation, the iPSC-derived CAR-NK cells demonstrated successful expansion profiles and maintained sufficient cell number and stable viability. Furthermore, the remaining iCAR-NK cells could still effectively suppress tumor growth in vitro and in vivo without evidence of NK-mediated toxicity and iPSC-mediated tumorigenicity. Fate Therapeutics has announced FDA clearance of their investigational new drug (IND) application FT596, an off-the-shelf, iPSC-derived CAR NK-cell product candidate that was engineered to target multiple tumor antigens for the treatment of B-cell lymphoma (NCT04555811) and chronic lymphocytic leukemia (NCT04245722). CAR molecules on the surfaces of iPSC-derived macrophages mainly facilitate target-specific phagocytosis activity against tumor cells. These iPSC-derived macrophages expressing CAR expanded, persisted well in vivo, and significantly reduced the tumor burden.^[33] Other potential CAR-expressing, iPSC-derived immune cells, including dendritic cells (DCs), γδT cells, and invariant natural killer T cells, have rarely been mentioned in current studies and require further exploration. Registered clinical trials of genetically engineered iPSC-derived immune cell products are listed in Table 4.

Table 4 - Registered clinical trials of genetically engineered iPSC-derived immune cell products.

IND	NCT identifier	Disease	Design	Clinical trial phase	Status
FT500	NCT03841110	Advanced solid tumors, lymphoma	Off-the-shelf (allogeneic), iPSC-derived NK cell	Phase 1	Active, not recruiting
	NCT04106167	Advanced solid tumors, lymphoma	Off-the-shelf (allogeneic), iPSC-derived NK cell	Phase 1	Recruiting
FT516	NCT04551885	Solid tumors, adult	iPSC-derived NK cells with high-affinity non-cleavable CD16a	Phase 1	Active, not recruiting
	NCT04630769	Ovarian cancer	iPSC-derived NK cells with high-affinity non-cleavable CD16a	Phase 1	Completed
	NCT04023071	AML B-cell lymphoma	iPSC-derived NK cells with high-affinity non-cleavable CD16a	Phase 1	Recruiting
	NCT04363346	COVID-19	iPSC-derived NK cells with high-affinity non-cleavable CD16a	Phase 1	Completed
FT596	NCT04245722	B-cell lymphoma, CLL	iPSC-derived NK cells with NK-CAR	Phase 1	Recruiting
	NCT04555811	NHL, DLBCL HGBL	iPSC-derived NK cells with NK-CAR	Phase 1	Recruiting
	NCT03407040	Solid tumors	iPSC-derived cancer antigen-specific T cells	Phase 1	Terminated

AML: Acute myeloid leukemia; CAR: Chimeric antigen receptor; CLL: Chronic lymphocytic leukemia; COVID-19: Coronavirus disease 2019; DLBCL: Diffuse large B cell lymphoma; HGBL: High-grade B cell lymphoma; IND: Investigational new drug; iPSC: Induced pluripotent stem cell; NCT: National Clinical Trial; NHL: Non-Hodgkin lymphoma; NK: Natural killer.

In summary, CAR design represents an efficient approach in translational medicine and proved effective in clinical practice. iPSC technology is now a popular research field in stem cells with relatively well-established protocols. Both methods involve extensive laboratory operations, while the final products of iPSC technology can be used as the initial material for CAR-armed products. Based on these facts, the strategy of combining CAR construction with immune cells derived from iPSCs can be attractive and theoretically feasible. Several studies have displayed their designs of iPSC-derived CAR-expressing immune cells and validation of their function. The core strategy was producing massive immune cells for multiplex manipulations at genetic levels that are necessary for reducing the immunogenicity of the final products. Genetic manipulations have included the knockout of HLA-A and HLA-B to evade recognition of host T cells, forced expression of the B2M/HLA-E fusion protein to evade recognition of host NK cells, and the upregulation of CD47 to evade recognition of host macrophages. All procedures were completed following a predesigned workflow to save the time that was previously used for purchase and transportation.

Possible Applications of New Breakthroughs in Gene-Editing Technologies

It is necessary to develop highly functional gene-editing tools when applying hypoimmunogenic iPSCs and CAR technology at the same time because of multiplex gene manipulations. Recently, Zhang et al^[12] isolated an engineered variant of the Cas12a nuclease, designated AsCas12a Ultra, with high multiplex genome editing efficiency comparable to SpCas9 and specificity similar to the original Cas12a. Both knockout and knock-in efficiency were elevated by this Ultra variant, and the single gene knock-in efficiency was up to 60% in T cells, 50% in NK cells, and 30% in hematopoietic stem progenitor cells. In the process of one-step generation of engineered immune cells, AsCas12a Ultra mediated nearly 40% double knock-in (enhanced green fluorescent protein[EGFP]/mCherry) efficiency and 93% simultaneous disruption of three genes (HLA-I, HLA-II, and TCR to evade host versus graft disease [HvGD] and GvHD) in T cells, while the conventional method usually resulted in only <5% of three-gene edited products in total cells. Researchers have emphasized that AsCas12a could have significant benefits as a clinical multiplex gene-editing tool due to its high activity and superior on-target specificities in ex vivo CAR engineering of HPSCs, NK cells, and T cells. The shorter gRNA for AsCas12a also presents a more economical way to promote this platform.

Metabolic reprogramming in immune cells themselves could also be an alternative approach to the improvement of in vivo persistence and antitumor activity. Zhu et al^[56] proposed that the deletion of cytokine inducible SH2 containing protein (CIS, encoded by the CISH gene) significantly improved expansion and antitumor activities in vivo. CISH^-/- (complete knockout) iPSC-NK cells exhibited delayed differentiation but almost the same mature phenotype as the primary NK cells. What is particularly interesting about the deletion of CISH is that the resulting iPSC-NK cells displayed increased sensitivity to interleukin-15 (IL-15), leading to better expansion and function than wild type iPSC-NK cells at low IL-15 concentrations. The advantages brought by the lower treatment doses of IL-15 are that they enable long-term administration of therapeutic NK cells with reduced systemic toxicity and better metabolic profiles during cryopreservation. Similar metabolic edits could be combined with CAR, hypoimmunogenic iPSCs, and immune checkpoint blockade to produce “all-in-one” cell products. Metabolic engineering against the immunosuppressive microenvironment also enhances the dynamics and therapeutic activities of CAR-equipped cells. Fultang et al^[57] demonstrated that CAR-T cells engineered to over-express functional argininosuccinate synthetase or ornithine transcarbamylase (the enzymes synthesizing arginine) were resistant to the low extracellular arginine concentration of the TME, significantly increasing cell proliferation, in vivo persistence, and antitumor efficiency without influencing the cytotoxicity.

Another important NK-cell-mediated killing effect, antibody-dependent cell-mediated cytotoxicity (ADCC), has also recently attracted attention. The same team generated a non-cleavable variant of CD16 (designated hnCD16), which binds to the Fc portion of IgG when attached to a target cell to mediate ADCC. The non-cleavable CD16 that cannot be removed from the cell surface of NK cells by a disintegrin and metalloprotease domain (ADAM) metallopeptidase domain 17 (ADAM17) endowed iPSC-NK cells with an enhanced ADCC response against tumor cells in the presence of mAbs.^[58]

Manipulations aiming to overcome the limitations of iPSC-derived immune cells are constantly innovating. Suicide genes, or the “off-switch” approach, were introduced to prevent the final products from forming teratomas caused by incorrect patterning and reprogramming factors.^[44,59] Notably, the heterogeneity across the different induced pluripotent stem cell lines due to the various morphologies, epigenetic expression,^[60,61] and the propensity of differentiation^[2] could also hamper the quality control. Factors that contribute to heterogeneity should be identified in the future.

The sequencing orders of operations in the generation of final products are also worth examining. The generation of CAR-expressing immune cells from CAR-iPSCs could serve as a more convenient way to produce any type of immune cell equipped with a specific CAR, while the production of CAR-expressing cells through derivation from iPSCs with subsequent transduction of the CAR construct circumvents the problem that CAR expression in iPSCs could influence endothelial-to-hematopoietic transition (EHT) and disturb immune cell differentiation.^[55] The corresponding protocols should be further explored.

The original cell source of iPSCs has been reported to play a role in the ultimate functionality of products. iPSCs derived from T cells (T-iPSCs) were reported to differentiate more efficiently into T cells than non-T-cell-derived iPSCs (non-T-iPSCs).^[62] Different subtypes of immune cells in the products also account for actual antitumor ability. A greater proportion of naïve T cells and memory T cells in the off-the-shelf product could possibly lead to better proliferation and more significant inhibition of tumors.^[63]

Conclusion

The advent of CAR engineering promoted the application of adoptive cell therapies. The great success of CAR-T cells in B cell malignancies has revealed the potential clinical significance of other CAR-based immunotherapies for not only hematological malignancies but also solid tumors. CAR-NK cells and CAR-macrophages have also been highlighted for their intrinsic killing capacity and sufficient infiltration into the TME, respectively. Despite the remarkable achievements of CAR therapies and CAR-based research platforms, the distinct drawbacks to current CAR-based therapeutic immune cells, such as limited ex vivo proliferation and in vivo persistence, unsatisfactory implementation conditions, and unaffordable costs, have hindered their function. The bottlenecks identified have catalyzed the development of “off-the-shelf” CAR-based therapeutic cells. The unique properties of rejuvenation and infinite multiplication in iPSCs provide a novel remedy for universal cell products. Previously, scientists established triple-homozygous iPSC cell line banks^[64] for matching as many patients as possible,^[65,66] but they still encountered obstacles due to the rarity of donors,^[67] and competition between banks.^[41] With the evolution of CRISPR-Cas technologies, synchronous edits for reducing immunogenicity and introducing CAR molecules into a single iPSC cell, combined with subsequent differentiation into any type of therapeutic cell, have become attractive approaches to generate a universal, “all-in-one” product for further commercialization of CAR-based immunotherapies. These strategies have been confirmed to be feasible, safe, and effective for T cells,^[3,68] NK cells,^[4] and macrophages^[33] in the laboratory, and they are undergoing clinical trials for the actual in vivo results.^[24] Although the complicated production could bring new problems with adverse mutations, relatively low efficiency, and possibly reduced antitumor activity compared with autologous CAR-equipped immune cells,^[69] the stability, standardized quality control, and the improvements in killing capacity could partially surmount these barriers. It should be emphasized that the long-term observation of these products is needed due to worries about the design itself, such as gene modification, the oncogenicity, and ethical issues with the grading of products. In the near future, promising studies on various types of cancers are worth anticipating since combining the aforementioned strategies with other immunotherapies, such as checkpoint blockade or even targeted photodynamic therapies,^[70] could inject new vitality into the current tumor treatments.^[31,34,35]

Acknowledgements

We thank all the faculty and staff in the Clinical and Laboratory Unit of the Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology for their clinical and technical support.

Funding

This study was supported by the Key Program of the National Natural Science Foundation of China (Nos. 81830008 and 81630006), the National Natural Science Foundation of China (No. 81570197), and the Natural Science Foundation of Hubei Province (No. 2018ACA140).

Conflicts of interest

None.