Making Potent CAR T Cells Using Genetic Engineering and Synergistic Agents

Abstract

Simple Summary

Although CAR T cells are regarded as a revolutionary drug in cancer treatment, however, it still has many limitations. To overcome the disadvantages of CAR T cell, many researchers have tried to optimize the CAR gene. In this review, I summarized the current studies regarding genetic engineering of CAR and synergistic agents to enhance the CAR T cell efficacy both in preclinical and clinical models.

Abstract

Immunotherapies are emerging as powerful weapons for the treatment of malignancies. Chimeric antigen receptor (CAR)-engineered T cells have shown dramatic clinical results in patients with hematological malignancies. However, it is still challenging for CAR T cell therapy to be successful in several types of blood cancer and most solid tumors. Many attempts have been made to enhance the efficacy of CAR T cell therapy by modifying the CAR construct using combination agents, such as compounds, antibodies, or radiation. At present, technology to improve CAR T cell therapy is rapidly developing. In this review, we particularly emphasize the most recent studies utilizing genetic engineering and synergistic agents to improve CAR T cell therapy.

1. Introduction

Researchers have tried to harness the immune system to attack tumors for several decades. The first attempt involved the use of heat-killed bacteria by William Coley, a surgeon [1]. After reading that tumor disappeared after a bacterial infection, he injected a mixture of heat-killed bacteria of species Streptococcus pyrogenes and Serratia marcescens, called Coley’s toxins, to sarcoma patients. Although his work remained controversial, he is regarded as the “Father of Cancer Immunotherapy” for his scientific contribution. In the 1980s, Rosenberg SA in NCI reported that tumor-infiltrating lymphocytes (TILs) were effective in eradicating tumors [2]. TILs were extracted from tumor lesions, expanded ex vivo, and then re-infused together with interleukin-2 (IL-2) into the patient. Although the manufacturing process is complicated, the clinical trial of TILs therapy is still active on melanoma [3].

In late 1980s, Eshhar Z in Weizmann institute suggested a fancy T cell model using an artificial receptor composed of a constant domain of the T-cell receptor fused to the variable domain of the antibody [4]. With the help of the variable domain of an antibody, these genetically modified T cells were redirected to antigen-expressing tumors. A chimeric receptor composed of a single chain fragment variable (scFv) and a signal transducing domain, which resembles the current first generation chimeric antigen receptor (CAR), was reported in 1993 [5]. In preclinical models, CAR T cells showed antigen-specific tumor elimination in vitro and in vivo [6,7,8,9]. In the early 2010s, complete remission with anti-CD19 CAR T cell therapy was reported in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL) [10,11]. The results were so overwhelming that researchers praised CAR T cell therapy with enthusiasm. Currently, two CAR T cell products, Kymriah and Yescarta, are approved by the US FDA for the treatment of pediatric ALL and adult large B-cell lymphoma (LBCL) [12]. However, it is still challenging to fabricate CAR T cells that work successfully on some types of leukemia, such as T cell lymphoma and acute myeloid leukemia (AML), and most solid tumors [13,14,15].

The CAR T cells currently under development have been engineered genetically to compensate for the weaknesses of existing CAR T cells. To overcome the obstacles of CAR T cell therapy, such as poor activity in solid tumors and severe side effects caused by activated CAR T cells, the current CAR constructs have been modified in multiple ways to express specific genes or to deplete immune-suppressive genes. In addition, compounds or radiation have been applied to CAR T cell therapy to synergize its effect. The ex vivo culture of CAR T cells has also been studied intensively to maximize the therapeutic effect of CAR T cells. This review is intended to provide a comprehensive overview for individuals who wish to invent potent CAR T cells using genetic engineering or combination agents.

2. CAR Construct

2.1. Basic Design of CAR

After molecular cloning of the CD3ζ clone [16], Eshhar et al. developed a CAR construct composed of a scFv domain fused to the CD3ζ domain [5]. This first-generation CAR T cell showed antigen-specific T cell activation in vitro (Figure 1A); however, the in vivo efficacy was modest [17]. The second-generation CAR construct was incorporated with CD28 domain and showed an enhanced ability to produce cytokine in CAR T cells (Figure 1B) [18]. Moreover, the proliferation of CAR T cell was improved by CD28-mediated signaling [19], and the cooperation of CD3ζ signaling and CD28 signaling improved CAR T cell activation [20,21]. In NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice models, CAR T cells with CD28 signaling had a much better antitumor effect than CAR T cells without CD28 signaling [22,23,24,25]. In clinical experiments, CD28 co-stimulation was proven to be important for T cell expansion and in vivo persistence in lymphoma patients [26]. In 2004, another costimulatory domain, 4-1BB, was introduced to CAR T cells, which enhanced the in vivo persistence of CAR T cells, leading to a superior antitumor effect in preclinical models [24,27]. In 2009, the June group first reported the third-generation CAR construct encoding both the CD28 and 4-1BB cytoplasmic domains as costimulatory domains (Figure 1C) [25]. After that, various third-generation CAR T cells, which utilized OX40 or inducible T-cell costimulatory (ICOS) as the second costimulatory domain, were developed [25,28,29,30,31,32,33,34]. Some reports claimed that third-generation CAR T cells persist longer than second-generation CAR T cells in vivo [35]. However, it is controversial whether the antitumor efficacy of third-generation CAR T cells is superior to second-generation CAR T cells. In a preclinical model, second-generation CAR T cells, including 4-1BB-based (bbz) or CD28-based (28z) CAR T cells, and third-generation CAR T cells, such as 4-1BB- and CD28-based CAR T cells (28bbz), showed a similar antitumor effect, whereas CAR T cells with only CD3ζ (z) showed much weaker effects than others [25]. However, other reports claimed that the combination of 4-1BB signaling and CD28 signaling enhanced the in vivo persistence of CAR T cells, leading to a superior anti-leukemic effect [33,34]. Researchers have also tried to reveal the best costimulatory domain for CAR T cell efficacy. 4-1BB co-stimulation of CAR T cells promotes longer persistence, greater ex vivo expansion, and better survival than CD28 co-stimulation [36]. In addition, 4-1BB signaling ameliorates T cell exhaustion by CAR tonic signaling, which is a signaling in an antigen-independent manner due to the CAR clustering, leading to a superior antitumor effect [37]. In an NSG mouse xenograft model, the ICOS-incorporated 4-1BB-based CAR construct—ICOS-BBz—showed a superior antitumor effect over BBz-based CAR construct by enhancing CAR T cell persistence [38]. Another study reported that CD28-based CAR T cells with 4-1BB exhibited higher in vivo efficacy than CAR T cells with OX40 [39]. Overall, it is regarded that CD28-based CAR T cells are CD45RO⁺ CCR7⁻, the same as effector memory T cells, and 4-1BB-based CAR T cells are CD45RO⁺ CCR7⁺, the same as central memory T cells. Therefore, 4-1BB CAR T cells are pushed toward a long-lasting central memory response, and CD28 CAR T cells are pushed toward a short-lived effector response [40,41]. However, in clinical studies comparing 28z and 4-1bbz, their antitumor effect was almost same, although bbz CAR T cells exhibited a more favorable safety profile than 28z [42,43]. Furthermore, it has been revealed that first-generation CAR (19z) T cells also eliminate the patient tumor as efficiently as second-generation CAR (1928z) T cells, even though second-generation CAR T cells persist for longer periods than first-generation CAR T cells in patients [44].

Figure 1.

Various constructs of chimeric antigen receptor. (A). First-generation CAR construct is composed of a single chain variable fragment fused with a CD3ζ endodomain. (B). Second-generation CAR construct has a costimulatory domain including CD28, 4-1BB, OX40, or ICOS. (C). Third-generation CAR construct has two costimulatory domains. (D). Coexpression of ligand of immune activating receptors, such as 41BBL or ICOSL-41BB, enhanced the anti-tumor effect of CAR T cell. (E). Recent report demonstrated that herpes virus entry mediator (HVEM) co-stimulatory signal domain (CSS) can be used for CAR constructs. (F). Myd88/CD40 signaling greatly potentiated the CAR activity in preclinical models. Myd88/CD40 signaling can be modulated by dimerization of FKBP12 F37V domain. (G). Toll-like receptor (TLR) domain and cytokine receptor domains (IL2Rβ or IL15Rα) were used as co-stimulatory domain to increase CAR T cell effect.

2.2. A Novel Design of CAR

Several groups have developed CAR T cells coexpressing ligand proteins of immune activating receptors, such as 41BB ligand (41BBL) or ICOS ligand (ICOSL), to produce costimulatory signalings (Figure 1D) [45,46]. Anti-CD38 CAR T cells expressing 41BBL exhibited a potent antitumor effect with improved proliferative capacity and enrichment of central memory T cells in an NSG xenograft model. [45] ICOSL or ICOSL-41BB expression also reinforced the expansion capability of CAR T cells and reduced the apoptotic events of CAR T cells [46]. A new costimulatory domain, the herpes virus entry mediator (HVEM) cytoplasmic domain, was incorporated into the CAR construct (Figure 1E) [47]. HVEM-based CAR T cell showed enhanced effector function, balanced differentiation into effector memory and central memory subsets, and reduced T cell exhaustion leading to superior antitumor effect in comparison to CD28-based or 41BB-based CAR T cells. The Spencer group used a completely different costimulatory domain, Myd88/CD40 (Figure 1F). Myd88 is the adaptor molecule for the IL-1 receptor family and Toll-like receptor (TLR) signaling. CD40 is a T cell costimulatory molecule found on antigen-presenting cells. They designed a remote control system to turn on the Myd88/CD40 signaling through a compound. Myd88/CD40 signaling enhanced the cytolytic activity and proinflammatory cytokine secretion of CAR T cells and reduced the surface expression of PD-1 on CAR T cells. As a result, CAR T cells with Myd88/CD40 signaling demonstrated a superior antitumor effect over CD28-based CAR T cells in an NSG muse model [48]. This system is also applicable to CAR-Natural Killer (NK) cells [49]. The Toll/interleukin-1 receptor (TIR) domain of the Toll-like receptor, which plays a significant role in the innate immune system, was also incorporated into the CAR construct, and it potentiated the CAR T cells antitumor efficacy both in preclinical and clinical trials (Figure 1G) [50,51,52]. Kagoya et al. invented a second-generation CAR construct encoding additional truncated IL2 receptor β-chain (IL2Rβ) and a signal transducer and activator of transcription proteins (STAT) 3/5 binding motif (YXXQ) at the C terminus of CD3ζ [53]. These CAR T cells produced all three signals, including T cell receptor (TCR), CD28, and cytokines, which are required for T cell activation. In preclinical models, these CAR T cells displayed increased proliferation and attenuated terminal differentiation, which leads to an enhanced antitumor effect. Nair et al. developed a CAR construct encoding signaling motif of IL15Rα [54]. IL15Rα co-signaling exhibited enhanced T cell expansion and cytotoxic activity.

2.3. Transmembrane and Hinge Domains of CAR

Recently, it was found that hinge and transmembrane (TM) domains are essential for CAR T cell activation. T cells expressing CARs with CD8α hinge and transmembrane domain produced fewer cytokines and exhibited lower levels of activation-induced cell death compared to those with CD28 hinge and transmembrane domain, resulting in enhanced in vivo efficacy [55]. A successful clinical trial was performed using CAR T cells with a hinge domain to produce low levels of cytokines, and high levels of anti-apoptotic molecules [56]. The hinge and transmembrane domains of CAR determine the CAR expression level, which correlates with antitumor efficacy [57]. Several groups have used the mutant CH2CH3 domain of IgG4 (L235E, N297Q) for the CAR hinge domain to escape FcγR recognition by the innate immune system [58,59]. Interestingly, ICOS transmembrane has a unique ability to promote association with lymphocyte-specific protein tyrosine kinase (Lck) and subsequent phosphatidylinositol 3-kinases (PI3K) activation. Based on this, ICOS TM was incorporated into the CAR construct, which facilitated the interaction between CAR T cells and target cells [60].

2.4. CAR Contructs Designed to Exhibit Reduced T Cell Activiation Lead to the Superior Antitumor Activity

Recent CAR T cell studies demonstrated that CAR T cells designed to exhibit reduced activation are likely to achieve excellent preclinical results. CD3ζ has three immunoreceptor tyrosine-based activation motifs (ITAMs), which contributes to the downstream signalings in T cells. Although ITAM-mediated signalings are critical for T cell activation, they can also trigger T cell differentiation and exhaustion [61]. Feucht et al. developed CAR T cells with only one ITAM in the CD3ζ domain, and these CAR T cells exhibited far superior preclinical results over those with three ITAMs in the CD3ζ domain by balancing memory and effector programs (Figure 2A) [62]. Guedan et al. found that changing asparagine of CD28 (YMNM) to phenylalanine (YMFM) promoted the antitumor activity of CAR T cells (Figure 2B) [63]. As asparagine is important for CD28 interaction with growth factor receptor-bound protein 2 (Grb2), which is responsible for the nuclear factor of activated T cells (NFAT) activation and IL-2 production, CD28 with YMFM resulted in reduced cytokine production, subsequently leading to reduced T cell differentiation and exhaustion. In a syngeneic mouse model, anti-CD19 CAR T cells expressing IL12 with only CD3ζ signaling were shown to be superior over IL12 expressing CAR T cells with both CD3ζ and costimulatory signaling (Figure 2C) [64]. Thus, it can be said that optimized CAR T cell activation is critical for excellent CAR T cell activity.

Figure 2.

CAR constructs designed to exhibit reduced T cell activation are likely to achieve enhanced preclinical results. (A). A CAR construct containing single ITAM in CD3ζ endodomain demonstrates excellent anti-tumor effect. (B). Defect in binding with Grb2 by mutant encoding YMFM instead of YMNM in CD28 endodomain reduced T cell exhaustion, which leads to excellent anti-tumor effect. (C). First-generation CAR T cells secreting IL12 is superior to second-generation CAR T cells.

2.5. Antigen-Binding Domains of CAR

The standard approach for the antigen binding domain in developing CAR T cell is to use single-chain variable fragment of monoclonal antibody. Murine scFvs were widely used in the early days of CAR T cell research, and both CAR T cell products approved by the US FDA—KYMRIAH and YESCARTA—use the same murine scFv, FMC63 (Figure 3A). However, cellular immunity specific to peptides of murine scFv, resulting in reduced expansion and suppressed antitumor effect of CAR T cells, has been reported in patients [65,66]. Moreover, murine scFv-based CAR T cells sometimes caused the development of anaphylaxis [67]. To avoid these disadvantages of murine scFv, fully human scFv, which can be generated by transgenic mice or phage display, or humanized scFv-based CAR T cells have been extensively studied (Figure 3B) [55,68,69,70,71,72,73,74]. CAR T cells with humanized scFv showed successful results in clinical trials [75,76,77,78]. Another approach for the antigen-binding domain is to use naturally existing ligands (Figure 3C). IL13Rα is known to be overexpressed in the majority of glioblastoma tumors, not in normal brain tissue [79,80]. IL13-based CAR T cells, which are capable of recognizing IL13Rα, showed excellent efficacy in preclinical models [81,82]. Intracranial infusions of IL13-based CAR T cells induced the regression of tumors in glioblastoma patients [83,84]. Another example includes granulocyte-macrophage colony-stimulating factor (GM-CSF)-based CAR T cells targeting CD116, GM-CSF receptor, for the curative treatment of juvenile myelomonocytic leukemia (JMML). These CAR T cells have been shown to inhibit the proliferation of JMML CD34⁺ cells, but not normal CD34⁺ cells [85]. CD70 CAR T cells are other examples of ligand-based CAR T cells [86,87,88]. IL11-based CAR T cells were found to be effective against IL11Rα overexpressing osteosarcoma cells in a mouse model [89].

Figure 3.

Various antigen binding domains. (A) In early days of CAR T cell research, murine scFv was used as antigen binding domain. (B) To avoid disadvantages of murine scFv, humanized scFv was used in recent research. (C) Natural ligands, such as IL13, GM-CSF, CD70, and IL11, can be used for antigen binding domain. (D) As chlorotoxin can bind to most primary glioblastoma cells, toxin-based CAR T cells were developed. (E) Designed ankyrin repeat proteins (DARPin), which are composed of repeating ankyrin motifs, can be used for antigen binding domain.

Wang et al. paved the way to the use of toxins for the antigen-binding domain. As chlorotoxin, which is a 36-amino acid peptide found in the venom of the deathstalker scorpion, binds specifically to glioblastoma cells, not normal cells, chlorotoxin-based CAR T cells were invented and studied for their anti-cancer effect (Figure 3D) [90]. It has been demonstrated that chlorotoxin binds to most primary glioblastoma tumors, whereas antibodies to Her2, IL13Rα2, or EGFR binds to a small population of primary glioblastoma tumors. Furthermore, an in vivo xenograft assay demonstrated that chlorotoxin-based CAR T cells mediated complete tumor eradication.

Designed ankyrin repeat proteins (DARPin) can also be used for the antigen binding domain (Figure 3E). DARPins, which are composed of repeating ankyrin motifs, are known to be small in size, thermodynamically stable, less immunogenic and less prone to aggregate compared to scFvs [91]. CAR T cells designed with anti-Her2 DARPin or DARPins specific for Her2, epidermal growth factor receptor (EGFR), and epithelial cell adhesion molecule (EpCAM) showed potent tumor regression in preclinical models [92,93,94].

3. Genetic Engineering to Express or Delete Specific Proteins

3.1. Expression of Specific Proteins

An enhanced antitumor effect can be achieved by expressing or depleting specific proteins in CAR T cells (Table 1). CAR T cells expressing telomerase reverse transcriptase (TERT) have been designed [95]. As shortening of telomeric DNA is correlated with cell senescence, CAR T cells expressing TERT exhibited prolonged in vivo persistence, and superior antitumor efficacy in NSG mice models. Another approach is to target the tumor microenvironment (TME). Heparanase, which is secreted by immune cells, is an important protein for degrading heparan sulfate proteoglycans, one of the main extracellular matrix components [96,97]. Heparanase expressing CAR T cells showed improved tumor infiltration, leading to enhanced antitumor activity [98]. Lynn et al. invented c-Jun expressing CAR T cells. They found that T cell exhaustion was associated with defects in IL-2 production, and that IL-2 production was driven by activator protein (AP-1) heterodimer, c-Fos-c-Jun. The c-Jun expressing anti-GD2 CAR T cells exhibited greatly enhanced antitumor activity [99].

Table 1.

Proteins co-expressed or depleted in CAR T cell.

Target Proteins	Method	Effect	Reference
TERT	Expression	Escaping cell senescence; Prolonged in vivo persistence	[95]
Heparanase	Expression	Degrading heparin sulfate proteoglycan; Improved tumor infiltration	[96,97,98]
JUN	Expression	Improved IL-2 production; Escaping T cell exhaustion	[99]
PD1 or PDL1 scFv	Expression	Suppressing the immune inhibitory signaling	[100,101,102]
sPD1	Expression		[103]
Chemokine receptors (CXCR1, CXCR2, CCR2, CCR4, CCR2b)	Expression	T cell’s trafficking; Enhanced trafficking to tumor lesion	[104,105,106,107,108,109,110]
Interleukins (IL12, IL18, IL15, IL21, IL23, IL36γ, IL7)	Expression	Enhanced tumor infiltration of CAR T cell; Modulate tumor microenvironment to increase M1 macrophage; Generate T-memory stem cell	[111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130]
NR4A	shRNA	Inhibition of PD1, TIM3 expression	[131]
Tet2	shRNA	Driving T cells into central memory phenotype	[132]
PTPN2/22	shRNA	Upregulation of TCR signaling by Lck phosphorylation	[133,134,135,136]
TGFβ signaling	SD-208 (kinase inhibitor)	Increasing CAR T cell viability & suppressing PD-1 expression	[137]
	KO of TGFβ receptor	Suppressed Treg conversion and CAR T cell exhaustion	[138]
	Expressing of dominant negative (DN) TGFβ receptor II	Enhanced cytokine secretion & Increased CAR T cell proliferation	[139]
Cholesterol esterification enzyme	shRNA	Elevated cholesterol level triggers the proliferation and cytotoxic activity of T cell	[140]
	inhibitor		[141]
DGK	Knockout	Reinvigorate the function of hypofunctional tumor-infiltrating immune cells	[142]
PD1	Knockout	Suppressed the immune inhibitory signaling	[143]
	PD1 decoy receptor		[144,145,146,147,148,149,150,151]
A2aR	shRNA	Increased CAR T cell efficacy	[152]

3.2. Depletion of Specific Proteins

Contrary to the above-mentioned approach, some researchers have achieved enhanced antitumor activity by depleting specific genes (Table 1). Chen J. et al. analyzed RNA sequencing data from CAR T cells and found that the nuclear receptor subfamily 4A (NR4A) is involved in the expression of the inhibitory receptors, programmed cell death protein 1 (PD1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3). CAR T cells depleted of three NR4A genes promoted tumor regression [131]. The fact that Tet methylcytosine dioxygenase 2 (TET2) depletion improves the antitumor efficacy of CAR T cells was discovered accidentally. In a clinical study, Fraietta et al. found that anti-CD19 CAR T cells in one leukemic patient, who experienced complete remission, originated from one single T cell in which the TET2 gene was disrupted by lentiviral vector-mediated CAR gene integration into 4q24 loci [132]. They proved that TET2 depletion drives CAR T cells into a central memory phenotype. Protein phosphatases were also targeted for depletion in CAR T cells. Protein tyrosine phosphatase N2 (PTPN2) is known to suppress TCR signaling by dephosphorylating Lck [133]. Depletion of phosphotyrosine phosphatase, non-receptor type 22 (PTPN22) in CD8⁺ T cells is known to enhance their capacity to eradicate tumors [134]. Enhanced tumor rejection was achieved by CAR T cells depleted with PTPN2 or PTPN22 [135,136]. As elevated levels of cholesterol triggers the proliferation and cytotoxic activity of CD8⁺ T cells, the cholesterol esterification enzyme was also studied to enhance CAR T cell activity [153,154]. The activity of the cholesterol esterification enzyme was suppressed by either knocking down or using the inhibitor [140,141]. As expected, CAR T cells lacking cholesterol acyltransferase 1 showed improved antitumor effects in preclinical models. Some researchers have focused on A2a receptor for combination therapy with CAR T cells. A2aR binds to adenosine and leads to increased production of intracellular cyclic adenosine monophosphate (cAMP), which suppresses T cell activation [155,156]. A2aR in CAR T cells was upregulated by stimulation with target cancer cells. A2aR depletion by shRNA, when combined with PD1 blockade, increased CAR T cell efficacy in a syngeneic mouse model [152].

3.3. Inhibition of TGF-β Signaling

As transforming growth factor-beta (TGF-β) is one of the important immunosuppressive cytokines, removing TGF-β signaling from CAR T cells could lead to enhanced antitumor efficacy (Table 1). TGF-β signaling in CAR T cell can be suppressed by using oncolytic adenovirus targeting TGF-β, genome-edited technology, expressing dominant-negative TGF-β receptor, or TGF-β inhibitors [137,138,139,157]. CAR T cells lacking TGF-β signaling showed an enhanced antitumor effect. SD-208, a kinase inhibitor that blocks TGF-β signaling, enhances the antitumor effect of CAR T cells by increasing the viability of CAR T cells and suppressing PD-1 expression [137]. Knocking out TGF-β receptor II (TGFBR2) reduced TGF-β signaling and suppressed T_reg conversion and exhaustion of CAR T cells [138]. Blocking TGF-β signaling by expressing dominant negative TGF-β receptor II enhanced cytokine secretion and increased CAR T cell proliferation [139].

3.4. Genome Editing Technology in CAR T Cell

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology is actively applied to CAR T cells to enhance antitumor activity. Contrary to retroviral integration, CRISPR-Cas9 technology enables the CAR gene to be targeted into a specific locus in the genome. Eyquem et al. targeted the anti-CD19 CAR gene to the T-cell receptor alpha chain (TRAC) locus through CRISPR/Cas9. The surface CAR expression of genome-edited CAR T cells is known to be regulated by antigen stimulation. In addition, these CAR T cells have relatively lower expression of immune inhibitory receptors, such as PD1, lymphocyte-activation gene 3 (LAG3), and TIM3. As a result, these TRAC-targeted CAR T cells exhibited superior antitumor efficacy over retrovirally encoded CAR T cells in a preclinical model [158].

Diacylglycerol kinase(DGK) inhibition is known to reinvigorate the function of hypofunctional tumor-infiltrating immune cells [159]. Based on this knowledge, Toolgen Inc. used CRISPR/Cas9 to develop anti-EGFRvIII CAR T cells depleted with DGK. These CAR T cells showed increased TCR signaling, leading to excellent efficacy in preclinical studies [142].

3.5. Inhibition of PD-1 Signaling

In genome-editing research of CAR T cells, immune suppressive receptors have been the most focused (Figure 4). PD-1 or Lag3 knockout CAR T cells by CRISPR-Cas9 exhibited excellent in vivo efficacy [143,160]. The PD1 expression level in CAR T cells has been regarded as a significant factor in determining the efficacy of CAR T-cell therapy. Clinical and preclinical studies have demonstrated that PD1 expression in CAR T cells is negatively associated with clinical outcome [161,162,163]. To downregulate the inhibitory signaling from PD1, some groups developed a smart system to use decoy PD1 receptors (Figure 4A). They made artificial proteins composed of the extracellular domain of PD1 fused to the cytoplasmic domain of CD28, which converted the immune suppressive signal to immune activating signal. Preclinical data showed that this decoy PD1 receptor enhanced the antitumor effect of CAR T cells by decreasing tumor-induced hypofunction and enhancing the tumor infiltration of CAR T cells [144,145,146,147]. The Barber and Zhu group developed chimera protein composed of PD1 extracellular domain fused to DNAX-activating protein 10 (Dap10) or natural killer group 2 member D (NKG2D) [148,149,150,151]. These PD1-based CAR T cells directly recognized and eradicated cancer cells expressing PD1 ligands, such as PDL1 or PDL2. PD1-based inhibitory chimeric antigen receptors have also been developed, in which the antigen-binding domain was fused with the PD1 or cytotoxic T lymphocyte antigen 4 (CTLA4) cytoplasmic domain to divert off-target response [164].

Figure 4.

Various approaches targeting PD1 signaling in CAR T cell. (A). Decoy receptors, composed of PD1 extracellular domain fused with CD28 or NKG2D endodomain, convert immune inhibitory signal to immune activating signal. (B). CAR T cell therapy combined with PD1 antibody demonstrated excellent anti-cancer effect in preclinical models. (C). CAR T cells secreting PD1 scFv or PDL1 scFv potentiates CAR T cell activity. (D). Depletion of PD1 protein by shRNA or CRISPR technology increased anti-cancer activity of CAR T cell.

It is well known that PD1 antibodies exhibit excellent clinical efficacy against numerous tumors [165]. Combination therapies of PD1 antibodies and CAR T cells have been actively studied in preclinical and clinical studies to achieve a synergistic effect (Figure 4B) [78,166,167,168,169,170,171,172,173]. Since PD1 expression in CAR T cells is elevated by PDL1 positive tumor cells, PD1 antibody treatment enhanced the antitumor activity of CAR T cells [167]. A case study showed a long term-response to combined PD1 antibodies and anti-CD19 CAR T-cell therapy in a patient with DLBCL [78]. In a humanized mouse model, PD-1 blockade by PD1 antibody showed a synergistic effect with CD28-based CAR T cells lacking the lck binding moiety [166]. PD-1 blockade increased the number of tumor infiltrating anti-EGFR variant III (EGFRvIII) CAR T cells leading to enhanced antitumor activity [171]. This synergistic effect can be achieved using different approaches. Synthetic biology technology has enabled the development of CAR T cells secreting PD1 antibody or PDL1 antibody (Figure 4C) [100,101,102]. In preclinical models, CAR T cells engineered to secrete PD1 scFv demonstrated superior antitumor efficacy compared to combined therapy with a checkpoint inhibitor and CAR T cells. Anti-Glypican 3 (GPC3) CAR T cells expressing a soluble PD1 protein showed a superior antitumor effect against hepatocellular carcinoma [103]. In addition, PD1 depletion by CRISPR/Cas9 or shRNA is an alternative method to achieve PD1 blockade (Figure 4D) [174,175,176,177,178,179,180]. In PD1-depleted CAR T cells, genes involved in DNA replication and cell proliferation were downregulated, whereas genes associated with cell metabolism were upregulated [174]. PD1 depleted CAR T cells produced elevated cytokines and exhibited superior cytotoxicity against PDL1-expressing cancer cells, leading to augmented antitumor activity [176]. A superior antitumor effect of CAR T cells has been obtained by knocking down triple genes, including PD-1, Lag-3, and TIM-3 [181]. Triple inhibitory receptor knockdown upregulated CD56, which contributes to cell–cell adhesion through homophilic interaction and enhanced tumor infiltration, IFNγ secretion, and survival of CAR T cells. PD1 expression can be modulated by targeting glycosylation, because glycosylation of PD1 is indispensable for PD1 stability in cells. It has been demonstrated that CAR T cell cytotoxicity is enhanced by inhibiting PD1 glycosylation [182].

3.6. Chemokine Receptors

Chemokine receptors have also been studied intensively for developing enhanced CAR T cell therapy. It is well known that tumor cells or stromal cells in the tumor microenvironment secrete chemokines such as C-C motif chemokine ligand 2 (CCL2), C-X-C motif chemokine ligand 1 (CXCL1), CXCL2, and CXCL5 [183,184,185]. As chemokines play a key role in T cell trafficking, chemokine receptors have been speculated to contribute to T cell recruitment into the tumor. Several preclinical studies have demonstrated that CAR T cells expressing chemokine receptors, including C-X-C motif chemokine receptor 1 (CXCR1), CXCR2, CCR2, CCR4, and C-C chemokine receptor type 2 (CCR2b), showed enhanced trafficking to tumor lesions and superior antitumor efficacy (Table 1) [104,105,106,107,108,109]. The chemokine receptor, such as CXCR1, also enhanced the trafficking of CAR-NK cells to tumor lesion, subsequently augmenting the anti-tumor response [104]. Jin et al. published an interesting report that CAR T cell trafficking to tumor was enhanced by local X-ray irradiation. As X-rays induced IL-8 cytokine secretion from tumor cells, CAR T cells expressing CXCR2, which is a receptor for IL-8, migrated to tumor lesions irradiated by X-ray, leading to enhanced antitumor effects [110].

3.7. Interleukins

Cytokines are the most actively studied proteins in CAR T-cell research. Researchers found that cytokine-secreting CAR T cells exhibited a much superior antitumor effect to normal CAR T cells. These CAR T cells are referred to as TRUCKs, which means T cells redirected for antigen-unrestricted cytokine-initiated killing. The cytokines utilized in TRUCKs are IL12 [111,112,113,114,115,116,117,182], IL18 [118,119,120], IL15 [49,121,122,123], IL21 [124], IL23 [125], IL36γ [126], and IL7 [127] (Table 1). IL12 is known to play a key role in bridging innate and adaptive immunity. In both syngeneic and NSG xenograft mice models, IL12 secreting CAR T cells exhibited enhanced tumor infiltration, expansion, and longer persistence to prolong survival. IL18, which is produced by macrophage, stimulates interferon gamma (IFNγ) secretion in T cells. IL18-secreting CAR T cells not only exhibited long persistence and enhanced in vivo expansion, but also modulate the tumor microenvironment to increase M1 macrophages and NK cells and decrease suppressive CD103⁺ dendritic cells and M2 macrophages. IL15 is known to be crucial for generating T-memory stem cells, and its signaling is mediated by membrane-bound complex IL15/IL15Rα (mbIL15). MbIL15 coexpression increased the population of CD45RO^negCCR7⁺CD95⁺, which is similar to T_scm, to enhance the antitumor effect through the long-term persistence of CAR T cells [122]. In tumor growth, the role of IL-23 is controversial. Recent studies have reported that IL23 is upregulated in tumors to enhance angiogenesis and reduces CD8 T-cell infiltration [128,129]. Prostate-specific membrane antigen (PSMA)-targeted CAR T cells expressing an IL23 antibody eradicated prostate tumor in a preclinical model. [130]. On the contrary, IL-23 engineered CAR T cells showed a promising result in solid tumor models [125]. CAR T cells secreting IL-36γ showed enhanced expansion and prolonged in vivo persistence to produce superior tumor eradication compared to conventional CAR T cells [126]. IL-7 improved the CAR T cells infiltration and survival in the tumor lesion [127].

3.8. Artificial Cytokine Receptors

Recombinant cytokines produced by CAR T cells might cause significant toxicity in the body. To avoid side effects caused by excessive cytokines, several groups developed CAR T cells expressing artificial cytokine receptors [186,187]. As IL7 signaling primarily promotes T cell survival, Shum et al. developed a constitutively active IL7 receptor (C7R) composed of a CD34 extracellular domain fused to IL7Rα transmembrane and endodomain with an inserted disulfide bridge (Figure 5A). C7R expressing CAR T cells induced STAT5 signaling to significantly enhance antitumor effect in vivo [186]. By using the chimeric cytokine receptor, CAR T cells can invert immunosuppressive signaling to activate T cells [188,189]. Mohammed et al. invented 4/7 ICR composed of IL-7 receptor endodomain fused to IL-4 receptor exodomain (Figure 5B). 4/7 ICR convert immune-suppressive IL4 signaling to immune-stimulatory IL7 signaling. CAR T cells expressing 4/7 ICR showed superior antitumor effects in vivo under immune suppressive conditions. Tan et al. invented a CRS protection system by co-expressing non-signaling membrane-bound IL6 receptor (mbaIL6) in CAR T cells [190]. IL6 is known to be central to CRS pathogenesis. MbaIL6 is composed of scFv of the IL6 antibody fused to a transmembrane anchoring peptide (Figure 5C). In vitro and in vivo experiments have demonstrated that CAR T cells expressing mbaIL6 remove IL6. Thus, it is anticipated that CAR T cells expressing mbaIL6 prevent cytokine release syndrome (CRS) without a significant loss of anticancer activity.

Figure 5.

Artificial cytokine receptors potentiate CAR T cell activity. (A). C7R, which is composed of CD34 extracellular domain fused to IL7Ra transmembrane and endodomain with inserted disulfide bridge, produces active STAT5 signaling in CAR T cell. (B). 4/7 ICR, which is composed of IL7 receptor endodomain fused to IL4 receptor exodomain, convert immune-suppressive IL4 signaling to immune-stimulatory IL7 signaling. (C). mbaIL6, which is composed of IL6 scFv fused with transmembrane anchoring peptide, depleted IL6. Thus, it is anticipated that CAR T cells expressing mbaIL6 might prevent CRS.

4. Synergistic Effects of CAR T Cell Therapy with Compounds

4.1. Compounds Enhancing CAR T Cell Activity

Many attempts have been made to enhance CAR T cell activity by using small molecules. Several studies have shown the synergy effects of small molecules, including tyrosine kinase inhibitors, cytotoxic agents, histone deacetylase inhibitors, proteasome inhibitors, and immune-modulators, with CAR T cells (Table 2) [191,192,193,194,195,196,197,198]. Afatinib, a tyrosine kinase inhibitor, increased CAR-NK cell infiltration into the tumor [192]. The addition of lenalidomide, an immunomodulatory drug, to CAR T cells cultured ex vivo increased cytotoxicity, cytokine production, and memory phenotype of CAR T cells [193]. Bortezomib, a proteasome inhibitor, augmented the CAR T cell’s antitumor effect both in vitro and in vivo [195]. In a model of Ewing sarcoma, which is characterized by the aberrant expression of hepatocyte growth factor (HGF), AMG102, an HGF receptor neutralizing antibody, improved the antitumor activity of anti-GD2 CAR T cells by increasing CAR T cell trafficking into the tumor mass [196]. Myeloid-derived suppressor cells (MDSC) targeting compounds have been well investigated to enhance immunotherapy. MDSCs are immunosuppressive cells that suppress the immune cells in TME and also inhibit the expansion of CAR T cells [199]. It has been reported that the antitumor effects of immunotherapy using immune checkpoint antibodies, such as PD1 ab or CTLA-4 ab, were enhanced by suppressing MDSC [200,201,202]. CAR T cell activity is also enhanced by MDSC targeting agents [203,204,205,206]. Polyinosinic-polycytidylic acid diminished the suppressive activity and cell number of MDSCs in tumor-bearing mice [203]. In an NSG mouse model, all-trans retinoic acid (ATRA) eradicated monocytic MDSCs, whose expansion was induced by sarcoma and the antitumor activity of anti-GD2 CAR T cells [204]. For CD33 is overexpressed in MDSCs of cancer patients, combination therapy with gemtuzumab ozogamicin, a CD33 antibody conjugated with a cytotoxic drug from the class of calicheamicins, increased the antitumor activity of CAR T cells by depleting MDSCs [205]. Pharmacological targeting of A2aR improved CAR T cell efficacy [152,207,208]. SCH58261, an A2aR antagonist, elevated the proliferation rate and cytokine production of anti-CD19 CAR T cells [207]. As glycogen synthase kinase 3 (GSK3) is known to be inhibited by phosphorylation at the residue of Ser9 when T cells are activated [209,210], the effect of GSK3β inhibition on CAR T cell activity was investigated. The GSK3β inhibitor SB216763 increased T cell expansion and reduced exhaustion by downregulating PD1 and FasL expression in anti-IL13Rα CAR T cells [211].

Table 2.

Compounds enhancing CAR T cell activity.

Compounds	Function	Effect	Reference
Afatinib	Tyrosine kinase inhibitor	Increase NK cell infiltration into tumor	[192]
Lenalidomide	Immunomodulatory drug	Increase cytotoxicity, cytokine production of CAR T cell	[193]
Bortezomib	Proteasome inhibitor	Augment anti-tumor effect of CAR T cell	[195]
AMG102	HGF receptor neutralizing antibody	Increase CAR T cell trafficking into tumor	[196]
pI:pC (polyinosinic-polycytidylic acid)	Immunostimulation by interacting with toll-like receptor 3	Diminish suppressive activity and cell number of MDSC	[203]
ATRA (All-Trans Retinoic Acid)	Agonist for the retinoic acid receptors	Eradicate monocytic MDSC	[204]
Gemtuzumab ozogamicin	CD33 antibody conjugated with calicheamicins	Remove MDSC	[205]
SCH58261	A2aR antagonist	Elevate the proliferation rate and cytokine production of CAR T cell	[207]
SB216763	GSK3β inhibitor	Downregulating PD1 and FasL expression	[211]
MK2206	Akt inhibitor	Increase CAR expression; Induced CD62L expressing central memory phenotype	[212]
AKTi			[213]
JQ1	Bromodomain inhibitor	Maintain T cells with central memory and stem cell like phenotype	[214]
IPI-549	PI3Kγ inhibitor	Exhibit potent antitumor activity	[215]
CAL-101	PI3Kδ inhibitor
Combretastatin A-4 phosphate (CA-4)	Vascular disrupting agent	Anti-angiogeneic agent; Improve the infiltration of CAR T cell into solid tumor	[216]
Bevacizumab	VEGF-a antibody		[217]
Ibrutinib	BTK/ITK inhibitor	Enhanced one-year PFS; Reduced the serum level of CRS-associated cytokines	[218,219]
Dasatinib	ABL inhibitor	Complete remission in CML patient treated with CD19 CAR T cell	[220]
Decitabine	DNA methyltransferase inhibitor	Complete remission in B cell lymphoma patients treated with CD19 CAR T cell	[221]

4.2. Usage of Compounds for T Cell Ex Vivo Culture

Compounds can be used to induce ex vivo cultured T cells into undifferentiated T cells. It has been shown in preclinical and clinical studies that a high percentage of central memory CAR T cells leads to excellent antitumor efficacy (Table 2) [41,44,222,223]. Ex vivo treatment with PI3Kγ or PI3Kδ inhibitors, bromodomain and extraterminal (BET) proteins inhibitor, AKT inhibitor, or metformin has elevated the levels of CAR T cells with memory phenotype, leading to improved T cell expansion and enhanced antitumor potency [212,213,214,215,224,225,226,227]. The additions of MK2206 or AKTi, an Akt inhibitor, into CAR T cell culture increased CAR expression and induced a CD62L-expressing central memory phenotype without suppressing proliferation of CAR T cells [212,213]. Through screening of various inhibitors of epigenetic targets, JQ-1, a BET inhibitor, was found to maintain T cells with functional properties of central memory and stem cell-like T cells [214]. As a result, CAR T cells cultured in the presence of JQ-1 showed enhanced antitumor activity. T cells that were cultured in the presence of either PI3Kγ or PI3Kδ inhibitor exhibited potent antitumor activity, while those with the inhibition of both PI3Kγ and PI3Kδ were functionally impaired [215].

4.3. Anti-Angiogenic Agents for CAR T Cells

To improve CAR T cell efficacy in solid tumors, anti-angiogenic agents were combined with CAR T cells (Table 2) [216,217]. Anti-angiogenic agents promote leukocyte interaction, leukocyte extravasation, and tumor infiltration [228,229]. In combination with bevacizumab, a vascular endothelial growth factor (VEGF)-A antibody, anti-GD2 CAR T cells were massively infiltrated into the tumor mass to inhibit tumor growth in an orthotopic xenograft mouse model [217]. Combretastatin A-4 phosphate, a vascular disrupting agent, also significantly improved the infiltration ability of CAR T cells into solid tumors [216].

4.4. Safety System to Block the Side Effects of CAR T Cells

Due to severe side-effects, such as cytokine release syndrome (CRS), neurotoxicity, and on-target off-tumor effect, safety systems to block these side-effects were demanded (Table 3). The Spencer group invented a smart safety system composed of an artificial protein and a compound. This system consists of a dimerization inducer and an artificial protein, which is a caspase-9 protein tagged with mutant FK506 binding protein 12 (FKBP12) (F37V) [230]. Rimiducid, a dimer of the mutant FKBP12 ligand, induces the dimerization of caspase-9, leading to CAR T cell apoptosis [231]. This system has been proven successful in CAR T cell clinical trial [121]. Contrary to the inducible caspase 9 (iCasp9) system, there are several ‘reversible’ systems. In these systems, CAR T cell activity can be modulated ‘reversibly’ by compounds or peptides. A couple of mutant FKBP12 (mFKBP12) bearing polypeptides—scFv-CD28-mFKBP12 and mFKBP12-CD3ζ—were expressed in T cells. In this system, scFv-CD28-CD3ζ can be formed by a small molecule, rimiducid, so CAR T cell can be active only in the presence of rimiducid [48,232,233,234,235,236,237]. The proteolysis-targeting chimera (PROTAC) system can be used to modulate CAR protein levels by fusing CAR construct to bromodomain [238]. In this system, CAR T cell activity can be modulated by the PROTAC compounds of bromodomain, ARV-771 and ARV-885. In the presence of PROTAC compounds, CAR protein is degraded by the ubiquitin-proteasome pathway. Cellectis Inc. reported a CAR construct containing protease and degron domain [239]. In the absence of a protease inhibitor, asunaprevir, the degron moiety is cleaved from the CAR by protease. However, in the presence of asunaprevir, the degron domain is not cleaved from the CAR, so the CAR protein is degraded by proteolytic pathways. Cytochrome P450 family 4 subfamily B member 1 (CYP4B1) can be used as a CAR T cell suicide system. Optimized human CYP4B1, in which serine at position 427 is mutated to proline, converts prodrug 4-ipomeanol to a cytotoxic alkylating agent [240]. CAR T cells coexpressing CYP4B1 are eradicated by 4-ipomeanol. In the case of the folate receptor, we can eliminate folate receptor-overexpressing cancer cells with folic acid linked to fluorescein isothiocyanate (FITC) and anti-FITC CAR T cells [241,242]. TO-207, an mRNA 3′-end processing antagonist, blocks the side-effects of CAR T cells by inhibiting pro-inflammatory cytokine production in monocytes [243]. Dasatinib, a multi-kinase targeted inhibitor, was introduced as an efficient agent to control CAR T-cell activity [244,245]. Dasatinib downregulates CD3ζ signaling by suppressing lymphocyte-specific protein tyrosine kinase (lck). In a mouse model of CRS, dasatinib was able to block CRS by halting CAR T cell’s cytolytic activity and cytokine production.

Table 3.

Compounds and artificial genes used in safety system for CAR T cell therapy.

Compounds Plus Genes	Mechanism	Effect	Reference
TO-207	mRNA 3′-end processing antagonist	Block cytokine production in monocyte	[243]
Dasatinib	Multikinase targeted inhibitor	Block CRS by suppressing lymphocyte-specific protein tyrosine kinase (lck)	[244,245]
Rimiducid plus iCasp9	Dimer of mutant FKBP12 (F37V) ligand	Induce dimerization of caspase-9 leading to apoptosis	[48,231,232,233,234,235,236,237]
ARV-771 plus bromodomain containing CAR	PROTAC compound against bromodomain	Induce degradation of CAR protein leading to suppress the activity of CAR T cell	[238]
Asunaprevir plus CAR containing protease and degron	Protease inhibitor	Induce degradation of CAR protein leading to suppress the activity of CAR T cell	[239]
4-ipomeanol plus optimized CYP4B1	prodrug	Mutant CYP4B1 converts 4-ipomeanol to cytotoxic alkylating agent.	[240]

4.5. Compounds Inducing Antigen Expression

Compounds can induce a synergistic effect with CAR T cells by increasing target antigen expression (Table 4) [246,247,248,249,250,251,252,253]. Inhibitors of epigenetic targets, such as histone deacetylase, histone-lysine N-methyltransferase, and DNA methyltransferase, have been shown to upregulate target antigens. Sodium valproate, a histone deacetylase inhibitor, upregulated NKG2DL expression in ovarian cancer cells to enhance the lytic activity of NKG2DL-specific CAR T cells. [246] Enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) inhibition upregulated GD2 expression in Ewing sarcoma [247]. The expression of CD20 on B-cell leukemia or CD38 in adult T-cell leukemia is induced by histone deacetylase inhibitor or all-trans retinoic acid [248,249]. Decitabine, a hypomethylating agent, induced strong upregulation of NY-ESO-1, a cancer/testis antigen, to enhance the efficacy of NY-ESO-1-directed immunotherapy with CAR T cells [251,254]. Several protein kinase inhibitors have been reported to upregulate specific membrane proteins. Crenolanib, an fms-related tyrosine kinase 3 (FLT3) inhibitor, increased FLT3 expression specifically in FLT3-ITD⁺ AML cell lines, and enhanced the antitumor activity of anti-FLT3 CAR T cells [255]. Sunitinib, an inhibitor of multi-tyrosine kinase, has been shown to upregulate the expression of carbonic anhydrase IX (CAIX) and enhanced the efficacy of anti-CAIX CAR T cells against renal cancer [256]. Bryostatin, a protein kinase C modulator, was shown to upregulate CD22 to improve anti-CD22 CAR T cell efficacy [250,253].

Table 4.

Compounds upregulating specific antigens.

Compounds	Target Proteins	Reference
Sodium valproate	NKG2DL	[246]
Ezh2 inhibitor	GD2	[247]
Histone deacetylase inhibitor	CD20	[249]
ATRA	CD38	[248]
Decitabine	NY-ESO-1	[251,254]
crenolanib	FLT3	[255]
sunitinib	CAIX expression	[256]
Bryostatin	CD22	[250,253]

4.6. Usage of Vaccines to Enhance CAR T Cell Activity

Vaccines have been used to enhance CAR T cell activity in solid tumors. Vaccination targeting gp100, varicella zoster virus (VZV), Epstein–Barr virus (EBV), or Wilms tumor 1 (WT1) has been shown to enhance the antitumor activity of CAR T cells [257,258,259,260]. Injection of live recombinant vaccinia virus encoding gp100 significantly enhanced the antitumor effect of anti-Her2 CAR T cells which were manufactured with gp100-specific T cells. Vaccination with dendritic cells pulsed with antigen enhanced the antitumor effect in a xenograft model. In a clinical trial, anti-CD19 CAR T cells from EBV-specific T cells were administered to pediatric ALL patients with vaccination using EBV-transformed lymphoblastoid cell lines. The clinical results showed that the persistence of CAR T cells with vaccination was enhanced. Some groups have developed special agents to induce vaccine boosting. Ma L. et al. developed an amphiphile CAR-T ligand, which consists of an albumin-binding domain and a target peptide or protein domain [261]. This vaccine is trafficked to the lymph node due to albumin, and the target peptide is displayed on the surface of antigen presenting cells, such as dendritic cells, by which CAR T cells in lymph nodes are boosted. In a preclinical study of Reinhard, K. et al., liposomal antigen-encoding RNA, mRNA of claudin6 (CLDN6-LPX), was administered to mice, which led to the surface expression of claudin6 on DCs, and subsequent activation of CAR T cells [262].

4.7. Radiotherapy Combined with CAR T Cell Therapy

Recently, several groups have reported the synergistic effect of CAR T cells and radiation therapy in preclinical models [263,264,265]. In a clinical trial, radiation therapy prior to CAR T cell therapy reduced the side effects of CAR T cells and enhanced the overall response [266,267,268]. Interestingly, patients who experienced a relapse after CAR T cell therapy achieved complete remission with radiotherapy [266,269,270].

4.8. Compounds Used in Clinical Trials of CAR T Cells

In clinical trials, several compounds have been proven to exhibit synergistic effects with CAR T cells (Table 2) [218,219,220,221]. The clinical trial with ibrutinib, a dual inhibitor against bruton tyrosine kinase (BTK) and IL-2-inducible T cell kinase (ITK), has shown the improvement of T cell number and T cell function in CLL patients treated with ibrutinib for more than one year [271]. In addition, ibrutinib prevented CRS after CD19 CAR T cell treatment in a preclinical mouse model [272]. In patients with relapsed or refractory CLL, combination CAR T cell therapy with ibrutinib improved the probability of one-year progression-free survival and reduced the serum levels of CRS-associated cytokines [219]. A case study of a 56-year-old man with lymphoid blast phase chronic myeloid leukemia (CML) harboring the T315I mutation in the BCR-ABL fusion gene demonstrated that anti-CD19 CAR T cell therapy following dasatinib treatment induced complete remission [220]. Patients with B cell lymphoma treated with a combination of decitabine and anti-CD19 CAR T cells achieved complete remission [221].

5. Manufacturing Process/Ex Vivo Culture Method

The manufacturing method is critical for the success of CAR T cell therapy. The first step for CAR T cell generation is to collect peripheral blood mononuclear cells by leukapheresis [273]. In this step, the presence of monocytes suppresses the expansion of T cells [199,274]. In addition, T cells from healthy donors significantly expand and express less exhaustion markers compared to T cells from leukemic patients [162]. Moreover, the patients treated with cumulative chemotherapy cycles have low expansion potential of T cells [275]. After T cell purification, T cell activation and expansion are typically accomplished using anti-CD3/CD28 antibody-coated beads. CAR T cells expanded with anti-CD3/CD28 dynabeads demonstrate an enhanced antitumor effect in preclinical models compared to CAR T cells cultured with soluble CD3 antibody and high-dose IL-2 [276]. Several groups have used artificial antigen-presenting cells (aAPCs) expressing stimulatory receptors for T-cell expansion. Typically, irradiated K562, which is genetically modified to express OKT3 (CD3 antibody) and CD28 ligand (CD80 or CD86), has been used. CAR T cells expanded with aAPCs have shown decreased T cell exhaustion and superior antitumor activity in preclinical models compared to bead-activated CAR T cells [277,278]. The use of aAPCs platform to expand CAR T cells has been proven to be safe and successful in clinical trials [279]. IL-2 or IL7/IL15 are mostly used in CAR T cell manufacturing processes [280]. Several studies have reported that IL7/IL15 preferentially induced differentiation of CAR T cells into stem cell memory T cells (T_SCM), while IL2 induced the differentiation of effector memory T cells (T_EM) [39,162,281,282,283]. A high percentage of T_SCM by IL7/IL15 led to enhanced in vivo persistence and elevated antitumor effects in mice models. However, it is still controversial because some data demonstrated that IL7/15 is not superior to IL2 for the generation of less-differentiated subsets [284]. After two to three days of T-cell activation, the CAR gene is transduced into T cells. The most popular method to transduce the CAR gene into T cells is to use viral vectors, including lentivirus or gamma-retrovirus [285]. Due to the safety issues associated with viruses and the high cost of preparing viral particles, non-viral gene transfer methods such as sleeping beauty transposon/transposase system are employed for CAR T cell manufacturing [286,287,288,289]. Clinical trials of CAR T cell therapy with this transposon system yielded encouraging results [279,290]. Ex vivo culture time is also important for CAR T-cell function. Many reports have demonstrated that short-term culture improved antitumor effects by increasing the percentage of memory T cells and by reducing the exhaustion markers, including PD1, and TIM3 [291,292,293,294,295]. It is well known that T cells cultured ex vivo from non-responders to CAR T cell therapy exhibit high expression of PD1, TIM3, and LAG3 [296]. As most CAR T cell products are administered to patients as frozen, cryopreservation is an important step for maintaining a high quality of CAR T cells. Several studies have demonstrated that there is no significant difference in overall T cell function between cryopreserved CAR T cells and fresh CAR T cells [297,298,299]. Although the cytokine levels are slightly decreased in cryopreserved CAR T cells, antitumor function is well retained [300].

6. Conclusions

Although CAR T cells have shown clinical successes in hematological cancer, translating these successes to solid tumors remains a challenge. Recent studies have focused on ways to overcome the current drawbacks of CAR T cell therapy. Through the understanding of the biology of CAR T cells, many genes or signaling pathways have been revealed to enhance or decrease CAR T cell efficacy. CAR T cells genetically engineered to harness or deplete these genes or signalings have shown significantly improved antitumor effects. Combination therapy with compounds, antibodies, radiation, or vaccines has also been shown to be effective for CAR T cell therapy. However, it is still limited to using CAR T cell therapy in clinical trials for solid tumors or hematological cancers other than B cell malignancies. One of the most important issues in CAR T cell therapy against solid tumor is to uncover the tumor specific antigen. Unlike the therapy against B cell malignancies, on-target off-tumor side effects could cause a fatal problem in CAR T cell therapy for a solid tumor. One of the resolutions for this issue is to use oncolytic virus to deliver the antigen specifically to tumor cells [301]. CAR-Macrophage can be another resolution to attack solid tumor because macrophage cells are able to present the tumor antigen in an MHC-restricted manner, which boosts the adaptive immune response against tumor [302]. As the manufacturing process of autologous CAR T cell is so complicated, substantial efforts to develop allogeneic CAR T cells have been devoted. The clinical trial with allogeneic CD19 CAR T cells combined with TCRα knock out engineered T cell has shown promising result without severe graft-versus-host disease (GVHD) [303]. In addition, off-the-shelf CAR NK cell therapy has shown impressive clinical result in B-cell hematological cancer [304]. The problems of the complicated manufacturing process of CAR T cell could be solved if these cell therapies were available.

We have to admit that we still have numerous hurdles to improve CAR T cell therapy. However, we are certainly moving forward step by step to overcome these hurdles by using genetic engineering and synergistic agents. In the future, these technical advances will make it possible to conquer the cancer through CAR T cell therapy.

Author Contributions

Literature search, C.H.P.; Writing and image generation, C.H.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Korea Research Institute of Chemical Technology (BSK20-402).

Conflicts of Interest

The author declares no conflict of interest.