Optimization of CAR‑T Cell-Based Therapies Using Small-Molecule- Based Safety Switches
Yanjun Zheng, Kutty Selva Nandakumar, and Kui Cheng*

ACCESS
Metrics & More
Article Recommendations

⦁ INTRODUCTION
Anticancer therapy for pre-B cell acute lymphoblastic leukemia (ALL) or B cell non-Hodgkin lymphoma refractory to standard treatments has been transformed since the U.S. Food and Drug Administration approved anti-CD19 chimeric antigen receptor T cell (CAR-T cell) products as treatments for hematologic malignancies of the B-cell lineages.1,2 Cells frequently used in adoptive T cell therapies include tumor-infiltrating lympho- cytes (TILs), T cells expressing engineered T cell receptor (TCR-T cells), and CAR-T cells. Among them, CAR-T cell therapy is a promising treatment option with high response rates against hematological malignancies.3−5 CAR-T cells are genetically engineered to express a class of synthetic receptors. A chimeric antigen receptor (CAR) usually consists of a single- chain fragment variant (scFv), a CD3ζ region, and one or two costimulatory domains.6 The scFv derived from the variable light- and heavy-chain regions of a monoclonal antibody used in this therapy specifically recognizes a tumor membrane protein. The CD3ζ endodomain includes immunoreceptor tyrosine-based activation motifs (ITAMs), which are required for signal transduction for T cell activation. Costimulatory domains provide additional costimulatory signals required for
T cell activation. As shown in Figure 1, compared with unmodified T cells, CAR-T cells recognize surface-exposed tumor-associated antigens (TAAs) more specifically and are activated to eliminate tumor cells without any major histocompatibility complex (MHC) restriction.7
CARs have evolved through several generations of structural design refinement. The architectural differences found in
several generations of CARs are illustrated in Figure 1. The first-generation CARs were genetically engineered to combine only an scFv with a CD3ζ endodomain.8 However, the efficacy of these CARs was limited because of insufficient CAR-T cell expansion, proliferation, and survival due to the absence of costimulatory signals and insufficient cytokine production.9 To overcome these limitations, the second-generation CARs were developed by incorporating costimulatory domains such as CD2810 or CD137 (4-1BB),11 which provide additional signals to CAR-T cells. This improvement increased their proliferation and secretion of cytokines and delayed CAR-T cell death.12 Chimeric receptors encompassing multiple costimulatory endodomains, such as CD28 and 4-1BB13 or CD28 and OX40,14 were modifications implemented with the third- generation CARs, which are expected to have improved potency for cytokine release and cytolytic capacity. Most of the third-generation CARs achieved their intended targets, although a few studies have shown some unexpected results.15 The fourth-generation CARs incorporate an additional trans- gene for the secretion of stimulatory cytokines. One example of fourth-generation CARs was termed “T cells redirected for

Downloaded via UNIV OF GLASGOW on August 12, 2021 at 00:35:38 (UTC).
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
© 2021 American Chemical Society

9577

https://doi.org/10.1021/acs.jmedchem.0c02054

J. Med. Chem. 2021, 64, 9577−9591

Figure 1. Schematic representation of the differences between conventional CD8+ T cells and CD8+ CAR-T cells in killing tumor cells and the architectural evolution of CARs. Conventional CD8+ T cells specifically recognize the peptide−MHC class I (pMHC-I) complex on the surface of APCs through the TCR, which transduces activation signals into the intracellular compartment through CD3, enabling the initial activation of T cells. By recognizing tumor cells through an artificially introduced single-chain fragment variant (scFv), CD3ζ domain, and costimulatory domain, CAR-T cells are activated and kill tumor cells independent of MHC restriction. The structural differences of several generations of CARs are mainly located in the transmembrane and intracellular domains. The intracellular domain of the first-generation CAR lacked the costimulatory signal domain and contained only the CD3 domain. The second-generation CARs were equipped with only one of the CD28 or 4-1BB costimulatory domains to provide the second signal for the activation of CAR-T cells. The third-generation CAR structures had two costimulatory domains. The fourth-generation CAR structure may be designed to express separate cytokine transgenes (e.g., IL-12). The fifth-generation CAR is yet to be developed; it is not yet clear whether it specifically refers to the approach incorporating a truncated cytoplasmic domain of IL-2 receptor β and the transcription factor STAT3-binding motif. Abbreviations: APC, antigen-presenting cell; Costim, costimulatory domain; IL-2Rβ, IL-2 receptor β; TAA, tumor-associated antigen.

universal cytokine-mediated killing” (TRUCKs), in which a gene for the inducible or constitutive expression of IL-12 was inserted. This design helps T cells release pro-inflammatory factors into the immunosuppressive tumor microenvironment and achieve superior antitumor effects.16 Other cytokines were also explored for constructing such “armored” CARs, including IL-15, IL-18, and a combination of CCL19 and IL-7.17−19 The fifth-generation CARs, also called next-generation CARs, incorporate a truncated cytoplasmic domain of IL-2 receptor β and the transcription factor STAT3-binding motif between the cytoplasmic CD28 costimulatory domain and the CD3ζ signal transduction domain that deliver cytokine signals after antigenic stimulation.20,21 However, investigations of the fifth- generation CARs are still in the early stages and have not yet been well-recognized. Although efforts have been made to design various CAR constructs, the contributions of the last
two generations of CAR constructs to CAR-T cell effectiveness remain to be comprehensively evaluated.
CAR-T cell-based cancer immunotherapies are under intensive investigation because of their extraordinary potential for clinical application to combat B cell lymphoid malignancies. Tisagenlecleucel (Kymriah) and axicabtagene ciloleucel (Yescarta) are two approved anti-CD19 CAR-T cell products for the treatment of pediatric relapsed or refractory ALL and adult relapsed or refractory large B cell lymphoma, respectively.22,23 However, similar to other immunotherapies, many side effects have been observed with the currently approved CAR-T cell therapy used to treat malignant hematological diseases. When genetically engineered CAR-T cells are reinfused into patients, antitumor effects may be accompanied by cytokine release syndrome (CRS), which may sometimes threaten the patient’s life. Therefore, there is a need to control the quantity of activated CAR-T cells to execute the intended clinical effects without severe systemic cytokine- derived toxicities. Incorporating a safety switch for the CAR-T cells will lead to the development of safer immunotherapies. Small-molecule compounds targeting diverse pathways are

anticipated to provide a flexible measure to control CAR-T cells because they possess tunable and drug-like pharmacoki- netic properties and can easily be synthesized by chemical modifications. Progress in designing safety switches for CAR-T cells has been made using fluorescein isothiocyanate (FITC), folate, rimiducid (AP1903), rapamycin, proteolysis-targeting chimera (PROTAC) compounds, and other small-molecule compounds. FITC and folate conjugated with other substances mediate the formation of a pseudoimmunological synapse. Rimiducid is a homodimerizer that induces the dimerization of caspase-9 with consequent apoptosis of CAR-T cells. Rapamycin is the other single agent that can assemble an FKBP12−rapamycin−FRB tripartite complex to “turn on” CAR-T cells. PROTAC technology that is under development offers an alternative approach to control the lysis of CARs. Dasatinib is also a single agent that can directly prevent the activation of CAR-T cells. These successful examples have encouraged scientists to search for other small-molecule compounds with better and more suitable characteristics for the design of safety switches. The ability of such safety switches to titrate the activity of CAR-T cells toward tumor cells and therefore to strictly control toxicities after administration of CAR-T cells will expand the clinical application of CAR-T cell therapy and bring dual treatment benefits for patients. Here we discuss how safety switches offer toxicity control over CAR-T cells with their distinct regulatory mechanisms. We also describe how they are developed by combining the advantages
of genetic engineering and chemical methodologies.

⦁ CAR-T CELL THERAPY: TOXICITY ISSUES ACCOMPANY THEIR EFFECTIVENESS
Cytokine release syndrome, typically manifested as high fever, myalgia, unstable hypotension, and even respiratory failure, is viewed as the most common toxicity associated with CAR-T cell therapies.24−26 CRS is graded according to its severity, and the symptoms associated with CRS vary by the grade.27 In clinical trials involving CAR-T cells, severe CRS has occasion-

Table 1. Summary of Small-Molecule Safety Switches

ally caused serious complications that necessitated termination of the trial. In patients with severe CRS, abnormal amounts of cytokines are released by activated lymphocytes and/or myeloid cells.26,28 Cytokines such as IFN-γ and IL-2 are released by overactivated CAR-T cells, while elevated levels of IL-6 are the result of secondary activation of macrophages by
these cytokines.28,29 Moreover, CRS can also be induced by tumor cell pyroptosis: CAR-T cells release a large amount of perforin, which forms pores on the surface of tumor cells and causes granzyme B to enter the tumor cells, which in turn activates the caspase 3-gasdermin E pathway to induce cell pyroptosis and subsequent CRS.30 Tocilizumab, a recombinant

Figure 2. Schematic diagram of the FITC/folate-based safety switch. (A) The formation of pseudoimmunological synapses between tumors and CAR-T cells is mediated by safety switches. CAR-T cells are not activated to eliminate tumors without a FITC/folate-based safety switch, even in the presence of tumor-associated antigens. FITC/folate-based safety switches are anticipated to effectively regulate the activities of CAR-T cells to manage severe CRS. (B) The FITC/folate-based safety switch mediates the formation of pseudoimmunological synapses via two components of binding sites. These two components can combine with predesigned antigen receptors present on the surface of CAR-T cells as well as the abnormally expressed receptors or epitopes present on the surface of tumor cells.

humanized anti-IL-6 antibody, was used as the pharmaco- logical intervention for patients who experienced life-threat- ening CRS. When tocilizumab does not completely reverse severe CRS, corticosteroids may be included in the treatment regimen.24,31 Both of these treatments have been used after the development of severe CRS, but they cannot solve the inherent problem of CAR-T cell treatment-related toxicities. Thus, it is necessary to endow CAR-T cells with an ability to titrate its activity toward tumor cells precisely so that toxicities can be strictly controlled. In another case of off-target/off-tumor toxicity, CAR-T cells targeting GD2, which is abundantly

supplementation with immunoglobulins.25,36 Another case of on-target/off-tumor toxicity was reported in a patient with colon cancer that had metastasized to the lung and liver after administration of autologous CAR-T cells targeting the ERBB2 (Her-2/neu) antigen. This patient died because the CAR-T cells recognized low levels of ERBB2 expressed on lung epithelial cells, which triggered the release of large amounts of cytokines.37 Although the incidence of graft-versus-host disease (GvHD) is low, infusion of activated allogeneic T cells still poses the risk of GvHD. Fortunately, the risk can be reduced by an infusion of donor-derived T cells and an inducible

present on the surface of neuroblastoma cells, led to lethal central nervous system (CNS) toxicity along with enhanced antitumor efficacy.32 This neurotoxicity has recently been termed immune effector cell-associated neurotoxicity syn- drome (ICANS).33 ICANS typically presents itself as a toxic encephalopathy with several neurological symptoms that include confusion, delirium, occasional seizures, and cerebral edema.27,33 A compromised blood−brain barrier was asso- ciated with a higher level of cytokines in the blood and cerebrospinal fluid, in addition to endothelial activation, which
caspase-9 safety switch system.38,39 Since the activation and proliferation of CAR-T cells cannot be controlled in vivo, it is extremely important to remotely and reversibly regulate the functions of CAR-T cells using a small-molecule-based safety switch.
⦁ SMALL MOLECULES FUNCTION AS SAFETY SWITCHES TO REDUCE THE TOXICITY ASSOCIATED WITH CAR-T CELL THERAPY
A variety of technologies, such as FITC-labeled antibodies,

was demonstrated to be a link between CRS.34
neurotoxicity
and
rapamycin-induced protein heterodimerization, rimiducid- induced protein homodimerization, and PROTAC-regulated

When the targeted tumor-associated antigen is expressed on
normal host cells, CAR-T cells will attack nonmalignant cells as well, causing on-target/off-tumor toxicity. It is difficult to
protein degradation, have been employed to design novel safety switches. Such safety switches were designed by applying distinct strategies to minimize treatment-related toxicities

identify specific surface molecules, such as EGFR variant III
(EGFRvIII), that exist only on the surface of tumors but not on normal cells.35 Therefore, on-target/off-tumor toxicities are more likely to occur when CAR-T cells have mistakenly targeted specific antigens expressed on the surface of normal cells, such as B cells. CAR-T cells are called “living drugs” because they have unpredictable half-lives and can survive long-term in patients. Occasionally, B cell depletion was induced after CAR-T cell therapy, which required subsequent
while improving the therapeutic efficiency of CAR-T cells. Safety switches produced to date can be divided into “on” and “off” switches. “On” switches mediate the assembly of tripartite complexes: FITC and/or folate-based switches mediate the formation of tertiary complexes by acting as pseudoimmuno- logical synapses between antigen-expressing tumor cells and CAR-T cells, and rapamycin mediates the assembly of an FKBP12−rapamycin−FRB tripartite complex. “Off” switches can be subdivided into three types: rimiducid induces caspase-

9 dimerization with consequent CAR-T cell apoptosis; PROTAC compounds can control the stability of CARs; and dasatinib directly inhibits CAR-T cell activation. The structures of recently described small-molecule compounds used as safety switches and their distinct mechanisms are summarized in Table 1.
⦁ FITC/Folate-Based Safety Switches Mediate the Assembly of Pseudoimmunological Synapses. A novel bifunctional/bispecific “switch” has been designed to maximize the antitumor activities of CAR-T cells while controlling the
Scheme 1. Synthesis of the FITC/Folate-Based Safety Switch: (A) General Scheme for the Synthesis of Site- Specific FITC−Antibody Conjugates via Click Chemistry;
(B) Synthesis of FITC−Folate Conjugates

toxicities associated with life-threatening CRS. Appropriate
small molecules have been conjugated with other components so that they function together as an intermediate bifunctional/ bispecific switch. This type of safety switch contains two key modules: one is endowed with the ability of specific recognition and binding to TAA, and the other is capable of binding to the reprogrammed antigen recognition domain of the CAR. FITC and folate are commonly used to design this type of safety switch. This bifunctional/bispecific switch can turn CAR-T cells to the “on” state by serving as pseudoimmunological synapses between CAR-T cells and tumor cells in a spatiotemporal and dosage-dependent manner. It is only after administration of the small-molecule safety switch that the pseudoimmunological synapse can be formed and CAR-T cells be triggered to eradicate the target cancer cells.
Fluorescein is a synthetic, exogenous fluorescent tracer for
fluorescence imaging, and it is used in the treatment of
malignant tumors.40−42 FITC is generated by introducing an
isothiocyanate group into the structure of fluorescein. FITC

has the advantages of a high fluorescence quantum yield and susceptibility to microenvironmental changes without signifi- cant influence on the specificity of the conjugated antibody. Most importantly, the gene sequences of anti-FITC antibodies have been extensively investigated, facilitating the use of FITC in the design of safety switches for CAR-T cells. At present, FITC is the most widely used fluorochrome in modifying antibody fragments or conjugating with folate to synthesize FITC-based switches (Figure 2). The development of additional antibodies against small molecules and elucidation of their gene sequences will provide alternative small molecules that can be use in such safety switches. When FITC conjugates with the target protein, the isothiocyanate group of FITC usually forms a thiourea bond with the primary amine group of the protein. Recently, novel approaches have been devised to conjugate FITC with antibodies. After FITC is chemically modified by compounds containing terminal alkynyl groups, it is allowed to undergo a “click” reaction with antibodies incorporating p-azidophenylalanine (pAzF) and then form
strong antibody−fluorescent dye conjugates. The specific reactions and the general scheme are summarized in Scheme 1A.43 First, by the use of PEG4 and endo-BCN-NHS as substrates, BCN-PEG4-FITC, which introduces cycloalkynyl groups into FITC, is synthesized in a two-step reaction. Meanwhile, pAzF is incorporated into antibody fragments genetically. The antigen-binding fragments (Fabs) that have been investigated to date include but are not limited to CD19 (clone FMC63)-, CD22 (clone M971)-, and Her2 (clone 4D5)-specific monoclonal antibodies. Finally, the terminal
alkynyl moiety of BCN-PEG4-FITC and the azido moiety of the antibody fragment undergo a catalyst-free click reaction to generate the FITC-based safety switch.43
Investigators carried out in vivo and in vitro experiments to verify the efficacy of FITC-based safety switches. Comparisons between conventional CD19-specific CAR-T cells and anti- FITC switchable CAR-T (sCAR-T) cells equipped with the optimized anti-CD19 FITC-based safety switch are shown in Figure 3A.43 Anti-FITC CAR-T cells with 1 nM anti-CD19 AB-FITC and conventional CART-19 cells exhibited com- parable activities in lysing Nalm-6 (CD19+) cells. In vivo experiments were carried out in a Nalm-6 xenograft model. Tumor growth was quantified by detecting the radiance in the region of interest (ROI). The results showed that anti-FITC CAR-T cells with anti-CD19 AB-FITC cleared the tumors as effectively as CD-19 CAR-T cells. In addition, the data show that the conjugation site greatly influences the therapeutic efficacy. The proximal bivalent AB-FITC switch displayed the best antitumor efficacy, and the proximal monovalent B switch was more potent than bivalent EF and random switches. Moreover, the toxicities of CAR-T cell therapy can be theoretically controlled by varying the dosing regimen of the small-molecule-based safety switch. Anti-CD19 AB-FITC at a concentration of 0.5 mg/kg caused a significant weight loss in mice, while a concentration of 0.05 mg/kg did not cause weight loss to a similar extent. In dose-escalation studies, a dose of anti-CD19 AB-FITC started at 0.05 mg/kg and increased to 0.5 mg/kg achieved equivalent tumor clearance and milder weight loss than a high starting switch dose of 0.5 mg/kg. These results provide evidence that the small- molecule-based safety switch approach may be useful for controlling severe CRS in clinical settings. FITC labeling of the Fab fragments of trastuzumab can also be applied to redirect CAR-T cells to Her2-expressing tumor cells, which are present in Her2-positive breast cancer patients. This FITC-labeled trastuzumab safety switch mediated sCAR-T cells to

Figure 3. Comparison of the anticancer and cytotoxic effects of switchable and conventional CAR-T cells. (A) Comparison of in vitro cytotoxicities and in vivo radiance quantifications in the region of interest (ROI) of conventional CAR-T cells and anti-FITC CAR-T cells with an optimized anti-CD19 AB-FITC switch. Reproduced from ref 43. Published by the National Academy of Sciences. (B) Comparison of in vivo antitumor efficacies and in vitro cytotoxicities of conventional anti-Her2 CAR-T and sCAR-T cells. Reproduced with permission from ref 44. Copyright 2016 Wiley-VCH.

but this isoform is expressed in endometrial cancer, ovarian cancer, cervical adenocarcinoma, nonsmall cell lung cancer, and malignant mesothelioma (MM).50−52 Hence, the differ- ential distribution of FRα in normal and tumor tissues together with the high affinity and stability of folate make FRα and folate potential targets for the design of safety switches
Folate-based safety switches were designed to mitigate the toxicities associated with CAR-T cell therapy. Unlike antibody fragments that are genetically encoded to incorporate an azido moiety where click reactions can occur, the humanized catalytic antibody h38C2 contains an unprotonated lysine Lys residue, which is adapted for site-specific conjugation with small molecules.53 The Lys99 residue can be used to chemically program antibodies by forming a stable amide bond with small-molecule compounds containing β-lactam functions. More recently, a new switchable CAR-T treatment platform based on chemically programmed antibody fragments (cp-Fab) was developed by modifying the Fab fragments with a trifunctional β-lactam−biotin−folate compound.54 This novel safety switch system was called cp-Fab/CAR-T. The first type of cp-Fab was produced by fusion of a GCN4 polypeptide fragment to the light- or heavy-chain terminus of h38C2 Fab followed by coupling with folate. The second type of cp-Fab was formed by conjugation of folate with the GCN4 polypeptide followed by coupling with the wild-type h38C2 Fab scaffold. The GCN4 polypeptide fragment binds to scFv 52SR4-based CAR-T cells, and folate binds to FRα that is
overexpressed on cancer cells.
Unlike switches composed of a tumor-targeting antibody, safety switches using small-molecule ligands that recognize and bind to folate receptors overexpressed on tumor cells are more practical and readily available. In another approach, a FITC− folate conjugate was developed as a bifunctional small-

completely clear tumors in rodent xenograft models. The dosage regimen for mice consisted of an injection of 3 × 107 anti-FITC CAR-T cells iv followed by another iv injection of anti-Her2 Fab-FITC switch at 0.5 mg kg−1 every other day up to day 14 (Figure 3B).44 The FITC-based safety switch has been demonstrated to redirect the specificity of CAR-T cells by inducing distinct spatial interactions. In fact, FITC was applied to link unique tumor-specific ligands to target different tumor markers.45,46 Another small-molecule switch named FITC- HM-3 bifunctional molecule (FHBM) was designed to simultaneously bind dual-receptor-modified T cells and cognate tumor cells expressing both mesothelin (MSLN) and integrin αvβ3, thus endowing CAR-T cells with not only an improved ability to distinguish tumor and normal cells but also an enhanced ability to control their activity and cytotoxicity.47 Folate (or folic acid), which is a to water-soluble B vitamin,
is a low-molecular-weight compound with low immunogenic- ity. It occupies an important place in the design of this type of safety switch. Cancer therapeutics using folate as a targeting ligand have attracted significant interest, including folate-
molecule switch. The FITC−folate conjugate was synthesized by condensing folate with an ethylenediamine linker followed by direct coupling to FITC through a second condensation reaction (Scheme 1B).55 This small-molecule switch is bispecific for FR-positive cells and anti-FITC CAR-T cells.
FRα is also a suitable marker for nonsmall cell lung cancers because of its high expression in tumor tissues.56 Therefore, the FITC−folate conjugate is expected to function as an intermediate switch to reduce the toxicity while increasing the efficacy of CAR-T cells to treat nonsmall cell lung cancer.57
Indeed, FITC could redirect CAR-T cells toward various antigen epitopes of cancer cells by conjugation with different substrates, thereby eliminating the need to re-engineer CAR-T cells. In addition, the wealth of existing studies on anti-FITC antibodies and the high affinity of FITC to these antibodies provide advantages to the switch design that cannot be ignored. Although this type of small-molecule-based safety switch theoretically improves the safety profiles of CAR-T cells, the antitumor effect of the modified cells was not always better than that of the unmodified CAR-T cells. Therefore,

attached cytotoxic drugs and folate-laden nanoformula-
additional designs and techniques need to be developed to

tions.48,49 Abnormal proliferation of malignant tumors requires large amounts of folate because it is an important precursor for nucleic acid biosynthesis and methylation. Hence, tumor cells rely on endocytosis mediated by the folate-binding protein folate receptor (FR) to actively absorb exogenous folate into the cytoplasm to maintain elevated metabolic needs. Among all of the isoforms of FR, FRα is expressed in a restricted manner within normal tissues, such as relatively low level expression on the apical (luminal) cell membrane of various epithelial tissues,
navigate trade-offs between the safety and efficiency profiles of such treatment options.
⦁ Rapamycin Induces the Assembly of FKBP12 and the FRB Binding Domain to Control the Activation of CAR-T Cells. T cells are activated by recognition of the
antigenic peptide−MHC (p-MHC) complex present on the surface of APCs along with interactions with other receptors
and ligands that provide secondary stimulatory signals. Genetically engineered CAR-T cells possess an extracellular

Figure 4. Schematic representation of rapamycin as a molecular switch. Comparisons of the cytotoxicities and anticancer effects of modified CD19- DARIC T cells and conventional CAR-T cells are shown. (A) In the absence of rapamycin, the antigen recognition domain is separated from the signal transduction domain of the CAR structure. The formation of the tripartite complex bridges the antigen recognition and signal transduction domains, restoring the CAR structure as a whole molecule. Thus, rapamycin can be used to regulate the activities of CAR-T cells remotely to avoid potentially fatal toxic effects. (B) (left) The cytotoxicity of CD19-DARIC T cells was comparable to that of conventional CD19-CAR-T cells. (right) Bioluminescence data showing superior in vivo activity of CD19-DARIC T cells with non-immunosuppressive rapamycin concentrations compared with conventional CD19-CAR-T cells. Reproduced with permission from ref 63. Copyright 2019 American Society for Clinical Investigation.

antigen recognition domain and an intracellular signaling domain. Another strategy for generating small-molecule-based switches is to split these two structures and rejoin them in the presence of small-molecule drugs. Chemical inducer of dimerization (CID) has emerged as a potent tool for controlling protein dimerization. In this context, rapamycin and analogues of rapamycin (rapalogues) can be used as CIDs to assemble domains introduced into CAR structures. Rapamycin exerts potent immunosuppressive properties apart from its promising antitumor functions by inhibiting mTOR (TORC1).58 To improve the pharmacological properties of rapamycin, various rapalogues were synthesized by structural modification of the C8 or C40 hydroxyl group, six-membered pipecolate ring, and cyclohexane ring present in rapamycin.59 Among them, AP21967 is a derivative of the C16 structure modified by methylindole with less undesirable immunosup- pressive activities.60 Rapamycin simultaneously occupies two different hydrophobic binding pockets of two proteins: human FK506-binding protein (FKBP12) and the FKBP12-rapamy- cin-binding (FRB) domain of FKBP-rapamycin-associated protein (FRAP).61 AP21967 dimerizes the T2089L mutant
of the FRB domain of FRAP and the FKBP12 domain. Rapamycin, after binding to the FKBP12 and FRB domains, can directly induce heterodimerization of these two binding domains.60,61 Thus, inserting the FKBP12−rapamycin−FRAP/ FRB tripartite complex into the structure of CAR can constitute a small-molecule-based safety switch to control CAR-T cell activities. The specific insertion site can be varied flexibly according to the design ideas. In the presence of rapamycin, the chimeric antigen receptor executes both antigen recognition and signaling functions. However, in the absence of rapamycin, the antigen-binding subunit and the intracellular signaling subunit are disconnected, keeping CAR-T cells in the “off” state even though the scFv fragments of CAR-T cells bind to the target antigens on the surface of tumor cells (Figure 4A). This type of safety switch allows CAR-T cells to be specifically regulated after being injected into the patient’s body, which means that the activity can be gradually titrated to
the appropriate therapeutic level to avoid serious adverse reactions.
Inserting fusion proteins of FRB and FKBP12 into the intracellular region separates the extracellular scFv antigen-

binding domain from the CD3ζ downstream signaling subunit marker but also as a suicide gene, which can be triggered by

and costimulatory domain. “On-switch” CARs adopt a split- receptor design that mediates heterodimerization by rapamycin or a rapalogue and possess a dual role, i.e., achieving maximum reduction in toxicity while generating strong antigen-induced signals.62 The optimal conformation of the two fusion proteins (FKBP and FRB*) was determined by changing the domain composition and the order of the CAR splitting parts and orthogonally combining these disparate parts. A dimerizing- agent-regulated immunoreceptor complex (DARIC) was generated by insertion of the FKBP12−rapamycin−FRAP/ FRB tripartite complex into the extracellular domain of the second-generation CD19-CAR.63 This study also evaluated the in vivo antitumor efficacy of CD19-DARIC T cells in comparison to that of conventional CD19-CAR-T cells. Compared with conventional CD19-CAR T cells, CD19- DARIC T cells in the presence of rapamycin or the analogue compound AP21967 exhibited equivalent levels (∼80%) of cytotoxicity in a fluorescence-activated cell sorting (FACS)- based analysis and superior tumor clearance in a bio- luminescence analysis (Figure 4B). In another attempt to mitigate the toxicity, this small-molecule-based switch strategy was applied to a novel CAR architecture based on an FcεRI receptor scaffold.64 These cellular products, called controllable transient CAR T-cells, were expected to have improved safety profiles because of the use of rapamycin or its analogues to control the function and cytotoxicity of CAR-T cells. Because rapamycin can join FKBP12 and FRB, it can also mediate apoptosis by inducing caspase-9 dimerization (see section 3.3) as long as the inducible caspase-9 system contains FKBP12 and FRB domains. For example, rapamycin was used to dimerize rapamycin-induced caspase-9 (iRC9) by linking the FKBP12 domain of one iRC9 and the FRB domain of another iRC9 and to induce apoptosis of CAR-T cells in a rapamycin-dependent manner.65
However, rapamycin and its analogues are not panaceae for
eliminating toxicities because of the following concerns: First, FKBP12- and FRB-fused proteins are not exclusive receptors for rapamycin and its analogues. Competition with endoge- nous FKBP12 and the mTOR complex may lead to unpredictable CAR functions and interfere with accurate control of the dose of rapamycin and its analogues. Second, rapamycin exhibits potent immunosuppressive activity, which contradicts the underlying concept of CAR-T cell therapy to enhance the activity of the immune system in vivo. Therefore, rapamycin may partially reduce the robust therapeutic effects of CAR-T cells. This is supported by the observation that traditional CAR-T cell activity was slightly suppressed after the administration of rapamycin. Notably, there are rapalogues that have lower immunosuppressive activity than rapamycin.60 Therefore, these agents may be used as alternatives to rapamycin. However, few studies have investigated their safety, efficacy, and clinical applications.
⦁ Rimiducid Induces Caspase-9 Dimerization and
Promotes Apoptosis of Excessively Activated CAR-T Cells. Genetically targeted T cells combined with suicide genes possess considerable practical utility, and this method has been tested in several clinical trials. To date, approaches introducing a suicide gene can be categorized according to the dimer and CID. CIDs such as rimiducid (AP1903) and ganciclovir can induce apoptosis of CAR-T cells when CAR structures include a suicide-gene-encoding fusion protein. A human cell surface polypeptide CD20 antigen was used not only as a selection
rituximab (anti-CD20 monoclonal antibody) to execute its protective function.66 We herein mainly focus on introducing a suicide gene system using small-molecule-based safety switches. The suicide gene system using ganciclovir and herpes simplex virus thymidine kinase (HSV-TK) has drawbacks compared with those using rimiducid and inducible caspase-9. Ganciclovir is a phosphorylated purine nucleotide analogue that interferes with DNA synthesis and kills dividing cells, but it requires several days before therapeutic benefits are gained.67 Moreover, HSV-TK is an exogenous virus-derived protein with potential high immunogenicity, which may induce undesirable immune responses and lead to premature elimination.68 Thus, we mainly discuss the extensive studies and promising clinical benefits of safety switches composed of rimiducid and inducible caspase-9. To avoid undesired severe toxicities, the inducible caspase-9 safety switch system was developed as a practical approach with a defined clinical profile. This system could promptly terminate the therapeutic activity of CAR-T cells through small-molecule-induced apoptosis.69 This system fuses modified human caspase-9 to human FK506-binding proteins (FKBPs) and uses rimiducid/AP20187 as the chemical inducer of dimerization.70 Rimiducid acts as a dimerizer by cross-linking the F36V variant of the FKBP12 (FKBP12-F36V) domain. AP20187 is similar to rimiducid and can homodimerize FKBP12-F36V. These two small molecules are different from AP21967, which dimerizes the T2089L mutant of the FRB and FKBP domains (see section 3.2). Rimiducid/AP20187 can mediate the conditional dimerization of inducible caspase-9 with subsequent activation of caspases 3, 6, and 7, causing apoptosis of cells expressing the fusion protein (Figure 5A).
There are numerous examples of safety switches using this approach. AP20187, an effective CID in vitro, can modulate CD-19 CAR-T cell toxicities by inducing the dimerization of mutant human caspase 9 and subsequent apoptosis of T cells.71 Novel CD19 CAR-T cells that coinsert the interleukin-15 (IL- 15) gene and an inducible caspase-9-based suicide gene can balance the potentially increased risk of direct toxicity and the improved antilymphoma/leukemia effects. In these iC9/ CAR.19/IL15+ T cells, investigators were able to manage uncontrolled proliferation after the introduction of IL-15 by pharmacologically activating the caspase-9-based safety switch.72 As demonstrated in Figure 5B, iC9/CAR.19/IL15+ T cells showed improved in vivo antitumor effects. There was a 4.7−5.4-fold reduction in the tumor burden in animals treated with iC9/CAR.19/IL15+ T cells compared with the group treated with CAR.19+ T cells [(1.7 ± 5) × 108 to (9.3 ± 1.6) × 108 cells by day 38]. In a separate series of experiments, a CD20 chimeric antigen receptor and a suicide switch consisting of inducible caspase-9 and rimiducid or AP20187 (B/B homodimerizer) were introduced into CAR-T cells. These CAR-T cells with the safety switch were efficiently removed both in vivo and in vitro and improved the efficacy and safety of adoptive T cell immunotherapy for lymphoma.73
Bellicum Pharmaceuticals has applied the CID technology with inducible caspase-9 and MyD88/CD40 to build safety and GO switches, respectively. The inducible caspase-9 system was applied to trigger T cell apoptosis in the context of HSCT therapy, and the inducible MyD88/CD40 switch was used to trigger T cell activation and proliferation in CAR-T cell therapy.74,75 The feasibility of deploying rimiducid as an iC9 safety switch to control severe CRS also been tested in

Figure 5. Schematic diagram of the iCas9 system and the in vivo antitumor activity. (A) Schematic diagram of the iCas9 system inducing apoptosis of CAR-T cells. Rimiducid and iCas9 are part of a potent suicide gene system that efficiently induces apoptosis of CAR- T cells in the case of CRS. iCas9 is a fusion protein composed of FKBP12-F36V (an F36V mutant of FK506-binding protein) and ΔCaspase9. Rimiducid is a small-molecule dimerizing agent that can bind to FKBP12-F36V with high affinity. Once rimiducid induces homodimerization of iCas9, the caspase cascade is triggered to activate caspase 3, 6, and 7, leading to apoptosis of CAR-T cells. (B) Summary of the bioluminescence signal in severe combined immunodeficient (SCID) mice engrafted either ip or sc with 3 × 106 Daudi cells labeled with FFLuc. Reproduced with permission from ref 72. Copyright 2010 Springer Nature.

patients. As listed in Table 2, the inducible caspase-9 safety switch system has undergone multiple clinical trials. The safety profile of CAR-T cell therapy modified by rimiducid−inducible caspase-9 was validated in five recipients who received a stem cell transplant for relapsed acute leukemia.70 Treatment in 12 haploidentical hematopoietic stem cell transplant (haplo- HSCT) patients also proved that administration of rimiducid can control GvHD and severe CRS by eliminating iC9-T cells.74 Long-term follow-up of 10 patients after haplo-HSCT
showed that a single administration of rimiducid can provide permanent control of graft-versus-host disease (GvHD) without impeding immune reconstitution.76 More surprisingly, the allo-depleted T cells containing the iC9 safety gene benefited the patients by their long-term persistence, immune recovery, and resistance to opportunistic infections.
Another study showed that rimiducid-activated dimerization not only induced CAR-T cell apoptosis but also introduced new applications for CAR modification. For instance, designing novel rimiducid-regulatable domains may make it possible to provide an alternate activation signal in CAR-T cells. Rimiducid-induced dimerization of inducible MyD88/CD40 (iMC) led to activation of downstream TLR and CD40 costimulatory signaling pathways. As a consequence, rimiducid administration enhanced survival and promoted proliferation of CAR-T cells, while cells could become anergic after rimiducid was withdrawn.75 The data from the above studies and the proposed mechanisms demonstrate the need for additional experiments to compare the therapeutic effects of conventional CAR-T cells with those modified with the iCas9 or iMC system. Altogether, most of these safety switches can
induce ∼90% CAR-T cell apoptosis within 2 h, and therefore, rimiducid may be regarded as a potent “off” safety switch with
a definite effect on inducing CAR-T cell apoptosis. However, although these safety switches are highly effective and have undergone numerous clinical trials, the major limitation is the irreversible depletion of CAR-T cells from circulation. This could result in loss of therapeutic efficacy, which may require reinfusion of CAR-T cells.77
⦁ PROTAC Technology Controls the Lysis and Degradation of CARs. CAR-T cells remain in an activated state as long as CARs recognize and bind to predetermined tumor-associated antigens present on the surface of target tumor cells. Therefore, the quantities of CARs expressed on the surface of T cells are a key indicator to quantify the anticancer activities and potential toxicities of CAR-T cells. Remote and reversible regulation of surface presentation of CARs by precise control of their degradation represents a novel approach in designing a safety switch. Currently, a new technology named proteolysis-targeting chimera (PROTAC) has emerged as a potent therapeutic intervention to degrade specific proteins within cells.78 The N- and C-terminal residues of cellular proteins are major determinants of N- and C- degrons, respectively, which are signals controlling the degradation and stability of proteins.79,80 Proteins of interest containing degrons are degraded mainly through the ubiquitin
(Ub)−proteasome system (UPS), in which E3 ubiquitin ligase promotes protein ubiquitination and the proteasome sub- sequently executes protein degradation.81,82 Because CAR is an artificial and synthetic protein, by incorporation of a degron into CAR it should be theoretically feasible to degrade the entire CAR protein once the degron is recognized by a proteolytic enzyme (Figure 6). Various novel small molecules have been used to control the degradation of CAR via different methods.
The SWIFF-CAR system, in which an off switch is integrated into the CAR construct, was designed to regulate the expression of CAR on the cell surface reversibly at the protein level.83 This novel CAR construct has three main components: a protease target site, HCV NS3 protease, and a degradation moiety (degron) (Figure 6A). Asunaprevir, an HCV NS3 protease inhibitor, controls the self-cleaving moiety, and the HCV NS3 protease/degron component was

Table 2. Clinical Trials of the iCas9 System Used for Safety Switch

CAR-T cells test no. year phase no. of participants conditions
iC9-CAR19 NCT03016377 2018 I, II 54 ALL
iC9-CAR19 NCT03594162 2018 − − ALL
iC9-CAR19 NCT03696784 2019 I 30 lymphoma
iC9.GD2.IL-15 NCT03721068 2019 I 18 neuroblastoma
P-BCMA-101 NCT03741127 2018 I 100 MM
P-BCMA-101 NCT03288493 2017 I, II 220 MM
CD19-CAR-T NCT03373071 2017 I, II 32 ALL, CD19-NHL
P-PSMA-101 NCT04249947 2020 I 40 prostatic neoplasms
anti-SLAMF7 NCT03958656 2019 I 36 MM
anti-GD2 NCT02107963 2014 I 15 sarcoma
iC9-GD2 NCT01822652 2013 I 11 neuroblastoma

Figure 6. Schematic diagram of PROTAC technology controlling CAR degradation through the ubiquitin−proteasome system. (A) Asunaprevir, known as an HCV NS3 protease inhibitor, inhibits HCV NS3 protease from cleaving the degron from CAR. Thus, the degron together with CAR is degraded by the proteasome. (B) Administration of ARV771 or ARV825 induces CAR degradation by the E3 ligase−proteasome pathway. (C) Shield-1 displaces and exposes the degron to the proteasome by competitively binding to FKBP12-F36V. Abbreviations: Costim, costimulatory; BD, bromodomain; E2, E2 ligase; E3, E3 ligase; Ub, ubiquitin.

incorporated into the CAR architecture. In the presence of asunaprevir, it inhibits the cleavage of the degron from the CAR by the HCV NS3 protease. Therefore, while active HCV NS3 protease activity promoted the membrane expression of a functional CAR, the administration of protease inhibitor acted as an “off” signal, prompting the proteasome degradation of the full CAR−protease−degron structure. Administration of ARV771 or ARV825, PROTAC compounds against bromo- domain (BD), regulate a second safety switch system by degrading the BD-containing CAR protein (Figure 6B).77 This safety switch controls both cytokine secretion and lytic activity of CAR-T cells. ARV-771 and ARV-825 are heterobifunctional
compounds comprising an alkyl linker and a BETP-binding moiety, Von Hippel−Lindau (VHL) for ARV-771 and the E3 ligase cereblon-binding moiety for ARV-825.84,85 The ligand- induced degradation (LID) domain can be fused to the CAR construct to modulate its characteristics. Shield-1, a small- molecule ligand, was designed to displace and expose the cryptic degron present within the LID domain, which initiates
proteasomal degradation of the CAR−LID fusion protein and downregulation of the surface expression of CARs.86
However, application of the PROTAC technology to control the lysis and degradation of CARs is more complicated. First, the slow onset of small-molecule-induced degradation of CAR might not be sufficient to reverse severe and acute CRS rapidly. Theoretically, the degradation of CAR protein and the restoration of CAR expression are reversible. However, most of the studies to date have been carried out in vitro only, except for a single in vivo experiment that proved the inhibitory function of PROTAC compounds on CAR expression. Thus, the feasibility of this PROTAC-based safety switch approach needs additional investigation in the near future. Nonetheless, PROTAC-technology-mediated CAR degradation still holds promise as a means to control toxicities because of its flexible and innovative properties.
⦁ Dasatinib Directly Prevents CAR-T Cell Activa-
tion. Existing drugs can be used as pharmacological safety switches to temporarily inactivate CAR-T cells to control their toxicities. Moreover, the antitumor effects can be restored after

Figure 7. Schematic diagram depicting distinct mechanisms of small-molecule compounds applied in safety switches. FITC−folate conjugates act as safety switches that induce the formation of pseudoimmunological synapses between tumors and CAR-T cells. Rapamycin mediates assembly of the FKBP12−rapamycin−FRB tripartite complex that bridges the extracellular antigen recognition domain with costimulatory and CD3ζ domains. Rimiducid binds to FKBP12-F36V and induces caspase-9 dimerization with consequent apoptosis of CAR-T cells. PROTAC compounds in conjunction with degrons control the surface expression levels of CARs by inducing degradation of CARs. Dasatinib prevents the activation of CAR-T cells by inhibiting the phosphorylation of Lck, CD3ζ, and ZAP70 molecules. Costim, costimulatory domain.

discontinuing the safety switch drugs. Dasatinib, a small- molecule multityrosine kinase inhibitor against BCR-ABL and SRC, suppresses TCR-mediated signal transduction, cellular proliferation, cytokine production, and in vivo T-cell responses by inhibiting lymphocyte-specific protein tyrosine kinase (Lck).87,88 Therefore, dasatinib is a potential drug of choice as a pharmacological safety switch to attenuate the toxicities of CAR-T cells without compromising their therapeutic efficacy. Inhibition of the autophosphorylation of Lck by dasatinib reduces Lck-mediated phosphorylation of CD3ζ and the ζ- chain of T cell receptor-associated protein kinase 70 (ZAP70) by ∼90%, which further reduces signaling in CAR constructs.89 Therefore, dasatinib is potentially effective for all CAR structures because CD3ζ is an immobile part of the signal transduction module present in all types of CAR constructs. The effects of dasatinib on antigen-induced T cell activation, proliferation, and cytokine production have been assessed in preclinical studies, and the results provide evidence that

mitigating antigen escape, and enhancing the specificity of CAR-T cells. In order to achieve these goals, various modifications are introduced into chimeric antigen receptor constructs. Fourth-generation CARs have been developed that can deliver cytokines or chemokines to confer resistance to the immune-suppressive tumor microenvironment. In addition, dual and triple CARs are being developed to simultaneously bind to two or three tumor antigens to improve their specificity to tumor cells.
Despite the excellent clinical profiles of CAR-T cell therapy, it is still difficult to attain the optimal therapeutic levels without causing severe toxicities when the tumor load is unpredictable and the activity of T cells is uncontrolled. Severe CRS remains a potentially fatal side effect of CAR-T cell therapy. As a part of the improvement of this therapeutic approach, sufficient attention should be paid to managing the CAR-T cell safety profile. To control the toxicity, severe CRS needs to be

dasatinib can act as a potent pharmacological off switch to control severe CRS and may also contribute to the treatment of ICANS.90 However, the inhibitory effect of dasatinib is not as robust in activated CAR-T cells, which might result in inferior toxicity control.89 Moreover, dasatinib is a nonspecific immunosuppressive drug that can suppress all T cells, including T helper cells.91 Since dasatinib is not specifically designed as a small-molecule safety switch but used only as an immunosuppressive drug, it is controversial whether it could be called a small-molecule safety switch for CAR-T cells. Overall, notwithstanding its limitations, dasatinib can still
monitored appropriately using the CRS grading system as a
guide and modulated precisely using small-molecule-based safety switches. CAR-T cells equipped with small-molecule compounds should have definite pharmacodynamic and pharmacokinetic properties. Thus, small molecules are expected to endow CAR-T cells with functional flexibility by artificially switching between the “on” and “off” states. Additionally, small molecules can also be delivered selectively to target tissues using drug-directed transport technology, which will help further mitigate the on-target/off-tumor toxicity issues. Small-molecule compounds such as FITC,

potentially be used because of its safety profile.
⦁ CONCLUSION
Adoptive T-cell immunotherapy based on the forced expression of genetically engineered antigen receptors presents significant clinical promise in the treatment of malignant hematological tumors. Ongoing investigations on CAR-T cell therapies are mainly focused on improving the clinical efficacy,
folate, rapamycin, rimiducid, and PROTAC compounds have been successfully adapted to design novel safety switches for CAR-T cells to reduce life-threatening CRS through various mechanisms (Figure 7). Nonetheless, most of the small- molecule-based safety switches have not yet been tested clinically in the context of CAR-T cell-based therapies, and therefore, it remains to be seen how effective they could be in enhancing safety while reducing toxicity.

In the future, small-molecule compounds are expected to be equipped with the following properties for safety switch design. First, the small molecules should be safe enough and preferably have low immunogenicity to avoid superfluous treatment-
Kutty Selva Nandakumar obtained his Ph.D. in immunology from Madurai Kamaraj University in India in 1996 and his D.Sc. in medical inflammation science from Lund University in Sweden in 2006. He received his postdoctoral training in tumor immunology at the Center

related
toxicities
and also be directed toward endogenous
for Cellular and Molecular Biology in India and in rheumatoid

■
targets so that they do not compete with endogenous substances for binding sites. Second, the small molecules should maintain the stability and invariability of the chemical structure in the biological environment until their function is accomplished. Finally, the small molecules are supposed to titrate the activities of CAR-T cells toward tumor cells in a dose-dependent manner, and the activities of CAR-T cells with safety switches should be as potent as those of standard CAR- T cells. The combination of chemical methodologies and tools with genetic engineering techniques is expected to facilitate CAR-T cell-based immunotherapies and provide new applications for small-molecule compounds. In summary, equipping CAR-T cells with robust small-molecule-based safety switches that fulfill the criteria of titrating CAR-T cell activities against tumor cells in a nonlethal and spatiotemporal manner will eventually promote enormous progress with CAR- T cell-based therapies.
AUTHOR INFORMATION
Corresponding Author
Kui Cheng − Guangdong Provincial Key Laboratory of New Drug Screening and Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China; orcid.org/0000- 0002-4136-2070; Phone: +86 20 6164 7192;
Email: [email protected]; Fax: +86 20 6164 8533
Authors
Yanjun Zheng − Guangdong Provincial Key Laboratory of New Drug Screening and Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China
Kutty Selva Nandakumar − Guangdong Provincial Key Laboratory of New Drug Screening and Guangzhou Key
Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China;
orcid.org/0000-0001-7790-8197
Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.0c02054

Author Contributions
Y.Z. and K.C. wrote the original draft. K.S.N. reviewed and edited the manuscript.
Notes
The authors declare no competing financial interest.
Biographies
Yanjun Zheng obtained her B.S. in pharmacy from the University of South China in 2020. Since then, she has been pursuing a Ph.D. in medicinal chemistry at Southern Medical University in China. Her research interests mainly focus on the design and development of novel potent small-molecule-based safety switches for CAR-T cells and the incorporation of chemical methodologies into genetic engineering techniques to improve the therapeutic profile of cellular products.
arthritis at Lund University from 1996 to 2001. He worked at Lund University and the Karolinska Institute in Sweden for more than 10 years, up to the level of associate professor. In 2016 he became a professor in the School of Pharmacy at Southern Medical University. His current research focuses on exploring pathogenic pathways and developing therapeutics for inflammatory diseases. He has published more than 155 scientific papers and is a coinventor of four PCT international patents.
■
Kui Cheng received a joint Ph.D. in biology from Nanjing University and the University of Colorado Boulder and carried out his postdoctoral research in chemical biology at the University of Colorado from 2011 to 2014. Subsequently, he worked at Tsinghua University as an assistant researcher. In 2016 he moved to the School of Pharmaceutical Sciences at Southern Medical University and was appointed as a professor. His current research focuses on small molecules used in cancer immunotherapy, such as the discovery and development of new drug-inducing Toll-like receptor modulators, PD-1/PD-L1 blocking agents, and safety switches for CAR-T cells. He has published more than 20 scientific papers in peer-reviewed journals and has applied for four U.S. patents, two of which have been authorized.

ACKNOWLEDGMENTS
■
We thank the National Natural Science Foundation of China (81773558, 82073689), the Natural Science Foundation of Guangdong Province (2020A151501518, 2018B030312010), and the Science and Technology Program of Guangzhou (201904010380) for financial support for this work.
ABBREVIATIONS
■
CAR-T cell, chimeric antigen receptor T-cell; PROTAC, proteolysis-targeting chimera; ALL, acute lymphoblastic leukemia; TIL, tumor infiltrating lymphocyte; TCR, T cell receptor; scFv, single-chain fragment variant; TAA, tumor- associated antigen; MHC, major histocompatibility complex; APC, antigen-presenting cell; pMHC-I, peptide−MHC class I; Costim, costimulatory domain; IL-2Rβ, IL-2 receptor β; ITAM, immunoreceptor tyrosine-based activation motif; CRS, cytokine release syndrome; EGFRvIII, EGFR variant III; CNS, central nervous system; GvHD, graft-versus-host disease; FITC, fluorescein isothiocyanate; pAzF, p-azidophe- nylalanine; ROI, region of interest; MSLN, mesothelin; FR, folate receptor; FKBP, FK506-binding protein; FRAP, FKBP- rapamycin-associated protein; FRB, FKBP12-rapamycin-bind- ing; CID, chemical inducers of dimerization; HSV-TK, herpes simplex virus thymidine kinase; CARD, caspase recruitment domain; IL-15, interleukin-15; haplo-HSCT, haploidentical hematopoietic stem cell transplant
REFERENCES
⦁ Prasad, V. ⦁ Immunotherapy: Tisagenlecleucel – the first approved ⦁ CAR-T-cell⦁ ⦁ therapy:⦁ ⦁ implications⦁ ⦁ for⦁ ⦁ payers⦁ ⦁ and⦁ ⦁ policy⦁ ⦁ makers.⦁ Nat. Rev. Clin. Oncol. 2018, 15 (1), 11−12.
⦁ Yip, A.; Webster, R. ⦁ The⦁ ⦁ market⦁ ⦁ for⦁ ⦁ chimeric⦁ ⦁ antigen⦁ ⦁ receptor⦁ ⦁ T ⦁ cell therapies. Nat. Rev. Drug Discovery 2018, 17, 161−162.
⦁ Chrusciel, E.; Urban-Wojciuk, Z.; Arcimowicz, L.; Kurkowiak,
M.; Kowalski, J.; Gliwinski, M.; Marjanski, T.; Rzyman, W.; Biernat, W.; Dziadziuszko, R.; Montesano, C.; Bernardini, R.; Marek-

Trzonkowska, N. Adoptive Cell Therapy-harnessing antigen-specific T cells to target solid tumours. Cancers 2020, 12, 683.
⦁ Maude, S. L.; Frey, N.; Shaw, P. A.; Aplenc, R.; Barrett, D. M.; Bunin, N. J.; Chew, A.; Gonzalez, V. E.; Zheng, Z.; Lacey, S. F.; Mahnke, Y. D.; Melenhorst, J. J.; Rheingold, S. R.; Shen, A.; Teachey,
D. T.; Levine, B. L.; June, C. H.; Porter, D. L.; Grupp, S. A. Chimeric antigen receptor T cells for sustained remissions in leukemia. N. Engl. J. Med. 2014, 371, 1507−1517.
⦁ Schuster, S. J.; Svoboda, J.; Chong, E. A.; Nasta, S. D.; Mato, A.
R.; Anak, O.; Brogdon, J. L.; Pruteanu-Malinici, I.; Bhoj, V.; Landsburg, D.; Wasik, M.; Levine, B. L.; Lacey, S. F.; Melenhorst,
J. J.; Porter, D. L.; June, C. H. Chimeric antigen receptor T cells in refractory B-Cell lymphomas. N. Engl. J. Med. 2017, 377, 2545−2554.
⦁ Zhang, C.; Liu, J.; Zhong, J. F.; Zhang, X. ⦁ Engineering⦁ ⦁ CAR-T
cells. Biomarker Res. 2017, 5, 22.
⦁ Ramos, C. A.; Dotti, G. ⦁ Chimeric antigen receptor (CAR)- ⦁ engineered lymphocytes for cancer therapy. Expert Opin. Biol. Ther. 2011, 11, 855−873.
⦁ Eshhar, Z.; Waks, T.; Gross, G.; Schindler, D. G. ⦁ Specific
activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 720−724.
⦁ Sadelain, M.; Brentjens, R.; Riviere, I. ⦁ The⦁ ⦁ promise⦁ ⦁ and⦁ ⦁ potential
pitfalls of chimeric antigen receptors. Curr. Opin. Immunol. 2009, 21, 215−223.
⦁ Savoldo, B.; Ramos, C. A.; Liu, E.; Mims, M. P.; Keating, M. J.;
Carrum, G.; Kamble, R. T.; Bollard, C. M.; Gee, A. P.; Mei, Z.; Liu,
H.; Grilley, B.; Rooney, C. M.; Heslop, H. E.; Brenner, M. K.; Dotti,
G. CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients. J. Clin. Invest. 2011, 121, 1822−1826.
⦁ Milone, M. C.; Fish, J. D.; Carpenito, C.; Carroll, R. G.; Binder,
G. K.; Teachey, D.; Samanta, M.; Lakhal, M.; Gloss, B.; Danet- Desnoyers, G.; Campana, D.; Riley, J. L.; Grupp, S. A.; June, C. H. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol. Ther. 2009, 17, 1453−1464.
⦁ Govers, C.; Sebestyen, Z.; Roszik, J.; van Brakel, M.;
Berrevoets, C.; Szoor, A.; Panoutsopoulou, K.; Broertjes, M.; Van, T.; Vereb, G.; Szollosi, J.; Debets, R. TCRs genetically linked to CD28 and CD3epsilon do not mispair with endogenous TCR chains and mediate enhanced T cell persistence and anti-melanoma activity. J. Immunol. 2014, 193, 5315−5326.
⦁ Carpenito, C.; Milone, M. C.; Hassan, R.; Simonet, J. C.;
Lakhal, M.; Suhoski, M. M.; Varela-Rohena, A.; Haines, K. M.; Heitjan, D. F.; Albelda, S. M.; Carroll, R. G.; Riley, J. L.; Pastan, I.; June, C. H. Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 3360−3365.
⦁ Hombach, A. A.; Chmielewski, M.; Rappl, G.; Abken, H.
Adoptive immunotherapy with redirected T cells produces CCR7- cells that are trapped in the periphery and benefit from combined CD28-OX40 costimulation. Hum. Gene Ther. 2013, 24, 259−269.
⦁ Abate-Daga, D.; Lagisetty, K. H.; Tran, E.; Zheng, Z.;
Gattinoni, L.; Yu, Z.; Burns, W. R.; Miermont, A. M.; Teper, Y.; Rudloff, U.; Restifo, N. P.; Feldman, S. A.; Rosenberg, S. A.; Morgan,
R. A. A novel chimeric antigen receptor against prostate stem cell antigen mediates tumor destruction in a humanized mouse model of pancreatic cancer. Hum. Gene Ther. 2014, 25 (12), 1003−1012.
⦁ Chmielewski, M.; Abken, H. ⦁ TRUCKs:⦁ ⦁ the⦁ ⦁ fourth⦁ ⦁ generation⦁ ⦁ of ⦁ CARs.⦁ Expert Opin. Biol. Ther. 2015, 15, 1145−1154.
⦁ Alizadeh, D.; Wong, R. A.; Yang, X.; Wang, D.; Pecoraro, J. R.;
Kuo, C. F.; Aguilar, B.; Qi, Y.; Ann, D. K.; Starr, R.; Urak, R.; Wang, X.; Forman, S. J.; Brown, C. E. IL15 enhances CAR-T cell antitumor activity by reducing mTORC1 activity and preserving their stem cell memory phenotype. Cancer Immunol. Res. 2019, 7 (5), 759−772.
⦁ Avanzi, M. P.; Yeku, O.; Li, X.; Wijewarnasuriya, D. P.; van
Leeuwen, D. G.; Cheung, K.; Park, H.; Purdon, T. J.; Daniyan, A. F.;
Spitzer, M. H.; Brentjens, R. J. Engineered tumor-targeted T cells mediate enhanced anti-tumor efficacy both directly and through activation of the endogenous immune system. Cell Rep. 2018, 23 (7), 2130−2141.
⦁ Adachi, K.; Kano, Y.; Nagai, T.; Okuyama, N.; Sakoda, Y.;
Tamada, K. IL-7 and CCL19 expression in CAR-T cells improves immune cell infiltration and CAR-T cell survival in the tumor. Nat. Biotechnol. 2018, 36 (4), 346−351.
⦁ Kagoya, Y.; Tanaka, S.; Guo, T.; Anczurowski, M.; Wang, C.
H.; Saso, K.; Butler, M. O.; Minden, M. D.; Hirano, N. A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects. Nat. Med. 2018, 24, 352−359.
⦁ Tokarew, N.; Ogonek, J.; Endres, S.; von Bergwelt-Baildon, M.;
Kobold, S. Teaching an old dog new tricks: next-generation CAR T cells. Br. J. Cancer 2019, 120, 26−37.
⦁ Schuster, S. J.; Bishop, M. R.; Tam, C. S.; Waller, E. K.;
Borchmann, P.; McGuirk, J. P.; Jäger, U.; Jaglowski, S.; Andreadis, C.; Westin, J. R.; Fleury, I.; Bachanova, V.; Foley, S. R.; Ho, P. J.; Mielke,
S.; Magenau, J. M.; Holte, H.; Pantano, S.; Pacaud, L. B.; Awasthi, R.; Chu, J.; Anak, Ö.; Salles, G.; Maziarz, R. T. Tisagenlecleucel in adult relapsed or refractory diffuse large B-cell lymphoma. N. Engl. J. Med. 2019, 380 (1), 45−56.
⦁ Neelapu, S. S.; Locke, F. L.; Bartlett, N. L.; Lekakis, L. J.;
Miklos, D. B.; Jacobson, C. A.; Braunschweig, I.; Oluwole, O. O.; Siddiqi, T.; Lin, Y.; Timmerman, J. M.; Stiff, P. J.; Friedberg, J. W.;
Flinn, I. W.; Goy, A.; Hill, B. T.; Smith, M. R.; Deol, A.; Farooq, U.;
McSweeney, P.; Munoz, J.; Avivi, I.; Castro, J. E.; Westin, J. R.; Chavez, J. C.; Ghobadi, A.; Komanduri, K. V.; Levy, R.; Jacobsen, E. D.; Witzig, T. E.; Reagan, P.; Bot, A.; Rossi, J.; Navale, L.; Jiang, Y.; Aycock, J.; Elias, M.; Chang, D.; Wiezorek, J.; Go, W. Y. Axicabtagene Ciloleucel CAR T-Cell therapy in refractory large B-cell lymphoma. N. Engl. J. Med. 2017, 377 (26), 2531−2544.
⦁ Brudno, J. N.; Kochenderfer, J. N. ⦁ To⦁ x⦁ icities⦁ ⦁ of⦁ ⦁ chimeric
antigen receptor T cells: recognition and management. Blood 2016,
127, 3321−3330.
⦁ Kochenderfer, J. N.; Dudley, M. E.; Feldman, S. A.; Wilson, W.
H.; Spaner, D. E.; Maric, I.; Stetler-Stevenson, M.; Phan, G. Q.; Hughes, M. S.; Sherry, R. M.; Yang, J. C.; Kammula, U. S.; Devillier, L.; Carpenter, R.; Nathan, D. A.; Morgan, R. A.; Laurencot, C.; Rosenberg, S. A. B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood 2012, 119,
2709−2720.
⦁ Lee, D. W.; Gardner, R.; Porter, D. L.; Louis, C. U.; Ahmed, N.;
Jensen, M.; Grupp, S. A.; Mackall, C. L. Current concepts in the diagnosis and management of cytokine release syndrome. Blood 2014, 124, 188−195.
⦁ Lee, D. W.; Santomasso, B. D.; Locke, F. L.; Ghobadi, A.;
Turtle, C. J.; Brudno, J. N.; Maus, M. V.; Park, J. H.; Mead, E.; Pavletic, S.; Go, W. Y.; Eldjerou, L.; Gardner, R. A.; Frey, N.; Curran,
K. J.; Peggs, K.; Pasquini, M.; DiPersio, J. F.; van den Brink, M. R. M.; Komanduri, K. V.; Grupp, S. A.; Neelapu, S. S. ASBMT consensus grading for cytokine release syndrome and neurologic toxicity associated with immune effector cells. Biol. Blood Marrow Transplant. 2019, 25 (4), 625−638.
⦁ Davila, M. L.; Riviere, I.; Wang, X.; Bartido, S.; Park, J.; Curran,
K.; Chung, S. S.; Stefanski, J.; Borquez-Ojeda, O.; Olszewska, M.; Qu, J.; Wasielewska, T.; He, Q.; Fink, M.; Shinglot, H.; Youssif, M.; Satter, M.; Wang, Y.; Hosey, J.; Quintanilla, H.; Halton, E.; Bernal, Y.;
Bouhassira, D. C.; Arcila, M. E.; Gonen, M.; Roboz, G. J.; Maslak, P.; Douer, D.; Frattini, M. G.; Giralt, S.; Sadelain, M.; Brentjens, R. Efficacy and toxicity management of 19−28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci. Transl. Med. 2014, 6 (224), 224ra25.
⦁ Norelli, M.; Camisa, B.; Barbiera, G.; Falcone, L.; Purevdorj, A.; Genua, M.; Sanvito, F.; Ponzoni, M.; Doglioni, C.; Cristofori, P.; Traversari, C.; Bordignon, C.; Ciceri, F.; Ostuni, R.; Bonini, C.; Casucci, M.; Bondanza, A. ⦁ Monocyte-derived⦁ ⦁ IL-1⦁ ⦁ and⦁ ⦁ IL-6⦁ ⦁ are

differentially required for cytokine-release syndrome and neuro- toxicity due to CAR T cells. Nat. Med. 2018, 24 (6), 739−748.
⦁ Liu, Y.; Fang, Y.; Chen, X.; Wang, Z.; Liang, X.; Zhang, T.; Liu,
M.; Zhou, N.; Lv, J.; Tang, K.; Xie, J.; Gao, Y.; Cheng, F.; Zhou, Y.;
Zhang, Z.; Hu, Y.; Zhang, X.; Gao, Q.; Zhang, Y.; Huang, B. Gasdermin E-mediated Target Cell Pyroptosis by CAR T Cells Triggers Cytokine Release Syndrome. Sci. Immunol. 2020, 5 (43), No. eaax7969.
⦁ Maude, S. L.; Barrett, D.; Teachey, D. T.; Grupp, S. A. ⦁ Managing cytokine release syndrome associated with novel T ⦁ cell- ⦁ engaging therapies. Cancer J. 2014, 20 (2), 119−122.
⦁ Richman, S. A.; Nunez-Cruz, S.; Moghimi, B.; Li, L. Z.;
Gershenson, Z. T.; Mourelatos, Z.; Barrett, D. M.; Grupp, S. A.; Milone, M. C. High-affinity GD2-specific CAR T cells induce fatal encephalitis in a preclinical neuroblastoma model. Cancer Immunol. Res. 2018, 6 (1), 36−46.
⦁ Neelapu, S. S.; Tummala, S.; Kebriaei, P.; Wierda, W.;
Gutierrez, C.; Locke, F. L.; Komanduri, K. V.; Lin, Y.; Jain, N.; Daver, N.; Westin, J.; Gulbis, A. M.; Loghin, M. E.; de Groot, J. F.; Adkins, S.; Davis, S. E.; Rezvani, K.; Hwu, P.; Shpall, E. J. Chimeric antigen receptor T-cell therapy – assessment and management of toxicities. Nat. Rev. Clin. Oncol. 2018, 15 (1), 47−62.
⦁ Gust, J.; Taraseviciute, A.; Turtle, C. J. ⦁ Neuroto⦁ x⦁ icity
Associated with CD19-Targeted CAR-T Cell Therapies. CNS Drugs
2018, 32 (12), 1091−1101.
⦁ Li, G.; Wong, A. J. ⦁ EGF⦁ ⦁ receptor⦁ ⦁ variant⦁ ⦁ III⦁ ⦁ as⦁ ⦁ a⦁ ⦁ target⦁ ⦁ antigen ⦁ for⦁ ⦁ tumor⦁ ⦁ immunotherapy.⦁ Expert Rev. Vaccines 2008, 7, 977−985.
⦁ Pfeiffer, A.; Thalheimer, F. B.; Hartmann, S.; Frank, A. M.;
Bender, R. R.; Danisch, S.; Costa, C.; Wels, W. S.; Modlich, U.; Stripecke, R.; Verhoeyen, E.; Buchholz, C. J. In vivo generation of human CD19-CAR T cells results in B-cell depletion and signs of cytokine release syndrome. EMBO Mol. Med. 2018, 10, e9158.
⦁ Morgan, R. A.; Yang, J. C.; Kitano, M.; Dudley, M. E.; Laurencot, C. M.; Rosenberg, S. A. ⦁ Case report of a serious adverse ⦁ event following the administration of T cells transduced with ⦁ a ⦁ chimeric⦁ ⦁ antigen⦁ ⦁ receptor⦁ ⦁ recognizing⦁ ⦁ ERBB2. Mol. Ther. 2010, 18, 843−851.
⦁ Anwer, F.; Shaukat, A. A.; Zahid, U.; Husnain, M.; McBride, A.;
PerskY, D.; Lim, M.; Hasan, N.; Riaz, I. B. Donor origin CAR T cells: graft versus malignancy effect without GVHD, a systematic review. Immunotherapy 2017, 9, 123−130.
⦁ Tey, S. K. ⦁ Adoptive T-cell therapy: adverse events and⦁ ⦁ safety
switches. Clin. Transl. Immunol. 2014, 3, No. e17.
⦁ Acerbi, F.; Cavallo, C.; Broggi, M.; Cordella, R.; Anghileri, E.; Eoli, M.; Schiariti, M.; Broggi, G.; Ferroli, P. ⦁ Fluorescein-guided ⦁ surgery⦁ ⦁ for⦁ ⦁ malignant⦁ ⦁ gliomas:⦁ ⦁ a⦁ ⦁ review. Neurosurg. Rev. 2014, 37, 547−557.
⦁ van Dam, G. M.; Themelis, G.; Crane, L. M.; Harlaar, N. J.;
Pleijhuis, R. G.; Kelder, W.; Sarantopoulos, A.; de Jong, J. S.; Arts, H. J.; van der Zee, A. G.; Bart, J.; Low, P. S.; Ntziachristos, V. Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-alpha targeting: first in-human results. Nat. Med. 2011, 17, 1315−1319.
⦁ Leopoldo, M.; Lacivita, E.; Berardi, F.; Perrone, R. ⦁ Develop-
ments in fluorescent probes for receptor research. Drug Discovery Today 2009, 14, 706−712.
⦁ Ma, J. S.; Kim, J. Y.; Kazane, S. A.; Choi, S. H.; Yun, H. Y.; Kim,
M. S.; Rodgers, D. T.; Pugh, H. M.; Singer, O.; Sun, S. B.; Fonslow, B. R.; Kochenderfer, J. N.; Wright, T. M.; Schultz, P. G.; Young, T. S.; Kim, C. H.; Cao, Y. Versatile strategy for controlling the specificity and activity of engineered T cells. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E450−E458.
⦁ Cao, Y.; Rodgers, D. T.; Du, J.; Ahmad, I.; Hampton, E. N.;
Ma, J. S.; Mazagova, M.; Choi, S. H.; Yun, H. Y.; Xiao, H.; Yang, P.;
Luo, X.; Lim, R. K.; Pugh, H. M.; Wang, F.; Kazane, S. A.; Wright, T. M.; Kim, C. H.; Schultz, P. G.; Young, T. S. Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer. Angew. Chem., Int. Ed. 2016, 55, 7520−7524.
⦁ Lee, Y. G.; Marks, I.; Srinivasarao, M.; Kanduluru, A. K.; Mahalingam, S. M.; Liu, X.; Chu, H.; Low, P. S. ⦁ Use⦁ ⦁ of⦁ ⦁ a⦁ ⦁ single⦁ ⦁ CAR ⦁ T cell and several bispecific adapters facilitates eradication of multiple ⦁ antigenically⦁ ⦁ different⦁ ⦁ solid⦁ ⦁ tumors.⦁ Cancer Res. 2019, 79 (2), 387− 396.
⦁ Pellegrino, C.; Favalli, N.; Sandholzer, M.; Volta, L.; Bassi, G.; Millul, J.; Cazzamalli, S.; Matasci, M.; Villa, A.; Myburgh, R.; Manz,
M. G.; Neri, D. Impact of ligand size and conjugation chemistry on the performance of universal chimeric antigen receptor T-cells for tumor killing. Bioconjugate Chem. 2020, 31 (7), 1775−1783.
⦁ Zhang, E.; Gu, J.; Xue, J.; Lin, C.; Liu, C.; Li, M.; Hao, J.;
Setrerrahmane, S.; Chi, X.; Qi, W.; Hu, J.; Xu, H. Accurate control of dual-receptor-engineered T cell activity through a bifunctional anti- angiogenic peptide. J. Hematol. Oncol. 2018, 11, 44.
⦁ Xia, W.; Low, P. S. ⦁ Folate-targeted⦁ ⦁ therapies⦁ ⦁ for⦁ ⦁ cancer.⦁ J. Med. Chem. 2010, 53 (19), 6811−6824.
⦁ Low, P. S.; Kularatne, S. A. ⦁ Folate-targeted⦁ ⦁ therapeutic⦁ ⦁ and ⦁ imaging⦁ ⦁ agents⦁ ⦁ for⦁ ⦁ cancer.⦁ Curr. Opin. Chem. Biol. 2009, 13 (3), 256− 262.
⦁ Shen, J.; Hu, Y.; Putt, K. S.; Singhal, S.; Han, H.; Visscher, D. W.; Murphy, L. M.; Low, P. S. ⦁ Assessment⦁ ⦁ of⦁ ⦁ folate⦁ ⦁ receptor⦁ ⦁ alpha ⦁ and⦁ ⦁ beta⦁ ⦁ expression⦁ ⦁ in⦁ ⦁ selection⦁ ⦁ of⦁ ⦁ lung⦁ ⦁ and⦁ ⦁ pancreatic⦁ ⦁ cancer⦁ ⦁ patients ⦁ for⦁ ⦁ receptor⦁ ⦁ targeted⦁ ⦁ therapies.⦁ Oncotarget 2018, 9, 4485−4495.
⦁ O’Shannessy, D. J.; Yu, G.; Smale, R.; Fu, Y. S.; Singhal, S.;
Thiel, R. P.; Somers, E. B.; Vachani, A. Folate receptor alpha expression in lung cancer: diagnostic and prognostic significance. Oncotarget 2012, 3 (4), 414−425.
⦁ Boogerd, L. S.; Boonstra, M. C.; Beck, A. J.; Charehbili, A.;
Hoogstins, C. E.; Prevoo, H. A.; Singhal, S.; Low, P. S.; van de Velde,
C. J.; Vahrmeijer, A. L. Concordance of folate receptor-α expression between biopsy, primary tumor and metastasis in breast cancer and lung cancer patients. Oncotarget 2016, 7, 17442−17454.
⦁ Rader, C.; Turner, J. M.; Heine, A.; Shabat, D.; Sinha, S. C.;
Wilson, I. A.; Lerner, R. A.; Barbas, C. F. A humanized aldolase antibody for selective chemotherapy and adaptor immunotherapy. J. Mol. Biol. 2003, 332, 889−899.
⦁ Qi, J.; Tsuji, K.; Hymel, D.; Burke, T. R., Jr; Hudecek, M.;
Rader, C.; Peng, H. Chemically programmable and switchable CAR-T therapy. Angew. Chem., Int. Ed. 2020, 59, 12178−12185.
⦁ Kim, M. S.; Ma, J. S.; Yun, H.; Cao, Y.; Kim, J. Y.; Chi, V.;
Wang, D.; Woods, A.; Sherwood, L.; Caballero, D.; Gonzalez, J.; Schultz, P. G.; Young, T. S.; Kim, C. H. Redirection of genetically engineered CAR-T cells using bifunctional small molecules. J. Am. Chem. Soc. 2015, 137, 2832−2835.
⦁ Iwakiri, S.; Sonobe, M.; Nagai, S.; Hirata, T.; Wada, H.;
Miyahara, R. Expression status of folate receptor alpha is significantly correlated with prognosis in non-small-cell lung cancers. Ann. Surg. Oncol. 2008, 15, 889−899.
⦁ Chu, W.; Zhou, Y.; Tang, Q.; Wang, M.; Ji, Y.; Yan, J.; Yin, D.;
Zhang, S.; Lu, H.; Shen, J. Bi-specific ligand-controlled chimeric antigen receptor T-cell therapy for non-small cell lung cancer. BioSci. Trends 2018, 12, 298−308.
⦁ Romano, S.; Sorrentino, A.; Di Pace, A. L.; Nappo, G.;
Mercogliano, C.; Romano, M. F. The emerging role of large immunophilin FK506 binding protein 51 in cancer. Curr. Med. Chem. 2011, 18, 5424−5429.
⦁ Abdel-Magid, A. F. ⦁ Rapalogs⦁ ⦁ potential⦁ ⦁ as⦁ ⦁ practical⦁ ⦁ alternatives ⦁ to rapamycin. ACS Med. Chem. Lett. 2019, 10 (6), 843−845.
⦁ Bayle, J. H.; Grimley, J. S.; Stankunas, K.; Gestwicki, J. E.;
Wandless, T. J.; Crabtree, G. R. Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity. Chem. Biol. 2006, 13 (1), 99−107.
⦁ Choi, J.; Chen, J.; Schreiber, S. L.; Clardy, J. ⦁ Structure of⦁ ⦁ the
FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science 1996, 273 (5272), 239−242.
⦁ Wu, C. Y.; Roybal, K. T.; Puchner, E. M.; Onuffer, J.; Lim, W.
A. Remote control of therapeutic T cells through a small molecule- gated chimeric receptor. Science 2015, 350, No. aab4077.

⦁ Leung, W. H.; Gay, J.; Martin, U.; Garrett, T. E.; Horton, H. M.; Certo, M. T.; Blazar, B. R.; Morgan, R. A.; Gregory, P. D.; Jarjour, J.; Astrakhan, A. ⦁ Sensitive⦁ ⦁ and⦁ ⦁ adaptable⦁ ⦁ pharmacological⦁ ⦁ control⦁ ⦁ of ⦁ CAR T cells through extracellular receptor dimerization. JCI Insight 2019, 4, No. e124430.
⦁ Juillerat, A.; Marechal, A.; Filhol, J. M.; Valton, J.; Duclert, A.; Poirot, L.; Duchateau, P. ⦁ Design of chimeric antigen receptors ⦁ with ⦁ integrated controllable transient functions. Sci. Rep. 2016, 6, 18950.
⦁ Duong, M. T.; Collinson-Pautz, M. R.; Morschl, E.; Lu, A.; Szymanski, S. P.; Zhang, M.; Brandt, M. E.; Chang, W. C.; Sharp, K. L.; Toler, S. M.; Slawin, K. M.; Foster, A. E.; Spencer, D. M.; Bayle, J.
H. Two-dimensional regulation of CAR-T cell therapy with orthogonal switches. Mol. Ther. Oncolytics 2019, 12, 124−137.
⦁ Philip, B.; Kokalaki, E.; Mekkaoui, L.; Thomas, S.; Straathof, K.;
Flutter, B.; Marin, V.; Marafioti, T.; Chakraverty, R.; Linch, D.; Quezada, S. A.; Peggs, K. S.; Pule, M. A highly compact epitope-based marker/suicide gene for easier and safer T-cell therapy. Blood 2014, 124, 1277−1287.
⦁ Ciceri, F.; Bonini, C.; Stanghellini, M. T.; Bondanza, A.;
Traversari, C.; Salomoni, M.; Turchetto, L.; et al. Infusion of suicide- gene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the TK007 trial): a non-randomised phase I-II study. Lancet Oncol. 2009, 10, 489−500.
⦁ Berger, C.; Flowers, M. E.; Warren, E. H.; Riddell, S. R.
Analysis of transgene-specific immune responses that limit the in vivo persistence of adoptively transferred HSV-TK-modified donor T cells after allogeneic hematopoietic cell transplantation. Blood 2006, 107, 2294−2302.
⦁ Marin, V.; Cribioli, E.; Philip, B.; Tettamanti, S.; Pizzitola, I.;
Biondi, A.; Biagi, E.; Pule, M. Comparison of different suicide-gene strategies for the safety improvement of genetically manipulated T cells. Hum. Gene Ther: Methods. 2012, 23, 376−386.
⦁ Di Stasi, A.; Tey, S. K.; Dotti, G.; Fujita, Y.; Kennedy-Nasser,
⦁ ; Martinez, C.; Straathof, K.; Liu, E.; Durett, A. G.; Grilley, B.; Liu,
H.; Cruz, C. R.; Savoldo, B.; Gee, A. P.; Schindler, J.; Krance, R. A.;
Heslop, H. E.; Spencer, D. M.; Rooney, C. M.; Brenner, M. K. Inducible apoptosis as a safety switch for adoptive cell therapy. N. Engl. J. Med. 2011, 365, 1673−1683.
⦁ Diaconu, I.; Ballard, B.; Zhang, M.; Chen, Y.; West, J.; Dotti,
G.; Savoldo, B. Inducible Caspase-9 selectively modulates the toxicities of CD19-specific chimeric antigen receptor-modified T cells. Mol. Ther. 2017, 25, 580−592.
⦁ Hoyos, V.; Savoldo, B.; Quintarelli, C.; Mahendravada, A.;
Zhang, M.; Vera, J.; Heslop, H. E.; Rooney, C. M.; Brenner, M. K.; Dotti, G. Engineering CD19-specific T lymphocytes with interleukin-
15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety. Leukemia 2010, 24, 1160−1170.
⦁ Budde, L. E.; Berger, C.; Lin, Y.; Wang, J.; Lin, X.; Frayo, S. E.;
Brouns, S. A.; Spencer, D. M.; Till, B. G.; Jensen, M. C.; Riddell, S. R.; Press, O. W. Combining a CD20 chimeric antigen receptor and an inducible Caspase 9 suicide switch to improve the efficacy and safety of T cell adoptive immunotherapy for lymphoma. PLoS One 2013, 8, No. e82742.
⦁ Zhou, X.; Dotti, G.; Krance, R. A.; Martinez, C. A.; Naik, S.; Kamble, R. T.; Durett, A. G.; Dakhova, O.; Savoldo, B.; Di Stasi, A.; Spencer, D. M.; Lin, Y. F.; Liu, H.; Grilley, B. J.; Gee, A. P.; Rooney,
C. M.; Heslop, H. E.; Brenner, M. K. Inducible caspase-9 suicide gene controls adverse effects from alloreplete T cells after haploidentical stem cell transplantation. Blood 2015, 125, 4103−4113.
⦁ Foster, A. E.; Mahendravada, A.; Shinners, N. P.; Chang, W. C.;
Crisostomo, J.; Lu, A.; Khalil, M.; Morschl, E.; Shaw, J. L.; Saha, S.; Duong, M. T.; Collinson-Pautz, M. R.; Torres, D. L.; Rodriguez, T.; Pentcheva-Hoang, T.; Bayle, J. H.; Slawin, K. M.; Spencer, D. M. Regulated expansion and survival of chimeric antigen receptor- modified T cells using small molecule-dependent inducible MyD88/ CD40. Mol. Ther. 2017, 25, 2176−2188.
⦁ Zhou, X.; Di Stasi, A.; Tey, S. K.; Krance, R. A.; Martinez, C.;
Leung, K. S.; Durett, A. G.; Wu, M. F.; Liu, H.; Leen, A. M.; Savoldo,
⦁ ; Lin, Y. F.; Grilley, B. J.; Gee, A. P.; Spencer, D. M.; Rooney, C. M.; Heslop, H. E.; Brenner, M. K.; Dotti, G. ⦁ Long-term⦁ ⦁ outcome⦁ ⦁ after ⦁ haploidentical⦁ ⦁ stem⦁ ⦁ cell⦁ ⦁ transplant⦁ ⦁ and⦁ ⦁ infusion⦁ ⦁ of⦁ ⦁ T⦁ ⦁ cells⦁ ⦁ expressing ⦁ the inducible caspase 9 safety transgene. Blood 2014, 123, 3895−
3905.
⦁ Lee, S. M.; Kang, C. H.; Choi, S. U.; Kim, Y.; Hwang, J. Y.; Jeong, H. G.; Park, C. H. ⦁ A⦁ ⦁ Chemical⦁ ⦁ Switch⦁ ⦁ System⦁ ⦁ to⦁ ⦁ Modulate ⦁ Chimeric Antigen Receptor T Cell Activity through ⦁ Proteolysis- ⦁ Targeting⦁ ⦁ Chimaera⦁ ⦁ Technology.⦁ ACS Synth. Biol. 2020, 9 (5), 987− 992.
⦁ Liang, Y.; Nandakumar, K. S.; Cheng, K. ⦁ Design and ⦁ pharmaceutical⦁ ⦁ applications⦁ ⦁ of⦁ ⦁ proteolysis-targeting⦁ ⦁ chimeric⦁ ⦁ mole- ⦁ cules. Biochem. Pharmacol. 2020, 182, 114211.
⦁ Varshavsky, A. ⦁ N-degron⦁ ⦁ and⦁ ⦁ C-degron⦁ ⦁ pathways⦁ ⦁ of⦁ ⦁ protein ⦁ degradation.⦁ Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 358−366.
⦁ Bouchnak, I.; van Wijk, K. J. ⦁ N-Degron Pathways in⦁ ⦁ Plastids.
Trends Plant Sci. 2019, 24, 917−926.
⦁ Zheng, N.; Shabek, N. ⦁ Ubiquitin ligases: structure, function, ⦁ and regulation. Annu. Rev. Biochem. 2017, 86, 129−157.
⦁ Yesbolatova, A.; Tominari, Y.; Kanemaki, M. T. ⦁ Ligand-induced
genetic degradation as a tool for target validation. Drug Discovery Today: Technol. 2019, 31, 91−98.
⦁ Juillerat, A.; Tkach, D.; Busser, B. W.; Temburni, S.; Valton, J.;
Duclert, A.; Poirot, L.; Depil, S.; Duchateau, P. Modulation of chimeric antigen receptor surface expression by a small molecule switch. BMC Biotechnol. 2019, 19, 44.
⦁ Raina, K.; Lu, J.; Qian, Y.; Altieri, M.; Gordon, D.; Rossi, A. M.; Wang, J.; Chen, X.; Dong, H.; Siu, K.; Winkler, J. D.; Crew, A. P.; Crews, C. M.; Coleman, K. G. ⦁ PROTAC-induced BET ⦁ protein ⦁ degradation⦁ ⦁ as⦁ ⦁ a⦁ ⦁ therapy⦁ ⦁ for⦁ ⦁ castration-resistant⦁ ⦁ prostate⦁ ⦁ cancer.⦁ Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (26), 7124−7129.
⦁ Lu, J.; Qian, Y.; Altieri, M.; Dong, H.; Wang, J.; Raina, K.;
Hines, J.; Winkler, J. D.; Crew, A. P.; Coleman, K.; Crews, C. M. Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4. Chem. Biol. 2015, 22 (6), 755−763.
⦁ Richman, S. A.; Wang, L.-C.; Moon, E. K.; Khire, U. R.;
Albelda, S. M.; Milone, M. C. Ligand-induced degradation of a CAR permits reversible remote control of CAR T cell activity in vitro and in vivo. Mol. Ther. 2020, 28, 1600−1613.
⦁ Weichsel, R.; Dix, C.; Wooldridge, L.; Clement, M.; Fenton-
May, A.; Sewell, A. K.; Zezula, J.; Greiner, E.; Gostick, E.; Price, D. A.; Einsele, H.; Seggewiss, R. Profound inhibition of antigen-specific T- cell effector functions by dasatinib. Clin. Cancer Res. 2008, 14 (8), 2484−2491.
⦁ Wu, B. X.; Song, N. J.; Riesenberg, B. P.; Li, Z. ⦁ Development⦁ ⦁ of
molecular and pharmacological switches for chimeric antigen receptor T cells. Exp. Hematol. Oncol. 2019, 8, 27.
⦁ Mestermann, K.; Giavridis, T.; Weber, J.; Rydzek, J.; Frenz, S.; Nerreter, T.; Mades, A.; Sadelain, M.; Einsele, H.; Hudecek, M. ⦁ The ⦁ tyrosine kinase inhibitor dasatinib acts as a pharmacologic ⦁ on/off ⦁ switch⦁ ⦁ for⦁ ⦁ CAR⦁ ⦁ T⦁ ⦁ cells.⦁ Sci. Transl. Med. 2019, 11, No. eaau5907.
⦁ Weber, E. W.; Lynn, R. C.; Sotillo, E.; Lattin, J.; Xu, P.; Mackall, C. L. ⦁ Pharmacologic control of CAR-T cell function ⦁ using ⦁ dasatinib. Blood Adv. 2019, 3, 711−717.
⦁ Blake, S.; Hughes, T. P.; Mayrhofer, G.; Lyons, A. B. ⦁ The⦁ ⦁ Src/
ABL kinase inhibitor dasatinib (BMS-354825) inhibits function of normal human T-lymphocytes in vitro. Clin. Immunol. 2008, 127 (3), 330−339.