Recent Updates on Chimeric Antigen Receptor T-Cell Approaches in Cancer Immunotherapy

Abstract

Chimeric antigen receptor (CAR) T-cell therapy is a revolutionary development in the field of cancer immunotherapy, offering a targeted approach to combat various hematologic malignancies. In this treatment, the patient’s genetically modified T cells are extracted and transformed to produce chimeric antigen receptors (CARs) that are exclusive to cancer cells. These altered T cells identify, attach to, and destroy cancer cells when they are reinfused back into the patient, offering a customized course of therapy. While the CAR T-cell therapy’s clinical success has been most evident in cases of acute lymphoblastic leukemia and certain types of lymphomas, ongoing research aims to extend its applicability to solid tumors. Despite its promise, challenges like cytokine release syndrome and the high cost of treatment remain. Nonetheless, CAR T-cell therapy heralds a new era in cancer treatment, offering a potentially curative approach for patients with otherwise refractory diseases.

1. Introduction

Cancer is a global health problem with considerable rate of mortality, so that international agency for research on cancer reported about 10 million mortality and more than 19 million new cases in 2020 [1]. Despite recent advancements in cancer therapy approaches, curing cancer remains a complex challenge. Conventional approaches such as surgery, radiotherapy and chemotherapy have limited therapeutic efficacy, underscoring the importance of developing innovative treatments [2].

Cancer cells possess the ability to escape from the immune system, leading to insufficient anti-tumor immune responses and allowing for the survival and progression of the tumor [3]. Recently, immunotherapy has become a crucial aspect of treatment for many types of diseases, notably cancer. Some immunotherapy approaches enhance the body’s immunity against cancer cells, and others specifically focus on attacking tumor cells and killing them [4, 5]. Several immunotherapy techniques have been adopted in order to eradicate cancer cells, including bispecific antibodies, cytokine-induced killers (CIKs), tumor-infiltrating lymphocytes (TIL), immune checkpoint blockers, monoclonal antibodies (mAbs), tumor vaccines, and genetically engineered immune cells that express the chimeric antigen receptors (CARs) [6]. Different immune cells such as natural killer cells, macrophages, and T cells can be engineered to develop CARs to better recognize and respond against tumor cells [7]. However, lymphocyte T-based chimeric antigen receptors are still the most commonly used CARs [2]. This approach addresses the immune escapes of tumor cells, producing significantly effective T cells to target tumors [8].

CAR T cells are produced through genetic engineering of T cells derived from either the patient or a donor [9, 10, 11, 12]. These cells are modified to express specific receptors to target a surface tumor antigen (TA) and kill cancer cells [4, 5]. Extracellular domain of CAR T cells usually is a single-chain variable fragment (scFv) followed by a hinge, a transmembrane part, and a cytosolic part that produce an intracellular signaling [13]. Unlike traditional T cells, CARs identify unprocessed antigens, along with glycolipids and structures of carbohydrates commonly found on the surface of tumor cells [14]. Also, they are completely independent of the major histocompatibility complex (MHC) [8, 15].

2. CAR T-cell therapy

Esshar’s successful demonstration of genetically engineering cytotoxic T cells toward cancer cells throughout the 1980s indicated that these cells have anti-tumor potential [16]. Since that time, a new cancer treatment revolution known as chimeric antigen receptor (CAR) T-cell therapy has appeared [17].

Genetically modifying T lymphocytes to produce CAR T cells with targeted immune therapies represents a cutting-edge approach to cancer treatment [18]. This method has been used to augment the immune system’s reaction against tumor cells in cases where the adaptive defenses have been ineffective [19]. Targeting tumor-associated antigens and genetically modifying T cells are two aspects of immune therapies based on CAR T cells [20]. T-cell receptors (TCR) in adaptive immunity can only recognize foreign antigens that bind to MHC. Immunotherapy based on CAR T-cells bypasses this mechanism without relying on MHC presentation and is being explored in various clinical trials for its effectiveness against different types of cancers [21]. Furthermore, this method is specific and adaptable, as the TCR domain in CAR T-cell could be modified to recognize specific antigens [22]. Additionally, treatment based on CAR T cells can form memory within patients [23].

Several solid cancers such as pancreatic, colorectal, lung, thyroid, breast, and prostate cancers, as well as hematologic malignancies like Hodgkin’s and non-Hodgkin lymphomas, B-cell and T-cell lymphomas, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and multiple myeloma (MM) have all been studied in CAR T-cell therapy [21, 24, 25]. Nevertheless, treating hematologic disorders has been one of the most prominent applications of CAR T-cell treatments. The Food and Drug Administration (FDA) has approved some CAR T-cell therapies to treat hematologic malignancies that express BCMA or CD19 antigens [26, 27, 28].

The standard CAR T-cell production involves five stages (Figure 1). Initially, T lymphocytes are extracted from patients [29]. Next, these T lymphocytes are modified with CARs, enabling them to identify tumor cells and activate themselves, thereby forming CAR T cells [8, 30]. Gene transfer methods such as viral or non-viral vectors insert CAR genes into T-lymphocyte genomes [31]. Subsequently, the modified T-lymphocyte known as CAR T-cell undergo ex vivo cultivation and stimulation through cytokines to generate a substantial quantity of CAR T lymphocytes [32]. The next phase involves reintroducing the modified T lymphocytes into the patient at the proper dosage [30]. Then, the immune system based on CAR T cells can identify and remove cancer cells that express the specific target antigen after being reinfusioned into patients [33, 34]. Finally, patients require vigilant monitoring to oversee and manage various physical responses in the subsequent days [35]. This entire process spans around 3 weeks, with CAR T-cell preparation taking approximately 2 weeks, rendering it the most time-consuming phase [23]. Both autologous and allogeneic CAR T cells have made successful transitions from preclinical to clinical stages [36]. Meanwhile, among them, autologous CAR T cells have gained approval for treating cancer patients. Over 500 treatments based on CAR T cells have been the subject of global clinical trials [37, 38, 39].

Figure 1.

The schematic chart of standard CAR T-cell production.

A basic CAR includes four fundamental components: an antigen-binding domain, a hinge segment, a transmembrane domain, and an intracellular domain (Figure 2A) [40, 41]. The antigen-binding domain is responsible for recognizing specific target antigens, while the intracellular domain delivers activation and costimulatory signals [42]. The hinge and transmembrane domains connect these two sections, also impacting their functional traits [39].

Figure 2.

The composition of distinct generations of CARs. (A) This diagram illustrates the essential elements of a CAR’s extracellular, transmembrane, and intracellular domains (endodomains). (B) This diagram illustrates the progression of CAR development from the first to the fifth generation [43].

2.1 Antigen-binding domain

A CAR is a type of recombinant receptor that possesses the ability to both bind tumor antigen and stimulate T lymphocytes [44]. The antigen-binding region is the segment of the CAR T cells responsible for determining the specificity of the specific antigen [45]. The variable light chains (VL) and variable heavy chains (VH) present in antibodies (Ab) are the source of these domains [46]. These chains are connected together through a flexible connector of glycine and serine residues, creating a scFv [47]. The scFvs within CAR T cells have typically targeted tumor antigens on the extracellular surface, resulting in stimulation and growth of T cells independent of the MHC [48, 49].

The scFv has properties beyond just identifying and attaching to the target region [50]. The complementarity-determining regions’ placement and the interaction between the VH and VL are two factors significantly affecting the CAR’s accuracy and specificity in identifying its target [51].

The level of affinity is a crucial parameter within the antigen-binding domain, as it essentially controls the functioning of CARs [52]. The CAR’s antigen-binding affinity needs to be sufficiently high for optimal performance to identify target antigens, trigger CAR T-cell signaling, and stimulate T lymphocytes [50]. Yet, it should not be excessively high to cause apoptosis by interaction with a death factor and its receptor in CAR T lymphocytes or lead to potential toxic effects [53]. While affinity is significant, it is notable that different scFvs can have different effects on CAR T-cell performance, although they have identical affinity [53]. Hence, achieving optimal CAR binding to its specific antigen necessitates consideration of additional factors, including epitope position, specific antigen density, and avoiding scFvs linked to ligand-free tonic signaling mechanisms [47].

A transmembrane domain of protein transmembrane segments such as CD3, CD8, CD28, or FcεRI fuses the scFV to the T lymphocyte [50]. This section connects to the intracellular region, which includes the intracytoplasmic regions of proteins like CD8, CD28, or CD137, as well as CD3ζ [54].

2.2 Intracellular domain

The immune receptor tyrosine-based activation motif (ITAM), usually present on the CD3ζ chain in the TCR complex and starts the costimulatory signal transduction that activates T lymphocytes, is a part of the intracellular domain [52]. An scFv that identifies recombinant tumor-associated-antigens (TAAs) and an ITAM region is created in order to manufacture CAR T cells [55]. The standard method for delivering CAR molecules into T cells is in vitro transfection [56]. Transfection methods can involve viral methods such as retro or lentivirus or non-viral methods like transposon and mRNA electrotransfection [54]. These components are integrated into a recombinant plasmid in vitro during the manufacturing process [57]. Following this, T cells are exposed to the recombinant plasmid, which makes it possible for these cells to make the receptors required for antigens on the cell membrane of tumors [55]. Post-transfection, the T lymphocytes undergo expansion. CAR T cells possess the capability to recognize and eradicate tumor cells without relying on MHC molecules, thereby overcoming potential immune evasion strategies involving reduced MHC expression by tumor cells [58]. However, the targeting is highly selective as CAR T cells can recognize tumor-specific-antigens on the cell membranes of tumor cells [59, 60]. Alongside the intracellular signaling domain, costimulatory molecules like CD137 (4-1BB) or CD28 can enhance T-lymphocyte proliferation, cytokine production, T-lymphocyte activation, and prolong in vivo survival [58]. These additions also boost the anti-tumor effectiveness of CART cells [61, 62, 63, 64].

2.3 Transmembrane domain

The transmembrane domain links the intracellular and antigen-binding domains, functioning as the linker of the T-lymphocyte membrane [65]. This segment typically originates from a transmembrane receptor protein, and its selection affects the signal transduction and stimulation of the intracellular segment [48]. The transmembrane domain of CARs is likely the least understood of all their components [66]. Its main function is to attach the CAR to the membrane of the T lymphocyte [67]. However, data indicate that the transmembrane domain may be necessary for CAR T-cell function [68].

In particular, research indicates that the transmembrane domains of CAR T cells impact CAR expression levels, stability, potential signaling activity, synapse formation, and the ability to dimerize with natural signaling molecules [47, 69, 70, 71]. Numerous transmembrane domains, such as CD3ζ, CD4, CD8α, or CD28, are sourced from naturally occurring proteins [66]. As the transmembrane region is usually altered to align the requirements of the antigen-binding region or the intracellular domains, comparing the effects of different transmembrane domains on CAR function still needs to be better understood [47].

The CD3ζ transmembrane domain notably aids in CAR-mediated T-cell activation by promoting CAR dimerization and integration into native TCRs [70]. However, this advantageous effect of the CD3ζ transmembrane domain compromises the stability of CAR in comparison to those with the CD28 transmembrane domain [34]. The transmembrane domain and the hinge region seem to work together to influence CAR T-cell cytokine production as well as activation-induced cell death (AICD) [72].

Generally, research indicates that the intracellular domain with its associated transmembrane region may be the key to achieving optimum CAR T-cell signaling and activating [67]. The commonly used transmembrane domains of CD8α or CD28 may enhance CAR expression and stability [47].

2.4 An extracellular hinge or spacer domain

The hinge domain consists of members from the immunoglobulin (Ig), such as CD8, CD28, or IgG, contributing to signal transduction [73]. The antigen-binding domain extends from the transmembrane domain by extracellular segment known as the hinge or spacer domain [74]. It provides length and flexibility to cross barriers, allowing the single-chain variable fragment in the antigen-binding segment to reach the specific target antigen [75]. Crucially, due to alteration in length and composition within this area, the selected hinge seems to influence CAR functionality by changing signaling, flexibility, CAR production, epitope identification, activation strength, and epitope binding [76, 77]. Furthermore, it has been indicated that the hinge length is crucial in determining the proper intercellular spacing required to form an immune synapse [50]. The steric hindrance and the location of the specific target antigen are essential to determine the optimal hinge length [74]. While shorter spacers are better at binding epitopes distant from the membrane, longer hinges or spacers are more flexible and provide more efficient access to complex glycosylated antigens or epitopes close to the membrane [47, 78, 79]. In practical terms, the ideal spacer length is usually found through experimental methods and needs customization for each particular antigen-binding domain pairing [75]. The hinge domain frequently used the amino acid (AA) sequences found in IgG1, IgG, CD28, and CD8 [76]. However, due to potential interactions with Fcγ receptors, spacers originating from IgG may induce CAR T-lymphocyte depletion, limiting their durability in vivo [80]. To mitigate these effects, one can opt for an alternate hinge domain or perform further engineering of the hinge domain, considering functional or structural aspects [47].

3. CAR T-cell generations

In the lab, CAR T cells have undergone genetic modification [81]. These receptors are synthetic molecules based on TCR principles and costimulatory signaling [82]. CAR T cells are genetically engineered antibody/T-cell hybrids. The extracellular portion consists of antibody-derived heavy and light chains [83]. They are joined to form a single-stranded variable-length segment [83]. The transmembrane domain, which includes the intracellular and extracellular parts, is typically created by CD8 or IgG4 molecules [84]. The activation of T cells is mediated by an intracellular signaling domain that includes a costimulatory domain and a CD3ζ chain [85]. The intracellular domains within these structures serve as functional endpoints that trigger differentiation, cytokine production, and cytotoxic response of additional immune cells to improve the elimination of tumor cells [82]. These mechanisms enable the targeting of tumors without relying on MHC restriction [84]. Therefore, the primary focus of methods aimed at improving the effectiveness of CAR T cell therapy lies in fortifying and preserving the signaling pathways that play a crucial role in CAR T cell activation, expansion, and durability [86]. As a result, current research efforts are geared towards refining CAR structure, discovering new costimulatory molecules, and manipulating the tumor microenvironment to achieve optimal therapeutic outcomes with CAR T cells [86]. Since their introduction, the fundamental modular design of CAR T cells has not changed [86]. However, CAR T cells may be classified into five generations depending on how their intracellular signaling domain is organized (Figure 2B) [87].

3.1 First CAR T-cell generation

The first CARs contained only CD3 ζ-chain or an intracellular FcεRIγ domain without additional costimulatory domains [88]. The complexes were remarkably similar to the endogenous TCR but had one major limitation – they could not generate sufficient interleukin-2 (IL-2) because their response was weak, and the first-generation CARs had to be supported with exogenous IL-2 to ensure a successful response [89]. In addition, research has shown that these modified cells exhibit limited cell growth and have a short lifespan in vivo [90].

Following the successful integration of the CAR into the T cells, these were cultured and expanded to generate a population of CAR T cells [91]. After the patient’s reinfusion, the first-generation CAR T cells identified a particular antigen in cancer cells [91]. They attached themselves to it, which led to the T-cell activation and, eventually, to the elimination of the cancer cells [92]. Nevertheless, the first-generation CAR T cells had various limitations. Their presence in the patient’s body was limited, and they did not expand much, resulting in a shorter duration of response [89]. In addition, they often showed lower anti-tumor efficacy and were prone to T-cell exhaustion, leading to reduced functionality over time [92]. This has led to the need to develop the costimulatory domains further [93].

3.2 Second CAR T-cell generation

The second generation of CAR T cells aims to address the shortcomings of conventional CAR T cells, for instance, low cell proliferation, insufficient cytokine production, and short lifespan [94]. They achieve this by harnessing the power of two signals that stimulate the proliferation of T cells in the natural environment [94]. Second-generation CAR T cells have extracytoplasmic domains like CD28, 4-1BB, or OX-40 that can provide secondary signals when they encounter a tumor antigen [95]. Clinical and preclinical studies have shown that adding costimulatory signals can increase cell proliferation, cytotoxicity, and inflammatory responses by prolonging the in vivo half-life [96]. Further studies have also demonstrated that the composition of the costimulatory field is essential in influencing these events [96]. For example, studies have shown that 4-1BB z-CAR T cells persist in circulation more extended than CD28 z-CAR T cells [97]. On the one hand, the first factor may lead to a period of insufficient activity of CAR T cells [97]. In other words, the second factor is associated with continued activation even in the absence of antigen [98].

Treatment using second-generation CAR T cells has shown to be more successful than the first-generation CAR T cells, although it may still be linked to various side effects [50]. Cytokine release syndrome (CRS) is particularly common and has significant adverse consequence of second-generation CAR T-cell treatment [99]. The CAR T-cell activation can cause the overproduction of cytokines, leading to CRS [99, 100]. Releasing cytokines can cause a systemic inflammatory reaction, leading to symptoms similar to the flu, including fever, chills, tiredness, headache, and muscle pain [100]. In severe instances, CRS is capable of inducing high body temperature, reduced blood pressure, organ malfunctions, and potentially fatal complications [101].

Second-generation CAR T-cell treatment can cause immunological effector cell-associated neurotoxicity syndrome (ICANS), causing symptoms such as delirium, confusion, aphasia, seizures, and other neurological symptoms [102]. The cause of ICANS is believed to be caused by an immune system issue in the central nervous system; however, this is not entirely understood [103].

3.3 Third CAR T-cell generation

Third-generation CARs frameworks combine multiple costimulatory signal domains in the endodomain. Common examples of these structures are CD3ζ-CD28-OX40 or CD3ζ-CD28-41BB [104]. The CD28 costimulatory domain causes tumor cell removal, whereas the 4-1BB endodomain contributes to long-term CAR preservation [104]. However, they have been used to treat cancer with safety benefits, long-term efficacy, and increased efficacy potential [50]. However, sufficient effectiveness in contrast to second-generation CAR T cells has not yet been achieved [98, 105].

Third CAR T-cell generation therapy is founded on developing second CAR T-cell generation incorporating additional costimulatory domains [50]. Although third-generation CAR T cells aim to improve therapeutic efficacy, they may still be associated with specific side effects [6]. Third CAR T-cell generation can trigger CRS, causing systemic inflammation and flu-like symptoms like the second CAR T-cell generation [106]. The degree of side effects associated with CRS can differ from mild to very severe and threaten the patient’s life [63]. Accurate monitoring and prompt management are critical to mitigate the effects of CRS [107].

Additionally, neurotoxicity from third-generation CAR T-cell treatment may result in neurological symptoms such as delirium, disorientation, seizures, and aphasia [106]. The mechanisms underlying the neurotoxicity of third-generation CAR T cells are not fully understood but are likely to be an immune-mediated response affecting the central nervous system [108]. Third-generation hematologic toxicity from third-generation CAR T-cell treatment may include a decline in platelets, RBC, and WBC [87]. These conditions can increase the risk of infections, blood complications, and the need for blood transfusions [87]. Organ damage from CAR T-cell treatment includes hepatotoxicity or liver malfunction. Monitoring liver enzymes, kidney function, and other organ parameters is essential to detect and manage toxicity [109].

3.4 Fourth-generation of TRUCK CAR T-cell

The fourth CARs are based on the second design, as multiple costimulatory domains do not enhance the effectiveness of CAR T cells [110]. The difference between these two generations is that the latter is replaced by expression cassette containing a modified protein [110]. These T cells target CAR T cells for universal cytokine-mediated killing (T cells redirected for universal cytokine-mediated killing or TRUCK), designed to deliver transgene products to cancer cells [110]. This was achieved by engineering cells to carry the nuclear factor T-cell (NFAT) response cassette that contains transgenic cytokines like IL-12 [111].

Thus, transgene expression is induced when a CD3ze-containing CAR binds to its specific target [112]. Modifying TRUCK CAR T cells requires modifying two transcriptional systems, one for CAR production and the other for inducing cytokines [112]. In preclinical models, cytokine transgene increases the effectiveness of CAR T-cell therapy in contrast to second-generation CARs [108]. The approach also managed to avoid systemic toxicity, one of the most common issues with CAR T-cell treatment [113].

TRUCK CAR T cells have been modified to produce more cytokines or immune modulators that can lead to off-target effects [113]. These side effects can include inflammation in normal tissues or unintended immune responses that can damage healthy cells and tissues [114]. Adding additional components to TRUCK CAR T cells can increase their immunogenicity, meaning the patient and the immune system recognize them as foreign [113]. This recognition can trigger an immune response against CAR T cells, reducing perseverance and effectiveness [114]. TRUCK CAR T-cell treatment can also be associated with organ toxicity, such as hepatotoxicity (liver dysfunction) or nephrotoxicity (kidney dysfunction) [114]. Monitoring organ function and managing organ toxicity are essential aspects of patient care during TRUCK CAR T-cell therapy [110].

3.5 Fifth or next-generation CAR T cells

CAR T-cell therapies have advanced rapidly in recent years to increase perseverance, proliferation, safety, and efficacy [114]. On the other hand, reducing the toxicity of off-target tumors and CAR T cells is still challenging [114]. In this regard, the new generation, which experts have already called the fifth-generation, differs from the previous and has one additional membrane receptor [114]. TRUCKS and fourth-generation CAR T cells are modified T cells activated upon exposure to their target antigen and inducing secondary transgene [110]. Subsequently, transcription happens and leads to transgene secretion into the extracellular [110]. In this approach, the hidden signal can stimulate CAR T cells, promoting their development and the formation of memory T cells [114]. Additionally, it stimulates immunological function, enhancing its response to subsequent stimulations [115]. Fifth-generation CAR T cells utilize membrane receptors and operate on a distinct principle [73]. Currently, several approaches are under testing, with the most promising one involving increased IL-2 receptors [87]. The JAK/STAT signaling cascade is facilitated by these receptors in an antigen-dependent manner [116]. One fascinating advancement in the field is the design of switch receptors [117]. Recently, there have been reports of successful coupling of drug-dependent OFF switches, resulting in CAR exhaustion, as well as ON switches, leading to activation [117]. Building upon these principles, lenalidomide-gated CARs were developed and evaluated [118]. Although these cells displayed slight effectiveness in vitro, they showcased improved controllability compared to earlier generations of CAR T cells [87]. Consequently, they offer a better safety profile and a more comprehensive therapeutic effect [116].

4. CAR T-cell engineering

CAR T cells, initially known as T-bodies, were first described by Yoshihisa Kuwana and his team in Japan [87]. They combined segments of antibodies with the TCR. Gideon Gross and Zelig Eshhar conceptualized the first engineered T cell with a chimeric molecule, laying the foundation for CAR T-cell therapy in Israel (in 1989) [119]. Since its inception, CAR T-cell therapy has made significant advancements during the previous several decades, evolving over five generations to reach its most advanced fifth generation. This newest generation demonstrates improved efficacy while minimizing toxicity [120]. The CAR is a receptor design made of three distinct parts [6]. These include an ectodomain, transmembrane, and intracellular domain (Figure 2). The ectodomain corresponds to the extracellular portion of the CAR, and it serves as the region where a hinge joins together two components [121]. CAR design has a significant impact on CAR T-cell treatment as it shows the specificity and functionality of the engineered T cells [122]. A CAR’s design is made up of many essential components.

4.1 Extracellular antigen-binding domain

The extracellular domain is in control of identifying and binding itself to target antigens, which are expressed in cancer cells [52]. The scFv originates from an antibody or an alternative antigen-binding domain, such as a nanobody, and is designed to identify a particular antigen [52]. Its binding specificity determines the CAR T-cell’s specificity [123]. The CAR’s ability to recognize the target antigen is determined by the scFv’s affinity [124]. To stimulate T cells and identify tumor cells, scFv must have a strong enough affinity [124]. A correlation that is too high can lead to AICD and potential toxicity [125]. On the other hand, poor antigen affinity makes it impossible to target healthy tissue with little antigen [126]. A recent evaluation of CARs with various affinities for scFvs that bind to related epitopes and cross-react with mouse carcinoembryonic antigen glypican 3 (GPC3) showed that high-affinity CAR T cells in the body are toxic [127]. However, low-affinity CAR T cells do not cause tissue damage while still having cytotoxic effects on antigen-positive tumor cells [127].

4.2 Hinge region

The hinge region plays a crucial role in the connection between the antigen-binding domain and the transmembrane domain of the CAR [128]. Its main purpose is to provide flexibility, enabling optimal CAR binding to the target antigen [128]. This region, also known as the spacer, is a short segment of the ectodomain derived mainly from immunoglobulin G (IgG), with occasional contributions from CD8 and CD28’s hinges [87]. In addition to the antigen recognition domain, the ectodomain contains the hinge region, serving as a link between the endodomain and the ectodomain as well as the ectodomain and the TMD [87].

The primary purpose of the hinge region is to facilitate antigen attachment, enhance flexibility, and promote the formation of synapses between CAR T cells and target T cells [129]. The length of the hinge region determines the CAR’s flexibility and ability to reach membrane-proximal epitopes [129]. Longer hinges provide increased flexibility and improved access to these epitopes, while shorter spacers limit flexibility and target the antigen’s distal epitopes [130]. It is important to emphasize that differences in the hinge region’s length may have a substantial effect on CAR antigen binding and signaling [131]. The target epitope’s accessibility and location have the most effects on this [131]. While longer spacers supply more flexibility and more efficient access to membrane-proximal epitopes, shorter hinges enhance CAR T-cell activation [132]. By adding a CH2CH3 domain, for example, the distance between the T-cell surface and the CD22 epitope may be increased, boosting CD22-specific CAR activity [133]. There are substantial efforts undertaken to optimize the composition of hinges in addition to the hinge length [132]. Because of its interaction with Fcγ receptors (FcγRs), CD19-CAR built with a lengthy spacer of IgG4 hinge-CH2-CH3 has been reported to be related to a lack of in vivo anti-tumor efficacy [22]. However, by altering certain CH2 domain sections required for Fc receptor interaction, decreased persistence and anticancer activity may be recovered [23].

4.3 Transmembrane domain

The CAR is anchored in the T-cell membrane via the transmembrane domain [66]. This hydrophobic area stabilizes the CAR and makes sure it is positioned correctly on the surface of the T cell. The activity and stability of CAR T cells are greatly influenced by transmembrane domains, which often originate from type I proteins such as CD3, CD4, CD8α, or CD28 [70]. Interestingly, it has been shown that CARs with the CD28 transmembrane domain not only exhibit dimerization but also possess more excellent stability compared to CARs with the CD3 transmembrane region [66]. Furthermore, the transmembrane domain influences CAR expression levels directly by aiding in the removal and stabilization of the CAR inside the membrane [66, 134].

In comparing CARs with hinge and transmembrane sections made of CD28 or CD8α, recent studies have shown that the CD28 hinge can lower the antigen density cutoff needed to activate CARs [72]. Consequently, anti-CD19 CAR T cells incorporating the CD28 hinge and transmembrane regions have been found to generate more cytokine levels and exhibit increased AICD levels in contrast to CD8α hinge and transmembrane domains [72, 135]. Proline enhanced dynamic switching and local structural characteristics, such as disulfide bridges between dimeric molecules, according to further research into the biophysical and dynamic properties of CD8α, thereby enhancing signaling in a context of reduced antigen availability [72]. It is important to note that the isolated hinge domain can have a distinct function from the CAR as a whole [34]. Additionally, its interactions with the cell membrane surface might alter exchange dynamics and stabilize certain local structures [136].

4.4 Intracellular signaling domains

The CAR’s intracellular signaling domains are essential for sending stimulation signals to the T cell [137]. Commonly utilized signaling domains include the CD3ζ chain of the TCR complex, which is crucial for T-cell activation [138]. Costimulatory domains such as CD28, CD134, or CD137 are employed [139]. These costimulatory domains increase T-cell activation, persistence, and proliferation, thereby improving CAR T cells’ functionality and anti-tumor activity [140].

The choice and combination of intracellular signaling domains may critically impact the CAR T-cell’s biological properties [138]. CARs containing only the CD3ζ signaling domain are first-generation CARs and provide essential signaling for T-cell activation [141]. Second-generation CAR T cells possess one costimulatory domain and the CD3ζ domain, while third-generation CARs incorporate two costimulatory domains [142]. Fourth-generation CAR T cells are engineered to release specific cytokines upon target antigen recognition, enhancing their anti-tumor effects [143].

CAR design also takes into account factors like the persistence of CAR T cells, resistance to immunosuppression in the microenvironment of the tumor, and avoidance of off-target toxicity [53]. Researchers are actively exploring various modifications to the CAR design to overcome these challenges and increase the overall efficacy and safety of CAR T-cell treatment [87]. These modifications involve the use of alternative spacer and hinge regions, the incorporation of inhibitory receptors to counteract immunosuppressive signaling, and switchable CARs development for more effective CAR T-cell activity regulation [144]. In summary, CAR design is crucial for CAR T-cell engineering as it controls the specificity, activation, and functionality of the engineered T cells [73]. Ongoing research and innovations in CAR design continue to advance the CAR T-cell treatment field and aim to enhance the prospect for therapy of these promising immunotherapies [145].

5. CAR T-cell evolution

The field of molecular engineering has made significant advancements, offering new avenues for addressing challenges related to CAR T-cell therapy that have emerged since its implementation in clinical settings [146]. Researchers have created innovative next-generation CAR T cells, which address the limitations of therapies using second-generation CAR T cells, such as their limited effectiveness and high toxicity [53]. These advancements aim to enhance the efficacy and safety of CAR T-cell treatment, providing promising solutions for improving patient outcomes [146].

The challenge of tumor-associated antigen escape leading to high relapse rates has spurred researchers to develop CAR T cells with dual-targeting capabilities [147]. This approach has proven successful. Clinical studies indicate that dual systems, such as CD19/BCMA for multiple myeloma and CD19/CD22 for diffuse large B-cell lymphoma, may be effective in curing these respective conditions [148]. Another promising strategy for dual-targeting involves tandem systems when a single receptor is combined with two scFv domains that bind distinct antigens [147]. Studies using preclinical models of HER2/MUC1 (breast cancer) and HER2/IL13Ra2 (glioblastoma) have shown the effectiveness of tandem CARs [149]. In all cases, compared to single-targeted CARs, dual-targeting has shown superior anti-tumor responses. Researchers have found notable variations in the responses produced by these two designs, highlighting the need to optimize and select antigens to avoid recurrence.

5.1 Dual-targeting CAR T cells

Dual-targeting CAR T-cell therapy employs a dual CAR approach to selectively target two tumor-associated antigens on specific cells [150]. Two distinct CAR T-cell structures with different antigen-binding specificities can achieve this, or a single CAR T cell can target two different antigens [150]. It is worth noting that several tumor-associated antigens are expressed in normal cells and tissues at low levels, raising concerns regarding CAR T cells’ specificity in dual-targeting strategies [151]. Researchers focus on fine-tuning the antigen-binding domains to address this issue and identify specific tumor-associated post-translational modifications (PTMs) [151]. This approach aims to increase the CAR T cells’ specificity, ensuring that they selectively target cancer cells while minimizing off-target effects on healthy tissues [152]. This strategy holds promise as solid tumors are recognized for their tendency to overexpress truncated O-glycans [153]. Consequently, there is ongoing investigation into using scFvs that target these modified glycans at the preclinical stage [153]. Incorporating scFvs specific to these glycans is expected to decrease the likelihood of antigen escape and reduce the toxicity of off-target effects from CAR T-cell treatments [154]. By focusing on these modified glycans, this strategy presents a viable way to raise the safety and effectiveness of CAR T-cell treatments [155].

5.2 Controlling the toxicities using switchable CAR T cells

Switchable CAR T cells offer a promising alternative to traditional CAR T therapies, addressing the challenges they present [122]. The activation of CAR T cells may be regulated, providing a completely adjustable response by using a tumor antigen-specific recombinant Fab-based ‘switch’ [122]. This breakthrough technology offers a solution to overcome the translational barrier faced in CAR T-cell therapy [156]. The motivation behind the development of switchable CAR T cells stems from the treatment-related toxicities often observed with conventional CAR T therapies, particularly CRS and neurotoxicity, the most common adverse effects [157]. To enhance safety, researchers have harnessed new technologies that incorporate safety switches capable of inducing apoptosis and complement-dependent cytotoxicity to deplete CAR T cells when necessary [156]. Switchable CAR T cells are created by transducing genes encoding surface antigens that can be efficiently targeted by drugs or inducible intracellular antigen effectors [87]. Once these genes are expressed, the CAR T cells become sensitive to specific drugs, enabling their depletion as required [158].

5.3 Universal CAR T-cell

The traditional CAR design is fixed, and a CAR T cell targets only one antigenic epitope [159]. This rigid design restricts the use of the drug and makes the production cost unfavorable [159]. By separating the antigen binding domain from the normal CAR’s signaling domain, new CARs are created via a modular approach [160]. Therefore, the target antigen might be easily replaced or modified without the need to engineer CAR T cells [160]. Therefore, this CAR system works like a universal CAR (UniCAR) [161]. The UniCAR platform consists of a signaling module that binds to a particular epitope on the switching molecule and a switching module with an antigen-binding domain and epitope [162].

6. CAR T-cell FDA-approved

In August 2017, the United States granted its initial approval for the medical use of CAR T cells [163]. Subsequently, the FDA approved the application of CAR T cells specifically for the treatment of individuals with certain B-cell malignancies [164]. The most significant outcomes and approvals in the treatment of hematologic malignancies have been achieved through the utilization of CAR T-cell therapies targeting BCMA and CD19 [165]. The FDA has granted approval for six CAR T-cell treatments, all of which are second-generation structured, specifically for the treatment of patients with aggressive hematologic malignancies [166]. CAR T-cell treatment has shown its effectiveness in treating malignant B-cell tumors [167]. This achievement can be attributed to multiple factors, including the selected tumor cell and consistent CD19 or CD20 expression and the enhanced accessibility of CAR T cells to these targets [28]. This high rate of complete responses demonstrates CAR T-cell therapy’s strong anti-tumor effects in tackling CD19-positive malignancies [168]. It demonstrates how CAR T cells may successfully remove cancer cells expressing CD19, leading to substantial clinical improvements for a significant proportion of patients [169]. The trials demonstrating the success of CAR T-cell therapy provide hope to patients with limited treatment options. The study highlights the potential of CAR T-cell therapy in combating B-cell tumors, paving the way for further research of CAR T-cell treatments against various cancers. Figure 3 provides a summary of the key historical occurrences related to the advancements and developments in FDA-approved CAR T-cell product applications for cancer immunotherapy.

Figure 3.

Timeline of development for six CAR T-cell products authorized by the FDA in the past. A number of occasions have been identified as pivotal moments in the evolution of CAR T-cell research. Keys: ALL, acute lymphoblastic leukemia; NHL, non-Hodgkin’s lymphoma; FL, follicular lymphoma; MCL, mantle cell lymphoma; MM, multiple myeloma.

The FDA-approved CAR T-cell products until 2022 are listed in Table 1.

Trade name/generic name	Nickname	Structure	Target antigen	Target disease	Crucial research	Approval date
Tisagenlecleucel /Kymriah™	Tisa-cel	Endodomain: CDζ-4-1BB; ectodomain: anti-CD19	CD19	Follicular lymphoma, diffuse large B-cell lymphoma, and relapsed/refractory acute lymphoblastic leukemia in children and young adults	ELIANA trial [170]; JULIET trial [171]	August 2017
Axicabtagene ciloleucel/Yescarta™	Axi-cel	Endodomain: CDζ-CD28; ectodomain: anti-CD19	CD19	Relapsed or resistant large B-cell lymphoma in adult patients	ZUMA trial [172]	October 2017
Brexucabtagene autoleucel/Tecartus™	Brexu-cel	Endodomain: CDζ-CD28; ectodomain: anti-CD19	CD19	Adult patients suffering from B-cell-ALL and relapsed/refractory MCL	ZUMA trial [173]; TRANSFORM trial [51]	July 2020
Lisocabtagene maraleucel/Breyanzi™	Liso-cel	Endodomain: CDζ-4-1BB; ectodomain: anti-CD19	CD19	Relapsed or resistant B-cell lymphoma in adult patients	TRANSFORM trial [174]	February 2021
Idecabtagene vicleucel/Abecma™	Ide-cel	Endodomain: CDζ-4-1BB; ectodomain: anti-BCMA	BCMA	Relapsed or resistant multiple myeloma in adult patients	KarMMa trial [175]	March 2021
Ciltacabtagene autoleucel/Carvykti™	Cilta-cel	Endodomain: CDζ-4-1BB; ectodomain: anti-BCMA	BCMA	Relapsed or resistant multiple myeloma in adult patients	CARTITUDE-trial [175]	February 2022

Table 1.

All CAR T-cell products approved by the FDA until 2022.

Abbreviations: B-cell-ALL, B-cell acute lymphoblastic leukemia, BCMA; B-cell maturation antigen; MCL, mantle cell lymphoma; MM, multiple myeloma.

7. Challenges and opportunities

CAR T-cell therapy is a promising immunotherapy for certain cancer types and highlights that the research in this area is currently in a dynamic phase [8]. Due to the complexity of these biologic therapies, multiple approaches are required to provide the best possible treatment while minimizing toxicity [108]. But wider use and improved performance also face some challenges [176, 177]. Some of the main problems with CAR T-cell therapy include the following.

7.1 Cytokine release syndrome

Clinical manifestations of CRS include myalgia, hypotension, hypoxia, and fever [101]. It may produce severe fever, hemodynamic abnormalities needing vasopressor support, capillary leakage, and acute hypoxia necessitating mechanical breathing. It can also be moderate and self-limiting. Cardiovascular arrhythmias, pleural effusion, transaminitis, renal failure, coagulopathy, and hemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS) are additional clinical symptoms of CRS [51, 52, 53, 178]. The supraphysiological response of the immune system, initiated by T-cell activation and resulting in the production of various cytokines and chemokines, constitutes the fundamental pathophysiology of CRS [179]. Consensus criteria for assessing CART toxicity, including CRS, have been created lately by the American Society for Transplantation and Cellular Therapy (ASTCT) [178]. Treatment of immune cell therapy-related toxicities requires early identification and proper management [180]. In this case, the grading system is important and has been developed since the ASTCT’s current grading agreement for Adverse Events Criteria version 4.03 (CTCAE v.4.03) [100, 178].

7.2 Neurotoxicity

CAR T-cell treatment has been linked to potential neurological side effects, like ICANS [181]. Symptoms include confusion, delirium, seizures, and other neurologic abnormalities. Understanding and managing ICANS is crucial for patient safety [182]. Slow movements, tremors, lethargy, and an ataxic gait are among the usual signs of neurotoxicity [183].

CAR T-cell development is linked to high cytokine levels from immune cells, as well as increased levels of CSF and serum cytokines [99]. Therapeutic or preventive strategies that target important cytokines or preserve the integrity of the blood-brain barrier can be useful for neurotoxicity, according to cytokine profiles and autopsy investigations [184]. The consistency of neurotoxic effects linked to CAR T-cell treatment and other targeted, such as IL-2, cytotoxic chemotherapy, and blinatumomab, might be explained by this discovery [185, 186, 187].

7.3 Limited target antigens

The use of CAR T cells to treat hematologic malignancies by targeting CD19 has shown great potential [188]. On-target and off-tumor toxicities can occasionally be severe or fatal when CAR T-cell infusion is used to treat solid tumors [189]. Although both tumor and normal cells possess target antigens, not all CAR T-cell treatments exhibit appreciable off-tumor, on-target toxicities [92]. Examples of this include CAR T-cell therapies targeting carcinoembryonic antigen (CEA), mesothelin (MSLN), alpha2 (IL13Rα2), and interleukin 13 receptor [190, 191]. Moreover, preclinical trials have revealed varying levels of on-target and off-tumor toxicities among CAR T cells that target the same antigen [154]. While some toxicities were mild, others proved to be lethal, as seen in the case of fibroblast activation protein-α (FAP) targeting [192]. However, identifying appropriate target antigens for solid tumors has proven challenging [154]. Solid tumors often exhibit antigen heterogeneity, making it difficult to locate a single target capable of effectively eliminating all cancer cells [193]. CAR T cells pose a challenge in safely identifying and eliminating tumor cells due to the overexpression of targeted antigens on tumor cells [193].

7.4 Relapse and resistance

Despite initial success, some patients experience relapse or develop resistance to CAR T-cell therapy [194]. Tumor cells can evade CAR T-cell recognition by downregulating target antigens, altering antigen expression profiles, or creating other immune evasion mechanisms [24]. Overcoming resistance and preventing relapse are ongoing challenges in CAR T therapy [194]. The production of CAR T cells from leukapheresis lymphocytes, a type of patient cell, can significantly complicate drug production and potentially lead to failure [108]. In up to 10% of cases, this can occur due to either a reduction in the patient’s total T-cell count or the suppression induced by their monocytes [195]. Additionally, it may result from the autologous T cells not functioning correctly in patients undergoing intense pretreatment [195]. In most cases, failure results either from the primary inability to effectively stimulate an immunological response in vivo or from T cells’ inability to endure [196]. The mechanisms of early T-cell failure remain to be well understood [196]. Nevertheless, increasing the dose of CAR T cells cannot solve the problem, as they are only partially dose-dependent [197]. Conversely, enhancing the dose of CAR T cells may increase toxicity without increasing the efficacy of the response [123]. Combination therapies are used to stop resistance to chemotherapy or antibiotics [197]. In this approach, CAR T-cell therapy can utilize multiple antigens and immunomodulatory strategies to improve current outcomes [157]. Combining synthetic immunotherapy with checkpoint inhibitors allows for more effective treatment of recurrent and resistant B-cell malignancies, according to data from several bispecific CAR T cells evaluated in clinical studies [198]. Long-term monitoring of patients who have received CAR T therapy is necessary to understand possible relapses or secondary malignancies [157].

7.5 Manufacturing complexity

CAR T-cell production involves a complex process involving patient T-cell collection, manipulation, laboratory genetic modification, cell expansion, and infusion back into the patient [157, 199]. Standardizing and streamlining manufacturing processes is necessary to make CAR T-cell therapy more accessible and cost-effective [43]. Quality control measures are particularly important to autologous CAR T-cell therapy, as each “lot” of ingredients must be tested [43]. Timeliness is important to prevent the degradation of CAR T-cell products before infusion or cryopreservation [200]. According to the current European Union regulations, CAR T-cell therapy is classified as advanced therapy medicinal products (ATMPs) [201]. ATMPs are further categorized under EU Regulation 1394/2007, with gene therapy medical products (GTMPs) being a subset [202]. This classification includes four groups, among which CAR T cells, whether allogeneic or autologous, are encompassed, along with other treatments [202]. The Public Health Service Act (Section 351) regulates CAR T-cell therapy in the United States, requiring approval before it can be pre-marketed through conventional clinical trials [203]. The FDA and European Medicine Agency (EMA) have issued crucial guidelines for the advancement of gene and cell therapy [5].

8. Conclusion

CAR T-cell therapy is an immunotherapy that has been incredibly successful in treating certain types of cancer. Looking to the future, there are many promising and potential advances for CAR T-cell therapy:

• CAR T-cell therapy is expanding treatment options for hematologic malignancies such as leukemia and lymphoma, thereby broadening the range of cancers that can be successfully treated. The creation of CAR T-cell treatments that are successful in killing cancer cells is a current area of research focus.

• CAR T-cell therapy is a successful treatment, but it can occasionally cause serious adverse effects like neurotoxicity and CRS. Future advances to improve the safety of CAR T-cell therapy by modifying the CAR design. Given the specificity and design of CAR and virtually unlimited administration, control, and genome editing. Clinical trials are underway for CAR T-cell-based products for gene insertion, aiming to enhance tumor treatment and boost CAR T-cell production.

It will be important that these new measures capture as much information as possible from previous research. Clinical trials are underway for CAR T-cell-based products for gene insertion, aiming to enhance tumor treatment and boost CAR T-cell production.

Acknowledgments

We express our deepest gratitude to Dr. Piao Yang for her help in the Abstract preparation.

Conflict of interest

The authors declare no conflict of interest.

Acronyms and abbreviations

CAR	chimeric antigen receptor
CARs	chimeric antigen receptors
CIKs	cytokine-induced killers
TIL	tumor-infiltrating lymphocytes
mAbs	immune checkpoint blockers, monoclonal antibodies
TA	tumor antigen
scFv	single-chain variable fragments
MHC	major histocompatibility complex
TCR	T-cell receptors
AML	acute myeloid leukemia
ALL	acute lymphoblastic leukemia
CLL	chronic lymphocytic leukemia
MM	multiple myeloma
VL	variable light chains
VH	variable heavy chains
Ab	antibodies
ITAM	immune receptor tyrosine-based activation motif
TAAs	tumor-associated-antigens
AICD	activation-induced cell death
Ig	immunoglobulin
AA	amino acid
IL-2	interleukin-2
CRS	cytokine release syndrome
ICANS	immunological effector cell-associated neurotoxicity syndrome
TRUCK	T cells redirected for universal cytokine-mediated killing
NFAT	nuclear factor T-cell
GPC3	glypican 3
IgG	immunoglobulin G
FcγRs	Fcγ receptors
PTMs	post-translational modifications
ASTCT	American Society for Transplantation and Cellular Therapy
CEA	carcinoembryonic antigen
MSLN	mesothelin
IL13Rα2	alpha2
FAP	fibroblast activation protein-α
ATMPs	advanced therapy medicinal products
GTMPs	gene therapy medicinal products
EMA	European Medicine Agency