A review on car-t cell therapy- traditional strategies for cancer treatment

 Corresponding author: V. V. Rajesham Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. A review on car-t cell therapytraditional strategies for cancer treatment V. V. Rajesham *, K. Madhuri, M. Abhiram, B. Ashmitha and T. Rama Rao CMR College of Pharmacy, Kandlakoya (V), Medchal (M &D), Hyderabad 501401, Telangana, India. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(01), 114-125 Publication history: Received on 22 October 2024; revised on 12 December 2024; accepted on 14 December 2024 Article DOI: https://doi.org/10.30574/wjbphs.2025.21.1.0970 Abstract Cancer is the world's top cause of death. Numerous cytotoxic immunotherapies and traditional treatments have been created and introduced to the market. The development of a viable immunotherapy that targets cancers at both the cellular and genetic levels is necessary due to the complicated behaviour of tumors and the involvement of multiple genetic and cellular variables in tumorigenesis and metastasis. A new therapeutic T cell engineering technique called chimeric antigen receptor (CAR) T cell treatment involves in vitro altering patient blood-derived T cells to express synthetic receptors that are directed against a particular tumor antigen. The major histocompatibility complex is not involved in these; instead, the tumor antigen is directly identified. The use of this therapy in the last few years has been successful, with a reduction in remission rates of up to 80% for hematologic cancer, particularly for acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphomas, such as large B cell lymphoma. CAR therapy has the potential to offer a rapid and safer treatment regime to treat non-solid and solid tumors. CAR-T cell therapy's most significant benefit over other cancer treatments is its quick time intervention a single infusion of CAR T cells. Additionally, the patient only has to be properly cared for and observed for two to three weeks. CAR T cell therapy is considered a "drug of the present day," and because the cells may live in the host body for a long time and have the capacity to continuously identify and eliminate cancer cells after relapse, its effectiveness may last for decades. The present review insight into the structure and evolution of chimeric antigen receptors, we then report on the tools used for production of CAR-T cells. Finally, we address the challenges posed by CAR-T cells. Keywords: Cancer; CAR-T cell; Chimeric antigen receptor; Acute lymphoblastic leukemia 1. Introduction Chimeric antigen receptor (CAR T) cell therapy is a revolutionary new pillar in cancer treatment (1). Although treatment with CAR T cells produced remarkable clinical responsible with certain subsets of B cell leukemia or lymphoma many challenges limit the therapeutic efficacy of CAR-T cell in solid tumour and haematological malignancy (2). CARs are engineered synthetic receptor that functions to redirect lymphocytes, most commonly T cells, to recognize and eliminate cells expressing a specific target antigen. When we pioneered the first CAR design in the late 1980s and early1990s, it was widely known that T cell Are very powerful effectors in the fight against cancer, but the application of these cells to cancer patients suffered from two major limitation(3). First T cell recognition depends on the expressions of major histocompatibility complex molecules and antigen processing machinery, and many tumours silence these pathways as part of their escape from immune recognition(4). second many tumours that do not express costimulatory molecules required for triggering the full potency of T cell often render tumour-specific T cell nonfunctional researchers have designed CARs to offer an alternative to conventional T cell receptors (TCRs) and circumvent these hurdles(1,3). The unprecedented success of anti CD19 CAR-T cell therapy against B cell malignancies resulted in its approved by the US food and drug administration (FDA) in 2017.however, there are major limitations to CAR-T cell therapy that still must be addressed including life-threatening CAR-T associated toxicities, limited efficacy against persistence, poor trafficking and tumour infiltration, and the immunosuppressive ((2,5). World Journal of Biology Pharmacy and Health Sciences, 2025, 21(01), 114-125 115 1.1. Background Chimeric antigen receptors (CARs) are engineered receptors that provide immune effector cells (T cells) with a customized specificity (6). CARs consist of three components: an extracellular domain for antigen recognition derived from a single-chain variable fragment (scFv) of an antibody, a transmembrane segment, and an intracellular T cell activation domain known as CD3 (7). The purpose of CAR-T cell therapy is to guide a patient’s or donor’s T cells to precisely locate and eliminate cancer cells. This approach holds significant potential for treating hematologic cancers as well as solid tumors, without being restricted by major histocompatibility complex (8). Immunotherapy has revolutionized cancer treatment, offering a flash of hope to patients facing late-stage metastatic tumors. Science magazine acknowledged its impact, designating it as the “Breakthrough of the Year” in 2013(9). 1.2. Engineering of CAR-T cells Engineered receptors named chimeric antigen receptors (CARs) have the ability to transfer an arbitrary specificity onto an immune effector cell (T cell). CARs are composed of three components: a transmembrane domain, an intracellular T cell activation domain of CD3, and an extracellular antigen recognition domain of the single-chain fragment variant (astx), which is generated from an antibody(10). The goal of CAR-T cell therapy is to direct T cells from a donor or patient to specifically target and kill tumer cells. Solid tumers and haematologic malignancies without significant constraints on the major histocompatibility complex can benefit greatly from this treatment (11). 1.3. Structure of CAR-T cells: CARs involves mainly ectodomain, transmembrane domain and endodomain(12). Figure 1 Structure of CAR-T cell 1.4. Ectodomain The segment of a membrane protein that is exposed to the outside environment and not located within the cytoplasm is referred to as the ectodomain. In this instance, the signal peptide, the antigen recognition area, and the spacer constitute the ectodomain (7, 8). World Journal of Biology Pharmacy and Health Sciences, 2025, 21(01), 114-125 116 A signal peptide's role is to direct the developing protein into the endoplasmic reticulum (13). The variable regions of the heavy and light chains of an antibody are connected through a flexible linker to create the single-chain variable fragment (scFv), which acts as the signal peptide for the ectodomain in a CAR. Any singular antigen capable of binding to targets with high affinity can be detected by an antigen recognition domain, frequently found as a single-chain variable fragment (scFv) with a fundamental ectodomain and additional specialized recognition components (14, 15). The spacer acts as a bridge between the transmembrane domain and the antigen binding domain. The hinge region of IgG1 represents the simplest form of spacer and is sufficient for most scFv-based constructs. 1.5. Transmembrane domain The transmembrane domain, consisting of a hydrophobic alpha helix that extends across the membrane, represents the closest portion of the endodomain to the membrane (6, 8, 9). There is a relationship between the stability of the receptor and the transmembrane domain. The artificial TCR could integrate with the native TCR when the native CD3-zeta transmembrane domain is available (16, 17). At present, the most stable receptor is the transmembrane domain of CD28. 1.6. Endodomain The most commonly found component of the endodomain, which is the operational end of the receptor, is CD3ζ, which includes three immunoreceptor tyrosine-based activation motifs (ITAMs)(8,9). A signal is transmitted to the T cell when the receptors cluster and activate upon recognizing an antigen. Co-stimulatory signaling is essential during this phase (17). 1.7. Evolution of CAR-T cells Based on the endodomain's structure, CAR-T cells can be categorized into four generations since the first creation of CARs in 1989. A great illustration of how fundamental research can be applied in the clinic is the development of CAR treatment. Figure 2 Evolution of CAR-T cell 1.8. First generation In the first generation of CARs, signals from the endogenous T cell receptor (TCR) were primarily transmitted by a single structure derived from the CD3 ζ-chain or FcεRIγ from the intracellular domain (17, 18). However, to effectively eliminate tumor cells, exogenous IL-2 needed to be administered because these CAR-T cells couldn't produce sufficient interleukin-2 (IL-2) on their own. Therefore, the combined use of cytokines proved advantageous for the initial generation of CAR-T cell therapies that utilized single-chain receptors (19). Recent studies indicate that the apoptotic signal may be mitigated by removing phosphorylation from ITAM A and C in the CD3ζ signaling portion, which benefits the ongoing expression of the transgene (20, 21). Nonetheless, more extensive clinical research has been carried out with CD3ζ-chained CAR-T cells in comparison to FcεRIγ-chained CAR-T World Journal of Biology Pharmacy and Health Sciences, 2025, 21(01), 114-125 117 cells. This might be attributed to the presence of one ITAM in the FcεRIγ chain versus three in the CD3ζ chain. Conversely, although CAR-T cells utilizing the CD3ζ chain demonstrated lower expression levels in vitro, they proved more effective at activating T cells and eliminating tumor cells. The transmembrane domain of CAR-T cells comprises a homologous or heterologous dimer of CD3, CD8, and CD28. This receptor is capable of facilitating optimal cellular activation through CAR dimerization and its functional link with the endogenous TCR. Various cancers have been treated with CD10-CAR-T cells, scFv(G250)-CAR-T cells, GD2-CAR-T cells, alpha-folate receptor (FR)-CAR-T cells, and carcinoembryonic Ag-specific CD3ζ (MFEζ)-CAR-T cells(22–27). However, due to constrained proliferation, a limited lifespan in vivo, and inadequate cytokine release, most early experiments involving first-generation CAR-T cells did not produce the expected outcomes. 1.9. Second generation T cell activation is commonly described as a process requiring two signals. This process involves three primary types of receptors: co-stimulatory receptors, cytokine receptors, and T-cell antigen receptors. The initial signal is generated when the T-cell receptor (TCR) recognizes the antigenic peptide-MHC complex present on antigen-presenting cells. The secondary signal comes from a co-stimulatory molecule, such as CD28/B7, which promotes the production of IL-2, crucial for completing T cell activation and preventing cell death. Naïve T cells, even when stimulated by an antigen, cannot perform their normal functions without the co-stimulatory signal. Therefore, CARs that consist solely of the CD3ζ sequence cannot effectively activate CAR-T cells in the absence of this co-stimulatory signal. To provide additional signals to T cells, second-generation CARs incorporate intracellular signaling domains from various co-stimulatory protein receptors, such as CD28 or 4-1BB and OX40, into the cytoplasmic tail of the CARs. These modifications can improve the proliferation, effectiveness, and durability of CAR-T cells, as well as prolong their lifespan in vivo (28–30). In addition to being essential for the formation of memory and effector cells, CD28-mediated co-stimulation influences the proliferation and survival of lymphocytes. CD134 can boost IL-2 production and support proliferation. The ability of CD137 to maintain the signaling of T cell responses is vital for both the survival of T cells and the memory of CD8+ T cells (31–33). When used in the treatment of B cell cancers, the scFvCD19-CD137-CD3-CAR-T cells, MOv19-BBζ-CAR-T cells, and scFvCD19-CD28-CD3ζ-CAR-T cells demonstrated better outcomes compared to the first generation (34, 35). Although direct comparisons are lacking, it seems that 4-1BBζ-CAR-T cells have a longer persistence than CD28ζ-CART cells (36). CD28ζ-CAR-T cells are capable of stimulating, developing, and expanding in a consistent manner (37). Conversely, early exhaustion associated with 4-1BBζ-CAR-T cells may limit their antitumor effectiveness (38, 39). 1.10. Third generation To enhance potency through increased cytokine production and improved killing capabilities, various signaling domains, including CD3ζ-CD28-OX40 or CD3ζ-CD28-41BB, were combined to develop third-generation CARs (40). While scFv CD20-CD28-CD137-CD3ζ-CAR-T cells and HER2-CAR-T cells were used to treat lymphoma and colon cancer, the outcomes did not surpass those achieved with second-generation therapies (41, 42). It is possible that the limited number of cases studied contributed to this result. Therefore, further research is necessary to evaluate the effectiveness and safety of these treatments, and the selection of co-stimulatory molecules plays a vital role. 1.11. Fourth generation T cell redirected for universal cytokine-mediated killing (TRUCKs) refers to the fourth-generation CARs, developed by integrating IL-12 into the foundational design of second-generation constructs. TRUCKs enhance T-cell activation and also recruit innate immune cells to target and eliminate cancer cells that do not express antigens within the treated area. Exploring how TRUCKs influence the tumor microenvironment through the controlled release of transgenic immune modifiers could provide valuable insights. Additionally, these TRUCK T cells show efficacy in managing autoimmune disorders, metabolic issues, and viral infections (43). When taken as a whole, these subsequent versions of CAR-T cell therapy have greatly increased interest in cancer treatment (44). 1.12. Mechanism of action Host t cells are collected and are genetically modified ex vivo to express a CAR targeting a tumour specific antigen. The car construct consists of an extra cellular domain with a single chain variable fragment that targets the antigen of interest as well as an intra cellular domain that construct in the T cell membrane an initiate an intracellular signalling cascade (45). World Journal of Biology Pharmacy and Health Sciences, 2025, 21(01), 114-125 118 The patient’s mononuclear cells are collected, which is typically accompanied by leukapheresis. Manufacturers of CAR T cells is a complex, expensive, and time-intensive process, The T-cell subset is enriched, generally modified to express the CAR of interest, sub sequent expanded ex vivo, and stored until use (46). Genetic modification of t cells is accomplished by one of three major methods currently used in clinical practice: -retroviral vectors, lentiviral vectors, or the transposon system after immunophenotypic confirmation of successful genetic modification, a growth platform is used to rapidly expand the CART cells (47). Figure 3 Mechanism of CAR-T cell therapy 1.13. Tools of transduction for CAR-T cells To introduce a foreign gene into human cells, a specific tool is necessary. Currently, gene integration using vectors can be carried out through two primary methods: viral systems and non-viral systems. Viral vectors are preferred in gene therapy for both research and clinical applications due to their high efficiency in transferring genes, quick ability to achieve the necessary quantity of cultured T cells, and the variety of viruses available, each with distinct expression profiles. Most viral systems can incorporate genes from interesting and valuable cells and can provide the structural proteins and enzymes essential for the generation of infectious viral particles carried by vectors. Examples of viral vectors include retroviruses like lentiviruses, adenoviruses, and adeno-associated viruses. Among these, genetically modified retroviruses are the most commonly used for gene delivery (48). A group of retroviruses is known as Retroviridae. This group varies in aspects such as host range, pathogenic potential, structure of the genome, sequences of amino acids and nucleotides, and host range. Nevertheless, the viral vectors can pose health risks. The achieved titer is inadequate, the ability to carry is limited, and the insertion mutations used to stimulate the immune response may lead to cancer and toxicity (48, 49). The benefits of non-viral gene therapy compared to other cancer treatments include enhanced effectiveness, precise targeting, non-infectious nature, unlimited carrier capacity, customizable chemical composition, and high production availability. Examples of non-viral vectors comprise molecular conjugates, liposomes, polymerizers, and naked DNA(50,51). Minicircle DNA vectors represent a novel class of non-viral vectors created in bacteria from a parental plasmid, capable of producing high levels of transgene expression in vivo while lacking plasmid bacterial DNA sequences. This method is applicable in clinical settings (52, 53). 1.14. Production of CAR-T cell The process of creating CAR-T cells involves multiple steps. Additionally, conducting quality control assessments at every stage of the protocol is essential (54). Leukocytes are obtained from the patient or donor through a process called leukapheresis (55). Next, T cells are isolated and cleaned to distinguish them from the leukocytes. Then, specific antibody bead conjugates or markers are employed to separate the T cell subsets based on their CD4/CD8 composition. The final step involves culturing the T cells to activate them. This procedure includes purifying the autologous antigen-presenting cells (APCs) from the patient or donor, or utilizing beads that are coated with anti-CD3/anti-CD28 monoclonal antibodies, or just anti-CD3 antibodies, World Journal of Biology Pharmacy and Health Sciences, 2025, 21(01), 114-125 119 with or without feeder cells and growth factors such as IL-2, which is commonly used because it enhances rapid T cell proliferation; further adjustments to the culture conditions are made to direct T cells towards a specific phenotype (56). Viral vectors that are incorporated in CARs guide the reverse transcription of RNA into DNA, which then permanently integrates into the genome of the patient's cells. In the activation process, the viral vector is eliminated from the culture through media exchange and/or dilution. Due to their better profile for integration sites, lentiviral vectors are more commonly utilized in clinical trials compared to gammaretroviral vectors.(57) mRNA transfection and the Sleeping Beauty transposon system represent additional methods. However, many questions remain unresolved, such as the requirement for multiple infusion cycles with temporary mRNA transfection and the risks of insertional mutagenesis and transposon remobilization when using the Sleeping Beauty transposon system (58). CAR-T cells are cultivated using three distinct bioreactor culture systems: CliniMACS Prodigy, G-Rex, and WAVE Bioreactor (59). A significant drawback of the first two methods is that the flask must be opened during cell inoculation. Conversely, the CliniMACS Prodigy system serves as a comprehensive tool that effectively enriches, activates, transduces, and cultivates the cells (60). Once the cells reach the necessary quantity for therapeutic use, they are extracted and administered to the patients. 1.15. The clinical success of CAR-T cell therapy A patient at NCI suffering from advanced follicular lymphoma and patients at MSKCC with refractory CLL and relapsed B-cell ALL both demonstrated progress following second-generation CAR T cell therapy (61, 62). A retroviral vector named MSGV was utilized to deliver a CD19-specific CAR as part of the treatment at NCI. This CAR was designed to target the CD19 protein found on the surface of B-lineage cells, using an anti-CD19 scFv derived from the murine monoclonal antibody FMC63. It incorporated both a CD3z endodomain and a CD28 costimulatory endodomain. Following lymphodepletion, the patient received two infusions of CAR T cells and eight doses of IL-2. As a result of this therapy, the patient underwent selective elimination of B-lineage cells and achieved a partial remission of the lymphoma (61). Autologous CD19-targeted CAR T cells featuring the second-generation CAR (19-28z) were evaluated for safety and long-lasting effects in patients with B-ALL and CLL that had either relapsed or were resistant to chemotherapy in the MSKCC Phase 1 trial. Patients who had previously received cyclophosphamide training exhibited a partial response, whereas those who had not been trained showed no measurable reactions to their disease (62). When Dr. Carl June and his team at the University of Pennsylvania shared their findings that three adult patients suffering from advanced chronic lymphocytic leukemia (CLL) achieved either complete or partial remission after undergoing CD19-specific CAR T cell therapy, it represented a major breakthrough in the application of CAR T cell therapy(63,64). The construct utilized in this trial included the 4-1BB costimulatory endodomain, the CD3z signaling endodomain, and an anti-CD19 scFv derived from FMC63. An EF1-a promoter-driven lentiviral vector was employed to express this construct. Following injection, the CAR T cell counts in patients increased significantly, often by a factor of 1,000. These results enabled the treatment of advanced cases of CLL and other B-cell malignancies using second-generation CAR T cell therapy. The outcomes of these clinical studies revealed that lymphodepletion prior to treatment—specifically a type of chemotherapy that reduces the immune cell count—is essential for the success of CAR T cell therapy. Conversely, it appears that IL-2 is not necessary. Dr. Steven Rosenberg's team was the first to demonstrate that lymphodepleting chemotherapy is effective. They found that the combination of cyclophosphamide and fludarabine led to the in vivo growth and movement of injected tumor-reactive T cells toward tumor locations (65–67). The process of lymphodepletion might include reducing the count of native lymphocytes that compete with the infused T cells and increasing the circulating levels of T cell growth factors such as IL-15. This would promote more effective growth of the administered T cells within the host's body (68). 1.16. Challenges for CAR-T cell therapy Although CAR-T cells have been utilized in clinical settings, several questions persist, including the most suitable vector and the safety profile over the long term. A crucial element of the CAR-T cell product is the viral vector responsible for introducing the CARs into the T cells. Large quantities of the CAR-encoding viral vector can be produced and stored at - 80 °C for as long as nine years (69). Since these cells will be infused into the patients, it is crucial for the vector to be sterile. The third generation appears to be the safest minimum lentiviral vector found so far (70). It is essential to World Journal of Biology Pharmacy and Health Sciences, 2025, 21(01), 114-125 120 conduct quality control testing on safety, sterility, titer, purity, and potency. Given that there are multiple sources for vectors, it is vital to evaluate their function, stability, and purity. Ideally, CAR-T cells should be produced using a single vector while monitoring these variables. A concern that arises is the potential for insertional mutagenesis resulting from the integration of vector DNA into host cells. In comparison to other vectors, lentiviral vectors might pose a lower risk of mutagenesis (71). On another note, retroviral and lentiviral vectors possess the potential to induce cancer. The longterm safety of using viral vectors in CAR-T cell therapy remains uncertain. Thus, it is vital to monitor any possible longterm negative effects linked to these vectors. The impact of durable CAR-T cells on future pregnancies is still not fully understood. To ensure effective handling of materials and patient scheduling throughout the therapy, there needs to be seamless coordination among the collection, manufacturing, and treatment locations. Consequently, establishing a standardized manufacturing process for CAR-T cells is essential to identify the critical quality attributes and desired product characteristics. Furthermore, since various products exhibit differing CAR viability, phenotype, and positivity, gaining deeper insights into these processes is important (72). Leukapheresis produces a diverse array of starting materials, making it difficult to compare the resulting products. While CAR-T cell therapy has demonstrated encouraging results in treating hematological cancers, solid tumors present ongoing challenges due to immune-suppressive tumor environments, the loss of antigens in tumor cells, and a shortage of specific antigens (73, 74). Consequently, notable progress has been achieved by integrating CARs with various effector molecules; including PD1 switch receptors, anti-oxidant enzymes, matrix degradation enzymes, and others, to enhance the efficacy of CAR-T cell therapy (75–79). Severe cytokine release syndrome is the primary side effect associated with CAR-T cell therapy (CRS). It is vital to minimize side effects without compromising therapeutic effectiveness, even though most adverse reactions following CAR-T cell therapy can be managed with existing treatments. Similarly, improving the safety of CAR-T cell therapy is essential for increasing the specificity of the modified T cells (80). 2. The major challenges include 2.1. Severe adverse events CAR T cell therapy has shown great promise in the treatment of hematological cancers, but one major concern with this approach is the potential for life-threatening adverse events. Two of the most common adverse events are cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) (81).CRS is mediated by the cytokines IL-1 and IL-6, which can cause fever, hypotension, and other systemic symptoms. To mitigate the risk of CRS and neurotoxicity, the FDA approved the use of the humanized anti-IL6 receptor antibody tocilizumab in 2017 for CAR T cell therapy. Other potential treatments for these adverse events include the IL-1 receptor antagonist anakinra and the anti-IL6 chimeric antibody siltuximab(82). The severity of adverse events associated with CAR T cell therapy is influenced by several factors, including the pretreatment tumor burden, lymphodepletion regimen intensity, and CAR T cell dose (83). During CD19-CAR T cell therapy, elevated cytokine levels have been associated with prior tumor burden (84, 85). Tumor load can impede the rates of full remission and potentially overall survival, but it does not seem to influence CD19-CAR T cell growth peaks (86). 2.2. High cost of manufacturing autologous CAR T cells One drawback of the present CAR T cell therapy is the expensive charges of producing autologous CAR T cells, which can cause patients with severe CRS to spend up to $500,000 on treatment overall (87). It also takes between 21 and 35 days on average to manufacture autologous CAR T cells. During this waiting period, patients might need bridging therapy and occasionally pass away from quickly progressing illness without receiving the benefits of CAR T cell therapy. Additionally, T cells from healthy donors may be more active than T cells from sick patients due to tiredness. Therefore, a number of approaches are being investigated to make this treatment more accessible and economical, such as the use of commercially available allogenic CAR (allo-CAR) T cells and in vivo CAR T cell production. Generating CAR T cells in vivo is an intriguing cost-effective tactic. By delivering mRNA encoding the FAP-targeting CAR in lipid nanoparticles (LNPs), transient CAR T cells can be produced in vivo (88). 3. Conclusion CAR-T treatment for patients with tumors has shown promising outcomes; however, many remaining challenges need to be considered. The high quality of CAR-T products needs to be ensured through optimization of protocols, and the long-term safety requires further study. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(01), 114-125 121 Compliance with ethical standards Disclosure of conflict of interest No conflict of interest to be disclsoed. References [1] Parikh RH, Lonial S. 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