Cancer Genetics and Epigenetics, 2025, Vol.13, No.3, 136-144 http://medscipublisher.com/index.php/cge 137 point out the current challenges and future development directions. This study will also analyze the background and basic principles of CAR-T therapy, as well as its clinical therapeutic effects, adverse reactions and causes of drug resistance. Look forward to future technological innovations and their wider applications in the field of hematological tumor treatment. 2 The Mechanism and Technological Progress of CAR-T Cell Therapy 2.1 Structural composition and targeting mechanism of CAR-T cells Chimeric antigen receptor (CAR)-T cells are artificially modified T lymphocytes that can express a synthetic receptor for recognizing specific tumor-associated antigens. The basic structure of CAR includes extracellular antigen recognition regions (usually single-strand variable fragments, namely scFv), hinge regions, transmembrane regions, and one or more intracellular signaling regions (Clubb et al., 2020). scFv enables CAR-T cells to bind directly to the antigens on the surface of cancer cells without going through the MHC-mediated antigen presentation process, because in cancer cells, the MHC-mediated antigen presentation function is usually weakened (Gao et al., 2020; Zhou et al., 2021; Hashem et al., 2023). After antigen binding, the intracellular signaling region activates T cells, prompting them to release cytotoxic substances and cytokines, thereby eliminating the target cells (Gao et al., 2020; Clubb et al., 2020). The modular design of CAR can adjust its specificity and function as needed, thereby enabling it to target a variety of different antigens, such as CD19 in B-cell malignancies and BCMA in multiple myeloma (Zhou et al., 2021; Hashem et al., 2023). This direct targeting mechanism is the key to the remarkable efficacy of CAR-T therapy in hematological cancers. 2.2 Functional differences and technical optimizations of each generation of CARS CAR-T cell technology has developed for several generations, and each generation has new improvements to enhance therapeutic efficacy and safety. The first-generation CAR only contains the CD3ζ signaling region, has limited ability to activate T cells, and the survival time of T cells is relatively short. The second-generation CAR adds costimulatory domains (such as CD28 or 4-1BB), significantly enhancing the proliferation ability, survival time and anti-tumor activity of T cells (Clubb et al., 2020; Zhou et al., 2021). The third-generation CAR combines multiple costimulatory regions and further enhances the function of T cells (Hackett et al., 2019; Gao et al., 2020). The fourth-generation CAR, also known as the "armed CAR", is designed to secrete cytokines or immunomodulatory substances after activation, with the aim of breaking the immunosuppressive state of the tumor microenvironment and enhancing the anti-tumor response (Clubb et al., 2020; Gao et al., 2020). Technological improvements also include the development of bispecific Cars to address the issue of antigen escape, as well as the utilization of gene editing technology to enhance safety and reduce off-target effects (Hackett et al., 2019; Zhou et al., 2021; Hashem et al., 2023). These advancements have expanded the therapeutic scope and clinical application prospects of CAR-T cells. 2.3 Production process: from autologous collection, virus transduction to clinical infusion The preparation of CAR-T cells first requires the collection of autologous T cells from patients through leukocyte apheresis. Then, these T cells are activated and genetically modified. The most commonly used method is to introduce the CAR structure into the genome of T cells with the help of viral vectors (Lin et al., 2021; Hashem et al., 2023). The modified T cells will be cultured and expanded in vitro to obtain a sufficient quantity for treatment (Gao et al., 2020; Hashem et al., 2023; Ma et al., 2024). After undergoing strict quality inspection, the engineered CAR-T cells will be reinfused into the patients, where they will search for and eliminate cancer cells expressing the target antigen (Hashem et al., 2023). Although this treatment method has achieved remarkable results in clinical practice, the entire preparation process is complex, time-consuming and costly. There are also problems such as preparation delay and unstable product quality. Recent studies have been dedicated to simplifying the production process, developing general-purpose ("spot") CAR-T products, and improving production efficiency to enable more patients to benefit from this treatment method (Gao et al., 2020; Larson and Maus, 2021; Lin et al., 2021).
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