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Optimize the production of CART cells to improve the anti-cancer effect
Optimize the production of CART cells to improve the anti-cancer effect. CART cell therapy can achieve excellent response rates in patients with severe pretreatment of hematological malignancies. However, recurrence limits the effectiveness of this promising treatment.
The cellular composition and immunophenotype of the administered CART cells play a crucial role in the success of the treatment. Less differentiated CART cells are associated with improved expansion, long-term in vivo persistence and prolonged anti-tumor control. In addition, the ratio between CD4+ and CD8+ T cells has an effect on the anti-tumor activity of CART cells.
The composition of the final cell product is not only affected by the CART cell construct, but also by the culture conditions during the expansion of T cells in vitro. This includes different T cell activation strategies, cytokine supplementation and specific pathway inhibition for differentiation blocking.
The best production process has not yet been determined. In this review, we will discuss in detail the use and molecular background of different CART cell production strategies to generate improved CART cells.
Modern cancer therapies increasingly rely on immunotherapy. In particular, immune checkpoint inhibitors and adoptive cell therapy (ACT), including tumor infiltrating lymphocytes (TIL), T cell receptor (TCR) modified T cells and chimeric antigen receptor (CAR) T cells, represent The milestone treatment in the cancer innovation strategy. ACT shows limitations, because TILs therapy has only achieved encouraging results in certain highly immunogenic cancer entities (such as malignant melanoma) .
Human leukocyte antigen (HLA) restricted antigen recognition limits the application of TCR-modified T cells. Down-regulation of HLA expression can lead to tumor escape . CART cells combine the kinetics of T cells with the antigen specificity of antibodies. They can bind tumor antigens without antigen processing, and have nothing to do with HLA-mediated antigen presentation. CD19-specific CART cell therapy has shown very promising results in B-cell malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) .
Recently, the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) approved Kymriah® (Tisagenlecleucel) for the treatment of relapsed/refractory (r/r) B cell precursor ALL  or dual The used patient B-cell lymphoma (DLBCL)  and Yescarta® (Axicabtagene Ciloleucel) are used to treat patients with r/rDLBCL and primary mediastinal B-cell lymphoma (PMBCL) . Other tumor antigen targets for the treatment of multiple myeloma are currently being developed, such as B cell maturation antigen (BCMA) . In solid tumors, CART cells must still overcome the limitations of their therapeutic use .
Although the response rate is encouraging, it still relapses and limits the effectiveness of this promising treatment. Therefore, it is essential to understand the current limitations of CART cell therapy in order to fully utilize the potential of this modern anti-cancer therapy [9,10]. The in vivo efficacy of CART cells is related to their proliferation ability and long-term continuous maintenance of sufficient anti-tumor activity . In vivo expansion and persistence of CART cells are limited in some patients, and long-term anti-tumor control is prohibited. One way to improve the activity of CART cells is the further development of CAR constructs and gene transfer systems.
The reduced adaptability and dysfunction of T cells in the final cell product used by some patients may be another reason for impaired activity in the body. Therefore, improving mitochondrial fitness and biogenesis capacity may enhance the therapeutic effects of CART cell therapy and other ACTs . Another way to improve the efficacy of therapeutic CART cells is the selection or modification of CART cell subpopulations and subpopulations. The cellular composition of the final cell product has a significant impact on the proliferation ability of CART cells, and it is directly related to the efficacy in vivo [13-15].
The optimal T cell activation and culture strategy for CART cell generation is essential to produce high-efficiency CART cells with the preferred T cell immune phenotype and subpopulation. However, the manufacturing process of CART cells has not yet been standardized. In this review, different strategies for generating high-efficiency CART cells will be discussed.
2. The role of different T cell subtypes and subgroups in effective CART cell therapy
It is assumed that the number of input CART cells mainly determines the early treatment success rate of CART cell therapy. However, when a certain threshold is exceeded, the absolute number of CART cells infused is not directly related to in vivo expansion and treatment success . Therefore, in addition to the absolute number of CART cells infused, other factors may be more important for the effectiveness of CART cells. For example, the cellular composition and phenotype of adoptively transferred T cells, including T cell subtypes and subpopulations, have been identified as one of the most critical success factors for effective immunotherapy [16,17].
Although cytotoxic CD8+ CART cells can especially mediate the direct elimination of tumor cells, CD4+ T helper cells (Th cells) are considered to be highly effective and clinically important T cell subsets . Facts have proved that CD4+ CART cells have comparable cytotoxicity to cytotoxic CD8+ CART cells . In addition, a balanced ratio of CD4+ Th cells and CD8+ cytotoxic T cells can have a positive impact on tumor elimination . According to reports, a 1:1 ratio of CD4+ and CD8+ (CD4:CD8 ratio) CART cells can achieve a higher remission rate in the treatment of B-ALL patients . For example, Lisocabtagenemaraleucel (liso-cel; JCAR017) represents an anti-CD19 CART cell product that is administered in a specific ratio and has a specific ratio of CD4+ Th CART cells and cytotoxic CD8+ CART cells . The subsets must be isolated at the beginning of production and modified separately to obtain the defined CD4:CD8 ratio, which leads to a more complicated manufacturing process.
In addition, different Th cell subgroups also play an important role. The balance between TE cells and regulatory T (Treg) cells will affect the success of adoptive immunotherapy . The infiltration of CD4+ Treg cells into solid tumors can reduce the anti-tumor activity of CD28-CD3 signal transduction CART cells . In the presence of Treg cells, the deletion of the Lck binding part in the CD28 CAR domain of the CD28-CD3 signaling CAR can enhance the anti-tumor effect . According to reports, CART cells with inducible T cell costimulator (ICOS) intracellular signaling domain can stabilize Th17 cell function and enhance the persistence of CART cells in mice bearing human tumor xenografts . In addition, compared with CART cells based on 4-1BB, CART cells with ICOS and 4-1BB intracellular signaling domains show enhanced efficacy in solid tumors .
In addition to T cell subtypes, the differentiation state of CART cells also plays a crucial role in the success of treatment. Isolated and ex vivo expanded T cells have inherent characteristics that must be considered in cellular immunotherapy. The function, phenotypic characteristics of T cells and their appearance in peripheral blood (PB) of healthy donors and patients vary, depending on age, previous antigen exposure, and cytotoxic therapies applied due to their differentiation state [25 ].
In ACT, the terminally differentiated CD45RA+ CCR7 T effector cells (Teff cells) showed enhanced anti-tumor activity in vitro, while the activation, proliferation and persistence of T cells in vivo were impaired . These findings changed the method and criteria for selecting specific T cell subsets for ACT, and focused on less differentiated T cells: naive T cells (TN cells) defined as CD45RA+ CD45RO CD95RO CD95 T cells expressed Lymph node homing markers CCR7 and CD62L, as well as CD28 and CD27 . On the contrary, CD45RACD45RO+ CD95+ memory T cells can be divided into CD62L+ CCR7+ T central memory-like cells (T CM cells) and CD62LCCR7 T-segment memory-like cells (TEM cells) .
Stem cell memory-like T cells (TSCM cells) represent a recently described T cell subpopulation, similar to TN cells, because they are CD45RA+CD45RO CCR7+, and they express memory-related markers, such as CD95, thus exhibiting the characteristics of stem cells. Characteristics, including high proliferation and self-renewal ability [25,26]. After administration to patients, TN cells and TSCM cells have the ability to survive and proliferate in the body for a long time, and they may lead to improved clinical outcomes [16,26,27]. In particular, the ability of self-renewal and the ability to differentiate in all memory and effector subgroups allows TSCM cells to maintain long-term anti-tumor activity by providing more differentiated TEM cells and TE cells for immune attack.
Refresh the T cell pool with new TCM cells and TCM cells with a lower degree of differentiation . Therefore, a large reduction in the transfusion of differentiated CART cells is beneficial to the success of the treatment. The potential of a single T cell subpopulation is well described in the literature . However, there are few descriptions on how to form a more favorable cell composition and T cell phenotype in the final CART cell product during the production process.
3. Expression of exhaustion and homing markers on CART cells
Inhibitory tumor microenvironment combined with inhibitory receptors, such as PD-1, CTLA-4, LAG-3 and TIM-3 on T cells, may also lead to insufficient response rate of CART cells in certain tumor entities, thereby impairing immune attack [28,29]. The high expression of fatigue-related inhibitory receptors PD-1 and TIM-3 on CD8+ T cells is related to impaired T cell function . Tumor-specific T cell dysfunction is a dynamic process that leads to antigen-driven differentiation and is triggered in the early stages of tumorigenesis .
Transcriptome analysis showed that gene expression related to memory is enriched in CART cells of CLL patients, and complete remission can be achieved. In contrast, analysis of CART cells from non-responders revealed that genes that mediate T cell differentiation, glycolysis, exhaustion, and apoptosis are up-regulated . In addition, a group of CD8+ CART cells with weak differentiation and no PD-1 expression have been identified to play a vital role in tumor control . In addition, lower expressions of PD-L1, PD-1, LAG-3 and TIM-3 were observed in lymphoma patients who responded to CD19-specific CART cell therapy.
Non-responders express higher levels of immune checkpoint ligands on tumor cells and immune cell receptors . By transferring the target antigen to T cells, CART cells can cause reversible antigen loss through phagocytosis, resulting in a decrease in target density on cancer cells . In addition, T cell activity is reduced by promoting exhaustion and killing T cells . It has been reported that CART cells encoding a single immunoreceptor tyrosine-based activation motif (ITAM) show improved persistence of highly functional CART cells .
Strategies that lead to the destruction of the interaction between inhibitory T cell receptors and their ligands expressed on cancer cells may improve the therapeutic effects of cell-based therapies. The use of PD-1 blocking antibody improves the therapeutic effect of CART cells . In addition, it is reported that, compared with conventional CART cells in preclinical models, CART cells that produce anti-PD-1 single-chain variable fragments (scFv) mediate effective therapeutic effects . Although these strategies are aimed at optimizing in vivo CART cell therapy after administration to patients, there must be other strategies to improve fatigue, especially reducing the expression of inhibitory receptors on CART cells during the manufacturing process.
Another challenge is to improve the infiltration of CART cells into the tumor site. T cell homing is the result of multiple molecular interactions. The suppression of the anti-tumor immune response of CART cells at the tumor site can be mediated through the immune barrier . Different homing characteristics constitute another unique feature of different T cell subgroups. TN cells, TSCM cells and TCM cells tend to migrate to lymphoid tissues, while TEM cells and TE cells prefer peripheral tissues . The stronger expression of lymphatic homing markers CD62L and CCR7 on less differentiated T cells is related to the improvement of anti-tumor activity in preclinical models of ACT, which may be beneficial to CART cells .
T cell extravasation, homing and persistence in the tumor microenvironment are important aspects to overcome the current limitations of CART cell therapy in solid tumors. Facts have proved that CD28 costimulation can reduce the inhibition of T cell proliferation mediated by transforming growth factor (TGF) . Overexpression of CXCR2 can improve the migration of T cells to tumor sites . The overexpression of CCR2b on mesothelin-specific CART cells  and GD2-specific CART cells  resulted in enhanced T cell tumor infiltration.
It has been reported that CD30-specific CART cells expressing CCR4 can mediate enhanced tumor control in xenograft models . NKG2D-specific CART cells can recruit and activate endogenous antigen-specific cytotoxic CD8+ cells and CD4+ Th cells at the tumor site in a CXCR3-dependent manner, thereby improving tumor eradication . In the future, the regulation and effect of specific homing marker expression on CART cells must be further examined.
4. Optimization of CART cell manufacturing process
The main aspects of the CART cell manufacturing process are relatively standardized, and significant differences can be found in each manufacturing step (Figure 1).
The production process of CART cells includes the initial separation and enrichment of T cells , the preparation of CART cells, including T cell activation , T cell expansion , and the use of viral or non-viral vectors for gene transfer of CAR vectors . Viral vector system , and then CART cell amplification in vitro  (Figure 1). The final cell product requires post-processing and cryopreservation  (Figure 1). In order to ensure the integrity of the product, quality control tests are performed during the production process and in the final frozen CART battery products. Cancer patients usually receive lymphatic clearance therapy  before giving the final approved CART cell product  (Figure 1).
4.1 Separation and enrichment of T cells
Peripheral blood mononuclear cells (PBMC) are usually obtained from PB. Ficoll density gradient centrifugation is used to remove granulocytes, red blood cells and platelets . Alternatively, an automated cell scrubber can be used to separate T cells . Other tools have been developed to facilitate or combine manufacturing steps in a system, such as the Sefia cell processing system for the separation, harvesting and final formulation of cell products, or the CliniMACS Prodigy® type for automated GMP-compatible cell manufacturing [ 47,48].
The cell composition at the beginning of the production process affects the phenotype of CART cells, because patients with a large number of tumor cells in PB (such as untreated CLL patients) show fewer and less poorly differentiated T cells in PBMC . Endogenous cytokines can supplement cytokines and therefore can reduce the CART cell effect mediated by cytokines . Therefore, it may be necessary to choose CD3+ T cells in patients with a large number of circulating tumor cells in PB. Magnetic bead-based systems (such as the CliniMACS® system), such as systems with anti-CD3+, anti-CD4+ or anti-CD8+ microbeads, can be used to select or eliminate specific T cell types in PBMC supporting T cells with a defined CD4:CD8 ratio Amplify and administer the final cell product .
In order to enrich TN cells, TSCM cells and TCM cells, research focuses on the clinical scale selection, transduction and cell expansion of these low-differentiated T cells [51-53]. It seems very beneficial to generate CART cells from a defined T cell subpopulation. However, the previous selection process may complicate the manufacturing process, and the optimal cell composition at the start of CART cell production has not yet been determined.
4.2 T cell activation
T cell activation is an essential step for CART cell production. Optimal activation should result in sufficient T cell expansion without causing huge T cell differentiation or activation-induced cell death (AICD).
4.2.1 Anti-CD3/anti-CD28 antibodies
The established concept of activating T cells represents the use of unconjugated monoclonal antibodies. The coating of petri dishes or packaging bags can use anti-CD3 monoclonal antibodies (OKT-3) with or without anti-CD28 monoclonal antibodies. It is more common to use anti-CD3/anti-CD28 antibody-coated magnetic beads as artificial antigen presentation particles. Anti-CD3 antibodies can provide a strong proliferation signal, while anti-CD28 antibodies can provide an effective costimulatory signal .
The beads can continuously stimulate the cells and can be removed with a powerful electromagnet. It has been reported that compared with OKT-3 and interleukin (IL)-2 activation, beads have higher cytokine production and the resulting T cell activation [52,54]. In addition, compared with soluble stimulation, anti-CD3/anti-CD28 antibody-coated magnetic beads can induce less differentiation, possibly senescent T cells, and CART cells with enhanced proliferation ability and early anti-tumor response in vivo. OKT-3 and high-dose IL-2 . Other advantages involve the CART battery manufacturing process itself.
Enrichment and washing are simplified because the beads bound to the cells can be retained magnetically. In addition, beads that are not removed can be selected and activated until the end of the expansion and loss of expensive stimulating antibodies during the medium exchange process. Decrease . Therefore, anti-CD3/anti-CD28 antibody coated magnetic beads are considered to be a more promising activation strategy. In recent clinical trials, anti-CD3/anti-CD28 antibody-coated magnetic beads are often used, for example (Tisagenlecleucel; CTL019) [56,57] and Lisocabtagene maraleucel (liso-cel; JCAR017) , and for production (AxicabtageneCiloleucel; KTE-019) Anti-CD3 antibody with IL-2 .
According to reports, a dedicated polymeric nanomatrix product (TCell TransAct-) coupled with humanized recombinant CD3 and CD28 agonists, together with serum-free medium (TexMACS-), can be used for T cell activation during CART cell production. Compared to activation with plate-bound anti-CD3/anti-CD28 antibodies, this strategy resulted in an increase in TCM cells with high CCR7 and CD62L expression, an increase in IL-2 production, and a decrease in the level of exhausted CD57+ cells . However, the expression of PD-1 was not significantly affected. This strategy has no significant effect on the efficiency of gene transfer. According to reports, this activation strategy mediated lower expansion, supporting the expansion of CD4+ T cells with an average CD4:CD8 ratio of 4:1, and anti-CD3/anti-CD28 antibodies bound to the plate .
Another activation strategy involves the recombinant human fibronectin fragment RetroNectin®, which is mainly known to mediate increased gene transfer efficiency in retroviral transduction. Little is known about its use as a T cell activator. Retronectin combined with anti-CD3 or anti-CD3/anti-CD28 monoclonal antibodies used in the production of GD2-specific CART cells  and CD19-specific CART cells  for T cell activation can promote TN and TSCM cells Phenotype.
According to reports, similar effects to retroectectin-mediated T cell activation, as well as anti-CD3 antibody or anti-CD3/anti-CD28 antibody-coated microbeads against engineered T cells expressing AcGFP . Retronectin-based T cell activation can increase the number of cytotoxic CD8+ T cells and may shift the ratio of CD4:CD8 to 1:1, while activation with anti-CD3/anti-CD28 induces CD4+ Th cell expansion[60-62] .
The main disadvantages of retronectin-mediated T cell activation are weak T cell activation, insufficient T cell expansion, reduced gene transfer efficiency and reduced cytokine secretion. This is a report on GD2-specific CART cells  and CD19-specific CART cells . In addition, in patients with high PB tumor burden, the use of retronectin to activate T cells must be cautious, because it can activate and stimulate the persistent malignant B cells in the cell product, especially if the T cell selection process has not been performed before. Cells are activated at the beginning of production .
4.2.3 Artificial antigen presenting cells
In recent studies, non-surviving artificial APCs have been used to activate T cells produced by CART cells, which act as tumor-associated antigens (TAA) to activate T cells in a CAR-dependent manner . To this end, K-562 cells were genetically modified to co-express costimulatory molecules and TAA . These modified K-562 cells are irradiated and can be used for numerical amplification of CART cells. The advantage of this strategy is that it does not describe the expression of HLA-A or HLA-B molecules, and it can be cultured well in compliance with GMP . In addition, these artificial APCs only stimulate TAA-specific CART cells.
In conclusion, the applied activation strategy can positively affect the cellular composition and phenotype of cell products. In recent studies, the main T cell activation strategy is to use anti-CD3/anti-CD28 antibody-coated magnetic beads, followed by monoclonal antibodies. However, new alternative strategies are being developed. The best strategy depends on the tumor entity and tumor burden in PB.
4.3 Gene transfer system
The non-viral or viral gene transfer vector that transfers the corresponding genetic information to the T cell mediates the expression of CAR on the surface of the T cell. Plasmid-based transposon/transposase systems and viral vectors, including γ-retrovirus and lentiviral vectors, as well as genome editing and electroporation of naked DNA, can be used for gene delivery in CART cell therapy.
4.3.1 Viral transduction
Virus-based gene delivery systems are commonly used, which can achieve high transduction efficiency . Among the most commonly used viral vector systems, there are γ-retroviral vectors and lentiviral vectors, both of which belong to the retrovirus family . Retroviruses mediate stable long-term gene expression because the resulting viral DNA is integrated into the host DNA . Lentivirus needs to regulate genes to neutralize host cell defenses, weaken the immune response, and regulate virus replication .
The risk of insertional mutagenesis and carcinogenicity of lentiviral vectors seems to be low . There is almost no genotoxic effect of gene transfer to differentiated cells including T cells. In only a few cases, transgenic T cells have been reported in patients undergoing virus-mediated transformation therapy [65-67]. Lentiviral vector-mediated CAR transgene insertion was observed in CLL patients treated with CD19-specific CART cells, resulting in the destruction of the methylcytosine dioxygenase TET2 gene .
These CART cells that disrupted TET2 showed modified T cell differentiation, leading to a central memory phenotype at maximum proliferation . Although insertional mutagenesis is undesirable, the described TET2 modification can be used to optimize CART cell therapy. In lentiviral vector-mediated CBL gene integration, CD22-specific CART cell therapy treats CD22-specific CART cells, which is very important for the regulation of T cell response . In addition, insertional mutagenesis caused the patient to relapse after treatment with CD19-specific CART cells for CD19-negative leukemia, thereby allowing the tumor to escape .
In this case, the CAR gene was unintentionally introduced into a single leukemia B cell during the production of CART cells, thereby masking the meaning of recognition . To our knowledge, accidental insertion of γ-retroviral vectors for CART cell therapy has not been reported. Therefore, γ-retrovirus is still widely used and regarded as a safe carrier system for clinical ACT.
However, in order to produce retroviral vectors, stable packaging cell lines can be used, and the production of lentiviral vectors requires a large amount of plasmid DNA for transient transfection . The prerequisite for effective delivery of viral genes is the existence of dividing T cells after activating T cells, especially for retroviral gene transfer . Intensive and expensive vector production is the main disadvantage of viral gene transfer systems because of the need for proper clean room facilities and the performance of vector release tests for retrovirus or lentivirus transduction of cells . Even for large pharmaceutical companies in this field, this has become a major bottleneck.
Lentiviral transduction is the main viral gene delivery system. For example, it is used to produce Kymriah ® (Tisagenlecleucel) for the treatment of ALL  or DLBCL , and Lisocabtagene maraleucel (liso-cel; JCAR017) is used for the treatment of r /r Invasive NHL . For example, retroviral transduction was performed in the ZUMA-1 trial of Axicabtagene Ciloleucel for the treatment of r/r large B-cell lymphoma [58,70]. Viral vectors can not only regulate effective gene transfer efficiency, but also produce safe products. However, the production of viral vectors is still very laborious and therefore a cost-intensive aspect in the production of CART cells.
4.3.2 Plasmid-based gene delivery
The transposon/transposase system constitutes another strategy for non-viral CAR gene delivery. The “SleepingBeauty” transposon/transposase system is used for CART cell manufacturing . The system consists of two DNA plasmids, one containing the transposon encoding the CAR transgene, and the other expressing the transposase necessary for excision and insertion of the transgene [69,72].
Compared with electroporation of naked DNA, the use of the transposon system can increase the efficiency of gene transfer, showing promising results for CART cell therapy, and it represents an economically beneficial strategy . The advantage of this plasmid-based gene delivery for CART cell therapy is that it is cheap and labor-intensive, because it does not require the generation of GMP-grade viruses .
Plasmid electroporation is mainly used in first-generation (1G)  and third-generation (3G) CART cells . The first clinical application of the “Sleeping Beauty” transposon/transposase system in CART cell therapy has produced encouraging results .
At present, the analysis of applied gene transfer systems mainly focuses on transduction and clinical efficacy, safety and cost. The optimal gene transfer system has not yet been determined and further research is needed.
4.3.3 Genome editing
Genome engineering tools, especially CRISPR/Cas9-based gene editing, represent the field of development of CAR-based therapies, which can carry out effective sequence-specific interventions on human cells . CRISPR/Cas9 technology can achieve specific genome destruction of multiple gene loci. The CRISPR/Cas9 system is combined with the adeno-associated virus (AAV) vector repair matrix, and the CAR-encoding DNA is integrated into the T cell receptor constant (TRAC) locus, thereby causing the uniform expression of CAR, thereby improving the effectiveness of T cells.
And the inhibitory effect on T cell differentiation and fatigue . In addition, it is reported that CRISPR/Cas9-mediated genome editing and lentiviral transduction are used to produce PD-1 deficient CD19-specific CART cells, thereby enhancing anti-tumor and therapeutic effects . Despite concerns about multiple issues, including efficiency, safety, and scalability, CRISPR/Cas9 enhanced immune gene cell therapy may further improve CART cell therapy . However, in CART cell-based immunotherapy, the full potential of genome editing has not been fully utilized and must be further studied in human clinical research.
4.4 CART cell construct
The optimal composition of CAR is essential for effective CART cell-based cancer immunotherapy. CARs contain antibody scFv as the extracellular binding domain recognized by HLA-independent antigens, and the transmembrane (TM) domain and CD3 chain as the intracellular signal transduction domain  (Figure 2). The additional stability of CAR can be obtained by the non-signaling extracellular spacer domain between the scFv and TM domains . The length and composition of the spacer domain can affect CART cell function independently of the intracellular domain [80,81]. The spacer domain is usually composed of the IgG hinge domain and CH2-CH3 domain of IgG-Fc .
Since its launch, the CAR design has undergone several generations of further development (Figure 3). The first-generation CARs are designed to have no costimulatory domain and only induce T cell activation through the main signal of the CD3 signaling domain. CART cells that rely solely on CD3 for signal transduction show low cytokine production capacity, insufficient T cell expansion, and it quickly becomes unresponsive [82,83]. These CD3-based CART cells have sufficient antigen-specific cytotoxicity, but the expansion ability of T cells is weak . Therefore, the clinical results of patients with ovarian cancer , NHL  and neuroblastoma  treated with CD3-based CART cells are also limited.
The second generation (2G) CAR was developed to achieve long-term durability and scalability, as well as to prevent AICD and anergy . The far-reaching change lies in the integration of a costimulatory domain, such as CD27 , CD28 [89,90], CD134 (OX40)  or CD137 (4-1BB) [92,93]. This modification improves the in vivo characteristics of CART cells and protects CART cells protected by AIART . Persistence in vivo is significantly affected by the inserted costimulatory domain . It has been described that CD28 as a costimulatory domain can support stronger T cell expansion and improve tumor eradication [89,90], while 4-1BB as a costimulatory domain is associated with persistence and can Improve the development of fatigue failure . Facts have proved that phosphorylation of tonic CARCD3 can cause early failure of CART cells, thereby limiting their anti-tumor ability .
In addition, it has been shown that CD28 costimulation increases, while 4-1BB costimulation reduces fatigue induced by ongoing CAR signaling . The second-generation CAR contains only one costimulatory domain (CD28 or 4-1BB), while the third-generation CAR contains a second costimulatory signal [96,97]. The combination of two costimulatory domains in the 3rd generation CAR may have the potential to combine these two advantages. Simultaneous infusion of 2G (CD28) and 3G (CD28/4-1BB) CD19-specific CART cells into patients shows that 3GCART cells have excellent expansion and persistence .
In addition, it has been shown that the intracellular signaling activity of the third-generation CART cells is higher than that of the second-generation CART cells, and may lead to excellent cell proliferation . First, the clinical study of anti-CD19 third-generation CART cells proved the efficacy and safety of patients with B-cell malignancies .
The further development of CAR has led to the redirection of fourth-generation (4G) CARs and T cells to universal cytokine-mediated killings (TRUCKs). These new fourth-generation CARs express molecules through complementary genetic modifications in the CAR structure, thereby improving the therapeutic effect of CART cell therapy . The CAR structure is a vehicle for the redirection of CAR T, which can produce and release transgenic products at the tumor site, such as pro-inflammatory cytokines . Recruitment and activation of other components of the immune system can be achieved through additional expression of costimulatory ligands (such as 4-1BB-L  and CD40-L ) or pro-inflammatory cytokines (such as IL-15). IL-7 and IL-21  lead to excellent anti-tumor cytotoxicity. According to reports, CD19-specific CART cells that secrete IL-12 can eradicate established tumor diseases without prior regulation .
It must be mentioned that IL-12 secreting T cells targeting tumors are resistant to T reg cell-mediated suppression . CAR-induced integration of IL-12 cytokine cassettes leads to the secretion of IL-12 after CAR signal transduction, which leads to the accumulation and maintenance of therapeutic levels of cytokines in target tissues, leading to TAA-expressing destroyed cells and TAA-negative tumor cells [105,106 ]. The disadvantage is that only the tumor site that expresses the antigen can initiate the release of IL-12. This strategy must be applied with caution: cytokines with safe toxicity characteristics and controlled release of cytokines need to be used . In addition, it has been reported that armored CART cells modified to express degrading enzymes exhibit an enhanced ability to infiltrate tumor sites .
Extensive research is currently underway to further optimize the CAR structure. Most of the protocols used for CART cell generation are optimized for 2nd and 3rd generation CARs. If it is necessary to develop novel production protocols for these novel CART cell therapy methods, further analysis is required.
4.5 T cell expansion
During the expansion of CART cells, the number of cells continues to increase, so more or larger tissue culture flasks or plates must be used to change the volume of the medium. This greatly complicates the manufacturing process and is incompatible with mass production. Therefore, static culture bags have been developed to allow less manual opening operations because the tube connection can be performed under sterile conditions . An alternative method is swing motion bioreactors, such as Xuri-Cell Expansion System and WAVE TM Bioreactor System, which use perfusion schemes to add nutrients and remove growth inhibitory substances, thereby simplifying the manufacturing process [109,110].
4.5.1 Cytokine stimulation
In addition to CAR vectors and T cell activation strategies, in vitro stimulation with supplementary chain cytokines during CART cell production is another important factor that affects the composition, quality and phenotype of the final CART cell product. The receptors for chain cytokines, such as IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, have a common CD132 or -chain. The two most common strategies produced by CART cells are based on IL-2 or IL-7 (with or without IL-15). So far, IL-2 has been mainly used for T cell expansion in previous clinical studies [3,45,46].
For example, IL-2 is supplemented for production (Axicabtagene Ciloleucel) . However, in the presence of IL-2, the expansion of T cells in vitro can lead to a more differentiated and exhausted phenotype and reduce the persistence of T cells . Compared with IL-2, the amplification of IL-7/IL-15 shows enhanced activation and proliferation . In addition, it is reported that the combination of IL-7/IL-15 can promote the survival and maintenance of less differentiated T cells (such as TN cells and TSCM cells with high expression of CD62L and CCR7) [112-114]. In addition, compared with IL-2 amplification, supplementation of IL-7-/IL-15 mediates CD4+ and CXCR3+ CD19-specific CART cells  and NY-ESO-1-specific T cells  High amplification.
It has been reported that compared with IL-2, supplementation with IL-15 alone can lead to reduced expression of exhaustion markers, increased anti-apoptotic properties, improved proliferation, and preservation of TSCM phenotype . In addition, IL-15 induces a decrease in mTORC1 activity, a decrease in glycolytic enzyme expression and an increase in mitochondrial adaptability, leading to the prevention of T cell differentiation .
IL-21 is another important member of the -chain family. It is reported that TILs amplified with IL-21 show less CD27+ CD28+ differentiation phenotype and have enhanced cytotoxicity . In adoptive cell transfer, IL-21 can inhibit antigen-induced bidentateization of CD8+ T cells, while IL-2 and IL-15 can enhance the differentiation into terminally differentiated TE cells . In addition, compared with IL-2 and IL-15, IL-21-mediated CD62L expression is higher, and anti-tumor activity is enhanced . The adoptive transfer of CD19-specific CART cells stimulated by IL-21 leads to enhanced control of B-cell malignancies in preclinical models .
In short, supplementation of cytokines during the in vitro expansion of CART cells is indispensable and essential for the CART cell manufacturing program. Current research mainly relies on IL-2, IL-7, IL-15 and IL-21. The optimal cytokine composition and the role of other cytokines in the production of CART cells are still unclear.
4.5.2 Inhibit specific signal pathways
In the process of in vitro T cell expansion, supplementation of pathway inhibitors may interrupt T cell differentiation by inhibiting specific signal transduction pathways, thereby shifting the T cell phenotype in the final CART cell product to a less differentiated phenotype [ 120]. Possible targets include GSK3, mTOR, AKT and PI3K for specific pathway inhibition (Figure 4).
It has been reported that the WNT-βcatenin signaling pathway induced by inhibiting GSK3β may interrupt the T cell differentiation process and generate CD8+ TSCM cells . The PI3K-AKT-mTOR signaling pathway is essential for the activation, survival, expansion, migration, function and differentiation of T cells .
mTOR plays a central role in the formation of T cell memory. The mTOR inhibitor rapamycin can mediate, preclinical evaluation, more T memory cells, increased expression of lymph node homing marker CD62L, and the anti-apoptotic molecule Bcl-2 . It has been reported that supplementation of IL-15 during the expansion of CART cells in vitro may reduce mTORC1 activity and retain a less differentiated phenotype . The retention of IL-15-mediated T cell phenotypes with a low degree of differentiation is likely to be caused by the decreased activity of mTORC1, because CART cells were expanded with IL-2 in vitro, and the mTORC1 inhibitor rapamycin showed similar Phenotypic characteristics, such as CART cells only expand on IL-15 . Studies have shown that adoptively transferred T cells exhibit improved anti-tumor activity after AKT inhibition in vitro [124,125].
B-cell receptor (BCR) pathway inhibitor Idelalisib is an inhibitor of phosphatidylinositol 3-kinase p110δ (PI3Kδ) and has been approved for the treatment of patients with CLL and follicular lymphoma. In addition to eliminating malignant B cells, edelisib can also degrade Treg cells, thereby reversing the immune tolerance of cancer cells [126,127]. In vitro treatment of CART cells with PI3Kδ inhibitors can mediate a greater number of undifferentiated CCR7+ CD62L+ T cells, and improve the mesothelin specificity , CD33 specificity  and CD19 specific CART cells Functional capabilities .
In healthy donors, supplementation of IL-7/IL-15 during the production process results in a very balanced CD4:CD8 radiation. However, samples of CLL patients without pre-selected T cells showed an imbalance in the CD4:CD8 ratio, and the main CD4+ T cells can be close to a 1:1 ratio by supplementing with Idarisib during the manufacturing process . The decrease in the expression of failure markers is another positive effect of PIK3 inhibitors in the production of CART cells [49,128].
The use of antagonists against PI3K and vasoactive intestinal peptide (VIP) during the ex vivo expansion of T cells in DLBCL patients can inhibit terminal T cell differentiation, reduce PD-1 expression and improve persistent T cell deficient mice in the immune system . The addition of these antagonists improved the expansion and gene transfer efficiency of human anti-CD5 CART cells, as well as their cytotoxic ability to CD5 + lymphoma cells .
This indicates that cooperative blockade is also a promising strategy to improve the expansion and functional capacity of antigen-specific T cells expanded in vitro . In addition, it is reported that the B cell adaptor (BCAP) of PI3K is also a regulator of CD8+ T cell differentiation, and may be another target for inducing the formation of specific T cell subsets .
Inhibition of PI3K/AKT/mTOR pathway can lead to down-regulation of c-Myc. Down-regulation of c-Myc-dependent target gene therapy of T cells with bromodomain and extra-terminal motif (BET) bromodomain inhibitors  resulted in enhanced expansion of CD8+ TSCM cells and TCM cells, improved durability and anti-tumor The number of CART units in the active ALL model . The increase of TN cells and TCM cells in CD33-specific CART cells was also reported. These cells were treated with the BET inhibitor JQ-1 or iBET for four days after five days of T cell activation .
In short, in vitro treatment with specific pathway inhibitors during the manufacturing process of CART cells may have a positive effect on CART cells, and therefore can improve the final CART cell product. Optimizing the use of signal pathway inhibitors for clinical applications will be the next step to further enhance the efficacy of CART cell therapy.
For most of the currently used CART cell therapy methods, quality control testing must be mandatory for cryopreservation of CART cells at the end of production, and the final product can be transported from the manufacturing site to the clinical center. In addition, the administration of the product is more flexible, and patients may receive a variety of CART cell therapies. According to reports, cryopreservation of CART cells for up to 90 days does not hinder the viability, recovery ability and gene transfer efficiency of cryopreserved CART cells . Although the function of the CART cells frozen immediately after thawing was reduced, the overnight incubation at 37°C restored the function of the CART cells, thus recovering from the severe freezing and thawing process . In addition, CART cells cryopreserved and thawed immediately before blood transfusion showed in vivo persistence and efficacy similar to fresh CART cells . All in all, cryopreservation is a manufacturing step for routine applications, and obviously it will not significantly damage CART cell products.
5. Conclusions and future prospects
CART cell therapy represents a promising new treatment option for patients with hematological malignancies, and may soon become a treatment option for patients with solid tumors.
Although both the FDA and EMA have approved the first CART cell products, the expensive and highly variable manufacturing process is still a controversial issue. Improved CART cell therapy can be achieved by infusing CART cell products with good phenotypes (including CART cells with a lower degree of differentiation).
In addition, costs can be reduced through more effective production agreements. The potential to improve anti-cancer immunotherapy by optimizing ex vivo expansion conditions during the CART cell manufacturing process has not yet been fully exploited. Therefore, further efforts must be made to standardize and optimize the CART cell production program.
(source:internet, reference only)