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The development strategy of CAR-T in the latest cancer treatment
The development strategy of CAR-T in the latest cancer treatment. In 2017, CAR-T cell products represented by Kymriah (CTL019) and Yescarta (KTE-C19) have been approved by the U.S. Food and Drug Administration (FDA) for sale on the market. The therapeutic potential of CAR-T in hematological malignancies.
However, although CAR-T is a promising anti-tumor drug candidate, it still faces many problems because it continues to encounter follow-up obstacles in the treatment of solid tumors.
Due to the mismatch of chemokines between CAR-T and tumor cells  and the presence of abnormal blood vessels  and stroma  in the tumor, it is difficult for CAR-T cells to migrate correctly and infiltrate the microenvironment of the solid tumor site ( TME).
Subsequently, CAR-T cells entering tumor tissues and various immunosuppressive cells (for example, regulatory T cells and suppressor cells of myeloid origin) [6,7], immune checkpoints (for example, programmed death ligand 1 [ PD-L1]) competition , and metabolic checkpoints in TME (for example, hypoxia and glycolysis) [9,10].
Even after successfully entering the tumor tissue, these CAR-T cells must also solve problems such as tumor antigen heterogeneity and antigen escape [11,12]. Moreover, the reduced activity and persistent limitation of the injected CAR-T product may be the main reason for tumor recurrence in hematology and solid tumors .
The inevitable immune-mediated toxicity is manifested as cytokine release syndrome (CRS) and immune effector cell-related neurotoxicity syndrome (ICANS), which are also key issues in the clinical application of CAR-T . Although mild patients may only show self-limiting symptoms, such as fever and hypotension, life-threatening organ damage and death may also occur, similar to those reported in the P-PSMA-101 study or the UCARTCS1 product .
Although CAR-T products with Good Manufacturing Practice (GMP) grade can be used at present, the manufacturing process of CAR-T still needs to be improved to increase the clinical use rate . More effective and economical methods to obtain T cell isolation, activation, amplification and genetic modification are worth exploring [17,18]. The success of CAR-T therapy in cancer treatment put it into a barrier.
Recently, more effective solutions have been developed and reported. Therefore, in this review, we discussed and summarized recent significant improvements and breakthroughs in CAR-T treatment to meet existing challenges (Figure 1).
2. Enhanced durability and anti-tumor properties of CAR-T treatment
2.1 Optimization of CAR-T structure
2.1.1 Optimization of extracellular domain
The single-chain variable fragment (scFv) designed to identify tumor antigens is the main component of the extracellular segment of CAR-T. Antigen heterogeneity is an important factor hindering the role of CAR-T in cancer treatment. According to reports, different antigen expression densities and simultaneous presence of different antigens are crucial for tumor escape or recurrence . Therefore, promoting CAR-T specific antigen recognition may help enhance the efficacy of CAR-T in cancer treatment.
Optimization of extracellular domain
Some cells with stem-like properties in solid tumors are called cancer stem cells (CSCs) because they are related to the evolution of tumor heterogeneity and resistance to conventional anti-tumor therapies . In view of this, more and more CSC-related antigens have been selected to enhance CAR-T function.
Cluster of differentiation (CD) 133 is a common marker of CSC in a variety of aggressive solid tumors. Vora et al. constructed CD133 (CART133) targeting CAR-T and confirmed that it is in patient-derived glioblastoma ( GBM) The superior efficacy in the xenograft model further indicates that the therapeutic dose of CART133 will not induce toxicity in the normal CD133+ hematopoietic stem cells and progenitor cells of the humanized CD34+ mouse model . In addition, the major histocompatibility complex class I chain-related genes A/B (MICA/B) and UL16 binding protein (ULBP). Cells (GSCs) and natural killer group 2 member D (NKG2D) expressing CAR-T have been shown to have effective tumor clearance on GBM models, and the toxicity associated with treatment is minimal .
Target multiple antigens at the same time
Targeting two or more tumor antigens is an important strategy. First, the sequential administration of different CAR-T cells against a single tumor antigen was studied, and it has shown good results in clinical studies . A preliminary study (ChiCTR, code ChiCTR) in 89 patients with refractory/relapsed (r/r) B-cell malignancies (51 patients with acute lymphoblastic leukemia and 38 patients with non-Hodgkin’s lymphoma) -OPN-16008526) showed that -CD19 and anti-CD22 CAR-T cells (CAR19/22 T cell mixture) showed significant anti-tumor effects (median progression-free survival was 13.6 months and 9.9 months, respectively) and safe Sex (high CRS and neurotoxicity): 22.4% and 1.12% of patients, respectively).
In addition, only one patient experienced a recurrence of antigen loss during the follow-up period, indicating that CAR-T cocktail therapy can effectively reduce the recurrence caused by tumor antigen escape . Similar results were observed in 20 children with r/r B acute lymphoblastic leukemia (B-ALL). Facts have proved that CAR19/22 T cell cocktail therapy is effective and safe, and in the long term, it can improve the durability of remission (ChiCTR, number ChiCTR-OIB-17013670) .
It is also an effective method to construct a multi-target CAR on the same T cell. Dannenfelser et al. screened more than 2.5 million dual antigens and approximately 60 million triple antigens in 33 tumor types and 34 normal tissues, and predicted that the combined antigens in 2- and 3-antigens and NOR logic gates will be better than the current ones. The clinical choice to improve tumor discrimination in CAR-T therapy .
Corresponding pre-clinical studies for 2-target and 3-target CAR-T have been continuously conducted, and satisfactory results have been obtained. Ruella et al. designed CAR-T with dual expression of CD19 and CD123, and further confirmed that it has higher activity in the B-ALL model compared with single expression or combined CAR-T combination .
In addition, a novel CAR-T-expressing antibody mimic receptor (amR) for two different antigens was designed to avoid the potential limitation of protein folding damage caused by linearly assembled single chains and the large size of each scFv. For example, the bispecific epidermal growth factor receptor (EGFR)-human epidermal growth factor receptor 2 (HER2) amR-T has shown significant anti-tumor efficacy in a mouse model . Anti-
CD19/20/22 CAR-T is an example of triple target modification.
Fousek et al. established this CAR structure on a single T cell through tricistronic transgene and found that in addition to the CD19+ B-ALL model, it can also effectively kill the primary CD19 CAR-T therapy or CD19 gene knockout. CD19-blasts of patients who relapsed after the B-ALL model are in vivo or in vitro .
2.1.2 Optimization of intracellular domains
Improve intracellular activation domain
From the first-generation (1G) CAR that only contains the CD3ζ signal domain to the 2G and 3G CAR that combines one or more costimulatory domains, the CAR structure has been continuously improved. Today, 4G and 5G CAR structures with better regulatory capabilities have been designed to solve the persistence problem and enhance the effectiveness of CAR-T .
CD3 composed of six peptides (CD3γε-CD3δε-CD3ζζ) is a leukocyte differentiation antigen widely distributed on the surface of T cells. The αβT cell receptor (TCR)-CD3 complex is a well-known key determinant of T cell antigen recognition and signal transduction . CD3 diversity is considered to be an important regulator of CAR-T efficacy. Among them, the CD3ζ cytoplasmic domain containing three immunoreceptor tyrosine-based activation motifs (ITAM) has been widely used as a fixed module to transmit the main activation signal in the construction of CAR-T .
Recently, Feucht et al. proved that the number and location of CD3ζITAM have a significant impact on CAR-T function. They constructed different single CAR-T mutants containing ITAM, called 1XX, X2X and XX3, and confirmed that 1XX CAR-T cells have balanced memory and effector properties. Corresponding clinical trials using 1XX CAR-T cells are currently underway . In addition, CD3ε, δ and γ chains may also be effective choices for CAR design. Wu et al. first confirmed that the incorporation of CD3ε into the CAR structure can enhance the anti-tumor activity of CAR-T cells. Although the monophosphorylated ITAM of CD3ε can reduce CAR-T cytokine production by recruiting inhibitory C-terminal Src kinase (Csk) kinase, the basic residue-enriched sequence (BRS) of CD3ε can enhance CAR-T through p85 Persistent (regulatory subunit of phosphoinositide 3-kinase) recruitment .
Improve intracellular costimulatory domain
Another important intracellular determinant of CAR-T efficacy is the costimulatory domain. It is found that simple modification of a single costimulatory domain can increase the durability and function of CAR-T. It is reported that the change of a single amino acid residue in the CD28-based mesothelin CAR-T (changes asparagine to phenylalanine) can promote the durable anti-tumor control of CAR-T in pancreatic cancer xenograft models, and Reduce T cell differentiation and failure .
Another example is the CAR-T based on recoverable 4-1BB (also known as tumor necrosis factor receptor superfamily 9, TNFRSF9) designed by Li et al. They first confirmed that blocking CAR ubiquitination by mutating lysine to arginine in the intracellular domain can redirect the intrinsic CAR back to the surface of T cells, thereby improving the durability and resistance of CAR-T therapy. Tumor efficacy .
However, a combination of different costimulatory domains may be an advantageous strategy. The CAR synapse encoding CD28 recruits lymphocyte cell-specific protein tyrosine kinase (LCK) to induce antigen-independent phosphorylation of CAR-CD3ζ and enhance antigen-dependent CAR-T activation, while 4-1BB encodes CAR synapses recruit molecules expressed by thymocytes. Participate in the selection of the (THEMIS)-Src homology domain containing phosphatase 1 (SHP1) phosphatase complex to reduce CAR-CD3ζ phosphorylation. Based on these facts, CAR synapses that have been proven to recruit LCK to enhance the anti-tumor dynamics of 4-1BB CAR-T or SHP1 to down-regulate the cytokine release of CD28 CAR-T help to enhance CAR-T Function . Similarly, it has also been found that combining inducible costimulator (ICOS)  or its ligand (ICOSL)  with 4-1BB can enhance the efficacy of CAR-T.
2.1.3. Optimization of T cell selection
Taking into account the heterogeneity of CAR-T cells, the selection of T cell phenotype and subtype is also a key factor to ensure CAR-T function. Recently, single-cell RNA sequencing (scRNA-seq) has been used to obtain the transcription profile of CAR-T cells . Deng et al. used scRNA-seq to detect the transcriptomic characteristics of autologous CD19 CAR-T products in 24 large B-cell lymphoma (LBCL) patients, and found that the memory-expressing CD8 T cells of patients who achieved complete remission (CR) had a partial response compared with (PR) or progressive disease (PD) patients are three times more .
According to reports, NOTCH and its downstream target forkhead box M1 (FOXM1) are responsible for transforming normal CAR-T cells into stem cell memory-like CAR-T cells (CAR-iT) with excellent anti-tumor efficacy .
Regarding T cell subtypes, natural killer T (NKT) cells and γδT (γδT) cells engineered with CAR have shown significant anti-tumor potential. Vα24 invariant natural killer T cells (iNKT) are a unique subset of T cells, which express the classic invariant TCR-α chain (Vα24-Jα18) and the TCR-β chain with limited Vβ fragments (Vβ11), which can be identified CD1d presents lipid antigens and induces innate and adaptive immune responses .
Heczey et al. constructed a ganglioside GD2 NKT targeting CAR2. The CAR-iNKT targets a highly expressed antigen on neuroblastoma cells and proved its powerful effect in a mouse model of metastatic neuroblastoma. Anti-tumor activity. Unlike T cells, these CAR.GD2 NKT cells do not trigger graft-versus-host disease (GvHD) .
γδT is another T cell subset with innate and adaptive immune characteristics , which can act as professional antigen presenting cells (APC) or lyse tumor cells in an antigen-dependent/independent manner to enhance tumors Control and avoid tumor occurrence. Target extra-tumor toxicity. For example, CARVδ2T has been found to be able to present processed peptides to responder αβT (αβT) cells to enhance tumor control , and it has been shown that anti-GD2 CARVγ9Vδ2T can avoid the non-specificity of non-tumor cells that express GD2 but do not participate in Vγ9Vδ2TCR. Kill . In addition, as described by Rozenbaum et al., CARγδT may also help solve the problem of antigen loss. It was found that unlike ordinary CD19 CAR-T cells, CD19 CARγδT can also target CD19-negative leukemia cells to achieve complete tumor control, especially after zoledronate initiation .
2.2. Combination therapy strategy of CAR-T
In order to cope with the high tumor recurrence rate after CAR-T treatment and the limited efficacy of CAR-T in solid tumors, a combination therapy has been developed to compensate for the inherent defects of CAR-T cells and assist CAR-T to exert better anti-tumor effects influences. Many promising results were reported.
2.2.1. Combination strategies to enhance antigen recognition
Combine CAR-T with epigenetic modulator
In order to solve the tumor escape caused by the down-regulation or loss of tumor antigen expression, the regulation effect of conventional epigenetic modulators on antigen density and distribution has been extensively studied in CAR-T therapy. Anurathapan et al. first confirmed the antigen sensitization effect of epigenetic modulators in CAR-T therapy. They found that the application of Decitabine (a hypomethylating agent that can increase the expression of tumor antigens by demethylating DNA) can upregulate resistant CAP
The mucin 1 (MUC1) expression in AN1 pancreatic cancer cells, while the previous MUC1 CAR-T treatment induced lower antigen expression, making it susceptible to infection for CAR-T treatment . Driouk et al. proved that valproic acid is an effective histone deacetylase (HDAC) inhibitor, which can up-regulate the expression of NKG2DL in acute myeloid leukemia (AML) cells, thereby enhancing the anti-tumor efficacy of NKG2D CAR-T cells[ 48]. It has also been reported that the lysis of the B cell maturation antigen (BCMA) on the surface of multiple myeloma (MM) cells and the subsequent release of soluble BCMA (sBCMA) caused by the multi-subunit γ-secretase complex (GS) reduce CAR-T An important mechanism of efficacy. As a result, in preclinical models of MM, it was found that small molecule GS inhibitors (GSI) can simultaneously increase BCMA and decrease sBCMA levels, and improve the efficacy of CAR-T . A clinical trial is underway to study the feasibility and safety of applying GSI to BCMA CAR-T treatment (NCT03502577).
Combine CAR-T with bispecific antibodies
Bispecific T cell adaptor (BiTE) is a type of bispecific antibody composed of two scFvs. One recruits CAR-T and bystander T cells by recognizing the T cell surface protein CD3ε, and the other targets tumor cells. The second antigen on the surface. The function of BiTE enables it to physically connect tumors and T cells, thereby mediating the killing of different tumor cells by T cells . EGFRvIII-specific CAR-T cells cannot eliminate heterogeneous glioblastoma, but they can cause EGFRvIII-/EGFR+ glioblastoma cell proliferation . In view of this, Choi et al. Developed CART.BiTE (an EGFRvIII-specific CAR-T that secretes EGFRvIII) and confirmed its ability to eradicate heterogeneous tumor cells in a mouse model of glioblastoma .
Combining the sequential or simultaneous delivery of universal CAR-T products with bispecific adapters is another strategy. Each bispecific adaptor is composed of an antibody fragment conjugated to a tag (for example, fluorescein, isothiocyanate, and biotin) for each antigen, which can be bound by the corresponding anti-tag CAR-T cell. Based on this, Lohmueller et al. designed an anti-biotin CAR-T and confirmed the activation induced by tumor cells coated with biotinylated bispecific antibodies (anti-CD19 and CD20), which were further dose-dependent The method mediates the lysis of tumor cells and the production of interferon (IFN)-γ . Similarly, it has also been reported that the fluorescein-linked bispecific adaptor mixture can bridge anti-fluorescence CAR-T cells and tumor cells, and can effectively eradicate heterogeneous solid tumors .
2.2.2. Combination strategies to overcome inhibitory TME
Combine CAR-T with chemotherapy
The main purpose of combining CAR-T with chemotherapy is to achieve better tumor infiltration and regression by reshaping the tumor’s immune microenvironment. For example, it has been found that combining CAR-T with interleukin (IL)-12 plus doxorubicin can increase the deep penetration of injected T cells and enhance the function of CAR-T in a xenograft solid tumor model.
Considering the hypothesis that chemotherapeutics can stimulate tumor cells to produce chemokines that attract T cells, Hu et al. tested different drugs and found that doxorubicin can promote TME penetration into CXCL9/CXCL10 (produced by tumor cells) mediated T Cells penetrate, and IL-12 induces high interferon (IFN)-γ expression and synergistically induces the production of chemokines .
From a clinical point of view, it is also a good choice to pre-use chemotherapeutic drugs before CAR-T infusion. For example, in an expanded and parallel clinical trial of anti-EGFR CAR-T (NCT01869166), pre-drugs of Nabu-paclitaxel and cyclophosphamide showed excellent performance in enhancing the efficacy of CAR-T. This combination has been proven to be the best matrix depletion program, which can deplete tumor matrix by combining secreted acidic and cysteine-rich proteins (SPARC) to promote CAR-T infiltration .
Combine CAR-T with local therapy
Local treatment is often used as a typical method for remodeling TME. Combining different local therapies with CAR-T cells also shows great feasibility. For example, it is reported that in the advanced syngeneic in situ GBM model, the follow-up application of local radiotherapy after intravenous administration of GD2 CAR-T is necessary to obtain a complete therapeutic response. Intravital microscopy imaging further shows that radiotherapy is an important promoter of effective vascular extravasation of CAR-T cells and their expansion in TME, resulting in a more powerful and durable anti-tumor response . Considering the remodeling effect of mild hyperthermia on TME (for example, reducing dense matrix structure and interstitial fluid pressure and increasing blood perfusion and immune cell recruitment), the application of CAR-T and photothermal therapy at the same time also shows excellent CAR-T accumulation and tumor control efficacy in solid tumor models .
Combine CAR-T with Checkpoint blockade
As mentioned earlier, highly expressed immune and metabolic checkpoints are the main components of inhibitory TME. In cancer treatment, different checkpoint blockades have been used to maintain CAR-T function. Regarding immune checkpoints, the combination of CAR-T and immune checkpoint blockade has shown significant resistance both preclinically (for example, in combination with PD-1 and PD-L1) and clinically (for example, in combination with pembrolizumab and pembrolizumab). Tumor role durvalumab) aspect . For the abnormal metabolic microenvironment, the corresponding blockade may also maximize the efficacy of CAR-T . Adenosine is an immunosuppressive metabolite produced at high levels in TME. It can inhibit the immune response by binding to the adenosine 2a receptor (A2aR) expressed on immune cells. According to reports, the combination of CAR-T and A2aR specific small molecule antagonist SCH-58261 helps overcome TME-mediated drug resistance and enhance CAR-T function .
Similarly, Yazdanifar et al. Prove that CAR-T is combined with resistance factor inhibitors (such as indoleamine 2,3-dioxygenase-1 (IDO1), cyclooxygenase 1/2 (COX1/2) and galectin 9 (Gal -9) The combined use can also significantly enhance the efficacy of CAR-T in the treatment of refractory pancreatic duct adenocarcinoma .
2.2.3. Combine CAR-T with vaccines to promote expansion in vivo
Tumor vaccines, including whole cell and molecular vaccines, are also important boosters to improve the effectiveness of CAR-T. Whole-cell vaccines can be divided into tumor cells or dendritic cell (DC) sources. An example is a K562-derived whole-cell vaccine expressing cytomegalovirus (CMV)-pp65 protein, immunostimulatory molecules CD40L and OX40L (CD252), and the inducible safety switch caspase 9 (iC9). Caruana et al. confirmed that this vaccine has the ability to enhance the anti-tumor efficacy of CAR-redirected CMV-specific cytotoxic T cells (CTL) in xenograft tumor models .
For DC vaccines, Wu et al. designed a DC pulsed Eps8-DC vaccine with EGFR pathway substrate 8 derived peptides, and found that Eps8-DC can effectively promote the expansion of CAR-T cells in vitro, while increasing the proportion of central memory T cells And reduce the proportion of effect memory T cells, and enhance CAR-T function in relapsing leukemia models . In addition, molecular vaccines play an important role in promoting CAR-T expansion. The main challenge in the application of molecular vaccines is effective delivery to secondary lymphoid organs.
Irvine et al. considered the phenomenon of “albumin free-riding” (the lipid tail at one end of the molecule can bind to serum albumin, allowing the molecule to enter albumin and then enter the lymph nodes). Irvine et al. synthesized a mechanism that binds to lipophilic albumin. Amphetamine vaccine (AMP) composed of cargo (antigen or adjuvant) connected to the tail, which can smoothly enter the lymph nodes and induce a 30-fold increase in T cell activation, with better tumor control effects and lower systemic toxicity [69 ]. Subsequently, the AMP-CD19 vaccine was developed. In a variety of solid tumor mouse models, it was found that it can induce a 200-fold increase in the number of injected CAR-T cells and enhance anti-tumor efficacy .
2.2.4. Combine CAR-T and oncolytic virus to meet the challenge of solid tumors
Oncolytic viruses (OVs) are considered promising partners for cancer immunotherapy because they affect many key steps in the cancer immune cycle. OVs can enhance the anti-tumor immune response by directly lysing tumor cells, leading to the release of immune activation components (such as soluble antigens and oncoproteins). In addition, these OVs can also be modified to express different therapeutic genes, thereby further increasing the accumulation and function of innate and adaptive immune cells . Although CAR-T therapy has encountered many challenges in solid tumors, oncolytic viruses, especially modified oncolytic viruses, have shown significant synergistic and enhanced effects .
First, antigen delivery can fundamentally solve the problem of antigen heterogeneity. A typical example is the oncolytic vaccinia virus encoding a truncated CD19t protein (OV19t). Tumor cells infected with these OVs produce de novo CD19 on the surface, which enables specific and effective targeting of CD19 CAR-T cells. In turn, CAR-T-mediated tumor lysis leads to the release of OV19t and further promotes the expression of CD19t in tumor cells . Arming OVs with cytokines is another option.
As we all know, many cytokines (for example, IL-2, IL-15 and tumor necrosis factor [TNF]-α) are highly related to the activation and function of immune cells. Even in the immunosuppressive TME of pancreatic ductal adenocarcinoma (PDA), the combination of CAR-T with TNF-α and IL-2 (OAd-TNFα-IL2) expressing oncolytic adenovirus (OAd) also showed significant The anti-tumor effect of OAd-TNFα-IL2 not only increases the tumor infiltration of CAR-T and host T cells, but also reshapes TME with the increase of M1 polarization of macrophages and DC maturation .
In addition, oncolytic viruses modified with immune checkpoint blockers also showed significant synergy with CAR-T in solid tumors . For example, Tanoue et al. The OAd was constructed with a helper-dependent advertisement expressing PD-L1 blocking mini-antibody, and further confirmed its ability to significantly enhance the function of HER2-specific CAR-T in a HER2 prostate cancer xenograft model . As a promising partner for CAR-T therapy, the special “delivery system” provided by OVs provides a new perspective for solving the challenges faced by CAR-T cells in solid tumors.
3. Enhance the clinical safety and accessibility of CAR-T therapy
3.1. Safe maintenance of CAR-T therapy
Adverse reactions (AEs) are the main obstacle to the effective application of CAR-T therapy. In clinical trials, life-threatening toxicity and subsequent death or patient abandonment of treatment can usually be seen . Just like what Penack et al. said. According to review, the unique toxicity characteristics of CAR-T, including CRS, ICANS, cardiotoxicity, pulmonary toxicity, metabolic complications, secondary macrophage activation syndrome (sHLH/MAS) and cytopenia, are also frequently seen in clinical trials. The events that occurred were treated with CD19 CAR-T . In addition, the individualized response of cancer patients to CAR-T toxicity makes it a more serious problem .
CRS is the most common side effect of CAR-T, with characteristic clinical symptoms ranging from mild (such as flu-like fever, fatigue, and headache) to severe (such as life-threatening multiple organ system failure). In addition, the positive feedback loop between the inflammatory cytokines (such as TNF-α, IFN-γ) produced by the tumor antigen binding CAR-T and the subsequent activation of bystander immune cells (such as macrophages and endothelial cells) releases pro-inflammatory Cytokines (such as IL-1 and IL-6) are considered to be the main mechanism of CRS . ICANS is the second most common side effect of CAR-T therapy and CRS or subsequent therapies. It has a wide range of clinical manifestations, such as language/behavioral disorders, peripheral neuropathy, and acute cerebral edema . Although the pathogenesis of ICANS is not fully understood, the activation of endothelial cells induced by high concentrations of pro-inflammatory cytokines and the destruction of the blood-brain barrier (BBB) after the diffusion of CAR-T and cytokines to the central nervous system are considered to be key factors [78 ]. In order to solve these problems, many management strategies have been adopted, such as accurate classification and intervention, to minimize the toxicity of CAR-T . At present, more and more safety switches that can directly prevent or block the toxicity of CAR-T have been developed to help avoid avoiding AEs in clinical cancer patients without weakening the efficacy of CAR-T, and are both preclinical and clinical. Significant results were observed .
3.1.1. Endogenous switch
Inhibitory CAR (iCAR) consists of scFv targeting normal tissue specific antigens and a powerful acute inhibitory signal transduction domain of immunosuppressive receptors. It is a promising endogenous method that can limit CAR-T treatment Unwanted AE. Fedorov et al. constructed iCAR-T based on PD-1 and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), and confirmed its selective and reversible inhibition of T cell activity, which provides a dynamic safety switch, It can prevent potential off-target tumor effects .
Another example is iCAR (iKP CAR) based on killer cell immunoglobulin-like receptor (KIR)/PD-1. The anti-CD19 CAR-T integrated with iKP CAR not only shows obvious cytotoxicity to malignant B cells, but also avoids damage to CD19-positive healthy B cells in vitro and in Burkitt’s lymphoma xenograft models .
SynNotch (SynNotch) system is another typical internal source safety switch. When the SynNotch part is activated by an antigen on tumor cells, the CAR structure that recognizes the second tumor antigen begins to be transcribed , which can specifically activate these dual-receptor AND gate CAR-T cells in the following ways: Antigenic tumor cells . In a mouse model, for fatal bone marrow failure (including ROR1+ stromal cells) caused by ROR1 specific CAR-T, Srivastava et al. designed EpCAM or B7-H3 (antigen expressed on ROR1+ tumor cells, but not in ROR1 + antigen expressed on stromal cells) specific synNotch receptor CAR-T to improve selectivity, so that tumor cells that are sufficiently separated from normal cells can be safely targeted, and tumor occurrence can be prevented. Potential on-target non-tumor effects .
3.1.2. Exogenous switch
Small molecule drugs are the most common choice for exogenous safety switches because they can regulate cell function by regulating protein-protein interactions. Safety switches can be divided into two types: on and off according to their functions. To ensure the safety of CAR-T, these two types have been used at the same time. Regarding the switch, it is reported that the alicyclic protein-based switch is a suitable choice for CAR-T, because human retinol binding protein 4 (hRBP4, a member of the alicyclic protein family) binds to the engineered hRBP4 binding agent under the following conditions: interaction. The small molecule A1120, therefore, successfully modulated the activity of CAR-T cells .
Regarding the disconnect switch, Giordano-Attianese et al. developed STOP-CAR-T by incorporating a chemically destructible heterodimer (CDH) into a synthetic heterodimer CAR. CDH has a high-affinity protein interface and can be destroyed by small molecule drugs, so that CAR-T can be dynamically inactivated under timed administration . Another example is the Fms-related tyrosine kinase 3 (FLT3) specific CAR-T designed with rituximab response switch, which can effectively ensure the recovery of bone marrow after AML remission .
Suicide genes are also a popular choice for safety switches. Inducible Caspase 9 (iCasp9) is a well-known suicide gene that has been incorporated into CAR-T cells to eliminate inappropriately activated CAR-T cells. Gargett et al. Proved the application of AP1903 (a small molecule dimer drug) in triggering rapid apoptosis of CAR-T cells highly expressed by iCasp9 . Another suicide gene is the herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene. It has been found that the administration of the prodrug ganciclovir (GCV) can induce the suicide of transduced CAR-T cells. Murty et al. integrated CAR-T with a mutant version of the HSV1-tk gene (sr39tk). He established a B7H3-specific sr39tk CAR-T and confirmed its complete ablation in an osteosarcoma model after intraperitoneal administration of GCV by bioluminescence and positron emission tomography (PET) imaging .
In addition to these irreversible all-or-nothing switches, more and more adjustable switches have been designed to obtain more flexible and accurate modulation of CAR-T activity. Richman et al. fused CAR into the ligand-induced degradation (LID) domain. Invented a novel CAR-LID structure, and confirmed that in the presence of small molecule ligands in CAR-T-treated tumor models, it has the ability to down-regulate CAR expression as needed. This is due to the mysterious gel exposure in LID induced by small molecule ligands, which can further lead to the degradation of CAR-LID protein and the loss of CAR expression on T cells .
Peptide-specific switchable CAR-T (sCAR-T) is another promising option. The dual-function switch composed of tumor antigen-specific Fab and peptide neo-epitope can only bind to sCAR-T and regulate its activity in a dose-dependent manner. The dynamic regulation of CD19-specific sCAR-T activity in a B-cell leukemia xenograft model is a successful example . The dose of tyrosine kinase inhibitor dasatinib can be titrated and can also achieve dynamic regulation of CAR-T function. It can inhibit the phosphorylation of CD3ζ and T cell receptor-related protein kinase 70 kDa (ZAP70) ζ chain, induce the functional shutdown state in the CAR structure, and fully recover after drug withdrawal. The protective effect was confirmed in the CRS mouse model treated with CAR-T .
Based on the constantly updated design of safety switches, many platforms have been established to make CAR-T therapy more reliable, such as the CAR platform for affinity control (integrating inducible and controllable functions into the CAR structure) , humanized Artificial receptor platform (manipulating CAR-T through bispecific targeting molecules)  and different general CAR platforms [96,97,98].
3.2. Increased access to CAR-T treatment
3.2.1. Acquisition of T cells
Currently, autologous T cells are the main source of CAR-T cells. However, the high cost and complicated acquisition process and the limited availability of T cells from immunosuppressed or immunodeficient cancer patients limit the wide clinical application of autologous CAR-T therapy . One of the solutions is to develop a high-efficiency and low-cost technology to isolate T cells. For example, it is reported that DNA aptamers are effective tools for achieving high-purity T cell isolation.
Kacherovsky et al. have confirmed that DNA aptamers can help isolate pure and traceless CD8-T cells with low cost and high yield. The proliferation, phenotype and efficacy of CAR-T cells derived from these cells in a mouse model of B-cell lymphoma are comparable to CAR-T cells isolated from antibodies . Exploring allogeneic universal CAR-T cells is another possible strategy .
Currently, peripheral blood mononuclear cells (PBMC), umbilical cord blood (UCB) and induced pluripotent stem cells (iPSC) from healthy donors are the main allogeneic sources . Among them, the ready-made CAR-T product FT819 derived from iPSC has become a promising choice . It is a new type of CD19 CAR-T designed by Fate Therapeutics, which encodes a CAR with a single activation motif (1XX) based on immunoreceptor tyrosine to balance the effectors and memory programs of CAR-T . It was inserted into the T cell receptor α constant (TRAC) site to delay the differentiation and failure of CAR-T , and the biallelic destruction of TRAC was used to edit the TCR expression to reduce the risk of GvHD  . According to a report at the 2019 ASH Annual Meeting, in the pre-clinical model, FT819 CAR-T has better anti-tumor efficacy and survival rate than the main CD19 CAR-T, which is the first phase I afterwards. Clinical trials provided support .
3.2.2. Optimization of CAR-T manufacturing process
Currently, viral vector-mediated semi-random DNA integration is the main method to express CAR structure on T cells . However, the manufacturing process of viral vectors is complicated and expensive, cumbersome production, quality control steps of viral vector-transfected CAR-T and long-term monitoring of unexpected side effects also limit its wide clinical application. In view of this, a non-virus delivery system with low cost and high security has become a better choice .
PiggyBac (PB) and Sleeping Beauty (SB) transposon systems, CRISPR system (clustered regularly spaced short palindromic repeats)-mediated site-directed integration, and mRNA vectors are popular non-viral methods that have been proven in CAR -T is effective in transfection; the latest improvements of each product further optimize their efficacy in CAR-T manufacturing.
For PB, Bishop et al. It is reported that dbDNA is the smallest DNA vector lacking undesirable plasmid characteristics, and may be a viable alternative to PB-mediated CAR-T production, with lower clinical application risks and costs .
At the same time, Querques et al. constructed a highly soluble SB transposase (hsSB) based on the crystal structure of the overactive SB100X variant to overcome uncontrolled transposase activity and further confirmed that hsSB can be used in the absence of transfection reagents. Under the circumstances, CAR-T cells with excellent anti-tumor efficacy are produced .
CRISPR-Cas9-mediated CAR-T construction with endogenous gene disruption is another optimization. For example, CD19 CAR-T that integrates CD into the PD1 locus not only shows significant anti-tumor efficacy in preclinical models, but also in patients with r/r aggressive B-cell non-Hodgkin’s lymphoma (B-NHL) It also shows safety and effectiveness. Researcher initiated clinical trials (IIT) . In addition, compared with Cas9-based methods, the AAV-Cpf1 system (combining adeno-associated virus with CRISPR-Cpf1) is reported to be more effective in generating double knockouts in CAR-T cells.
An example is CD22-specific AAV-Cpf1 KIKO CAR-T, a CAR-T product with homology-guided repair knockout and immune checkpoint knockout. It shows a lower exhaustion index, but it is comparable to Cas9 CAR. -T has comparable tumor control effects . Targeted mRNA nanocarriers are absorptive due to their simplicity. By simply mixing them with lymphocytes, these mRNA nanocarriers can effectively mediate the genome editing of CAR-T cells. During this period, it can also achieve the destruction of TCR or the transformation of the central memory phenotype .
4. Conclusion and outlook
CAR-T therapy, as a promising option in tumor immunotherapy, has shown significant potential and clinical application prospects. From the modification and production of CAR-T to the combination of CAR-T, the latest breakthrough has undoubtedly eliminated the dark cloud hindering CAR-T and ignited the light of hope.
If these breakthroughs can be supported by subsequent large-scale clinical studies, then CAR-T may bring a new blueprint for cancer treatment. It is believed that with the continuous deepening and extensive exploration of CAR-T, this therapy will ultimately benefit clinical cancer patients to a large extent.
(source:internet, reference only)