Application of GADD34 inhibitor in preparation of medicine for enhancing CAR-T cell tumor treatment

Abstract

The invention relates to the field of biological medicine, and discloses application of a GADD34 inhibitor in preparation of a medicine for enhancing CAR-T cell tumor treatment. The invention provides an innovative strategy for inhibiting CAR-T cell depletion through targeted regulation and control of an endoplasmic reticulum stress pathway to solve the problems that in an existing CAR-T therapy, the anti-tumor activity is reduced, and the solid tumor treatment effect is insufficient due to T cell depletion. Researches find that an IRE1-XBP1 pathway is used as a core signal axis of endoplasmic reticulum stress, and excessive activation of the IRE1-XBP1 pathway can drive CAR-T cell depletion related phenotypes (such as PD-1 / LAG-3 up regulation and cytokine secretion reduction). Screening experiments show that inhibiting the upstream regulatory factor GADD34 of the IRE1 not only can effectively reduce the IRE1-XBP1 signal intensity, but also can cooperatively relieve the endoplasmic reticulum stress pressure by regulating protein translation recovery, so that the CAR-T cell steady state is more comprehensively maintained. Therefore, the GADD34 inhibitor can provide a new way for enhancing CAR-T cell tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of GADD34 inhibitors in the preparation of drugs for enhancing CAR-T cell tumor therapy. Background Art

[0002] CAR-T (chimeric antigen receptor T-cell) therapy is an immunotherapy technology that genetically modifies a patient's T cells to specifically recognize and kill tumor cells. Its core approach involves introducing a man-made chimeric antigen receptor (CAR) into T cells. The CAR molecule consists of an antigen recognition domain (single-chain antibody variable region, ScFv), a transmembrane domain, a signal transduction region (CD3ζ), and a costimulatory domain (such as CD28 or 4-1BB). This allows T cells to bypass major histocompatibility complex (MHC) restriction and directly target tumor-associated antigens. This therapy has demonstrated significant efficacy in hematologic malignancies such as acute lymphoblastic leukemia and lymphoma, and several commercial products have been approved. However, CAR-T therapy still faces multiple challenges in cancer treatment, notably the susceptibility of CAR-T cells to exhaustion in vivo, which reduces their anti-tumor activity and limits their therapeutic efficacy. This has become a major bottleneck in the development of CAR-T therapy technology.

[0003] CAR-T cell exhaustion refers to the gradual functional decline of CAR-T cells under continuous antigen stimulation or immunosuppressive microenvironment conditions, manifested by decreased proliferation, decreased cytokine secretion, and increased expression of immunosuppressive receptors (such as PD-1 and LAG-3). Existing methods to address CAR-T cell exhaustion have developed a variety of strategies to reduce CAR-T cell exhaustion, mainly by optimizing CAR design, improving the metabolic environment, regulating transcription factors and epigenetic status, and optimizing CAR-T cell subtypes to relieve tumor microenvironmental inhibition. However, these strategies generally have the following drawbacks:

[0004] (1) It is difficult to balance efficacy and safety: reducing the activity of CAR-T cells can reduce the cytotoxicity caused by activation (such as cytokine release syndrome (CRS)), but may lead to a decrease in their anti-tumor efficacy; on the contrary, enhancing the function of CAR-T cells can improve tumor killing ability, but may be accompanied by a higher risk of toxicity.

[0005] (2) Insufficient dynamic regulation: Existing methods are difficult to flexibly adjust according to real-time changes in the tumor microenvironment (TME), resulting in the inability to fundamentally improve CAR-T cell exhaustion.

[0006] (3) Poor applicability to solid tumors: Existing technologies lack targeted intervention in the complex inhibitory mechanisms of TME, resulting in poor effectiveness of CAR-T cells in the treatment of solid tumors.

[0007] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0008] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide the use of GADD34 inhibitors in the preparation of drugs for enhancing CAR-T cell tumor therapy, aiming to solve the problems of decreased anti-tumor activity and insufficient efficacy in solid tumors caused by CAR-T cell exhaustion in existing CAR-T therapies.

[0009] The technical solutions of the present invention are as follows:

[0010] In a first aspect, the present invention provides the use of a GADD34 inhibitor in the preparation of a drug for enhancing CAR-T cell tumor therapy.

[0011] In a preferred technical solution, the drug inhibits the exhaustion of CAR-T cells by inhibiting the IRE1-XBP1 pathway, thereby enhancing the killing effect of CAR-T cells on tumors.

[0012] In a preferred technical solution, the GADD34 inhibitor is selected from one or more of small molecule compounds that inhibit GADD34, nucleic acid drugs that target GADD34, and gene editing tools that knock out or knock down the GADD34 encoding gene.

[0013] In a preferred technical solution, the GADD34 inhibitor is Sephin1.

[0014] In a preferred technical solution, the tumor is a solid tumor.

[0015] In a further preferred technical solution, the solid tumor is pancreatic cancer.

[0016] In a preferred technical solution, the CAR-T cells are B7-H3 CAR-T cells.

[0017] Secondly, an in vitro production method of CAR-T cells with improved tumor treatment effect is provided, in which CAR-T cells are treated with a GADD34 inhibitor.

[0018] In a third aspect, a composition for tumor treatment is provided, comprising: a GADD34 inhibitor and CAR-T cells.

[0019] The technical solution of the present invention has the following beneficial effects:

[0020] (1) Breaking through the exhaustion bottleneck: The present invention blocks the core driving factor of CAR-T cell exhaustion from a molecular mechanism by targeting the IRE1-XBP1 axis, significantly prolonging its survival time and functional persistence in vivo.

[0021] (2) Safety: While maintaining the inherent tumor killing activity of CAR-T cells, the present invention avoids the potential risks of directly inhibiting IRE1 and has better safety.

[0022] (3) Broad-spectrum applicability: Considering the conservative regulatory mechanism of CAR-T exhaustion, the present invention has potential therapeutic effects on both solid tumors and non-solid tumors (such as blood tumors). Combined with the drugability advantage of GADD34 inhibitor (Sephin1), it may be applicable to various types of CAR-T and can promote broad-spectrum tumor killing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the results of detecting the expression level of the PPP1R15A gene in Example 2.

[0024] Figure 2 This is a graph showing the results of detecting the splicing of XBP1 mRNA in the PPP1R15A gene knockdown cell line and the negative control cell line under endoplasmic reticulum stress in Example 3.

[0025] Figure 3 This is a graph showing the results of detecting the phosphorylation level of IRE1 and the expression level of XBP1s in the PPP1R15A gene knockdown cell line and the negative control cell line under endoplasmic reticulum stress in Example 3.

[0026] Figure 4 This is a diagram showing the results of testing the effect of Sephin1 on the splicing of XBP1 mRNA in Example 4.

[0027] Figure 5 This figure shows the results of testing the effects of Sephin1 on IRE1 phosphorylation and XBP1s expression in Example 4.

[0028] Figure 6 This is a graph showing the effect of Sephin1 on XBP1 mRNA splicing in B7-H3 CAR-T cells during co-culture of B7-H3 CAR-T and BXPC3 in Example 5.

[0029] Figure 7 This figure shows the results of detecting the effect of Sephin1 on the secretion of INF-γ and TNF-α in B7-H3 CAR-T cells during co-culture of B7-H3 CAR-T and BXPC3 in Example 6.

[0030] Figure 8 This figure shows the results of testing the effect of Sephin1 on the depletion of CAR-T cells and the killing of BXPC3 cells during the co-culture of B7-H3 CAR-T and BXPC3 in Example 6. DETAILED DESCRIPTION

[0031] The present invention provides the use of a GADD34 inhibitor in the preparation of a drug for enhancing CAR-T cell tumor therapy. To make the objectives, technical solutions, and effects of the present invention clearer and more specific, the present invention is described in further detail below.

[0032] CAR-T cell exhaustion seriously affects its persistence and anti-tumor activity. To alleviate CAR-T cell exhaustion, existing research focuses on the following aspects:

[0033] (1) CAR design optimization

[0034] a. Using the 4-1BB co-stimulatory domain: The CD28 co-stimulatory signal is stronger than the 4-1BB co-stimulatory domain signal. Continuous antigen stimulation can easily lead to overactivation of CAR-T cells, leading to exhaustion. However, in the treatment of some solid tumors, the persistence of 4-1BB CAR-T cells is still poor, and the weak 4-1BB activation signal may reduce the initial killing efficiency of CAR-T cells.

[0035] b. Low-affinity CAR design: Researchers have designed low-affinity CARs using low-affinity scFvs to reduce overactivation of CAR-T cells and mitigate the risk of exhaustion. However, low-affinity CAR-T cells may have reduced proliferation and killing abilities, making it difficult to control tumor progression.

[0036] c. Inducible systems (e.g., Tet-on / off, SynNotch): Conditional activation of CARs is achieved through external chemical inducers or logic gating designs (e.g., AND gates). Major disadvantages include increased immunogenicity risk, increased operational complexity, and the inability to completely address TME-induced exhaustion.

[0037] d. Promoter optimization: Use weak promoters (such as EF1α and MND) to reduce the sustained expression intensity of CAR to reduce exhaustion induced by sustained signals. However, such methods may weaken anti-tumor activity and are difficult to dynamically regulate.

[0038] (2) Metabolic regulation

[0039] a. Enhancing mitochondrial function: For example, overexpressing PGC-1α and regulating AMPK / mTOR signaling can increase ATP production and oxidative phosphorylation in CAR-T cells, enhance their metabolic tolerance in the TME, and delay exhaustion. However, due to the complexity of intracellular metabolic pathways, compensatory changes in other metabolic pathways may be triggered, affecting cellular homeostasis.

[0040] b. Enhancing CAR-T cell adaptability to low-glucose or high-lactate environments: For example, regulating the expression of glucose transporter (GLUT1) and lactate transporter (MCT1) can enhance CAR-T cell survival in environments with intense nutrient competition or acidic conditions. However, intracellular metabolic regulation requires a delicate balance; excessive regulation can lead to metabolic imbalance or disrupted energy supply.

[0041] (3) Transcription factor regulation

[0042] a. Gene editing to knock out exhaustion-related transcription factors: For example, using the CRISPR / Ca9 system to knock out exhaustion-related transcription factors such as TOX and NR4A directly blocks the exhaustion signaling pathway, thereby improving the anti-tumor activity and persistence of CAR-T cells. The disadvantage is that these transcription factors may play an active role in maintaining CAR-T cell homeostasis, and complete knockout may cause cellular homeostasis imbalance.

[0043] b. Enhancing anti-depletion transcription factors: For example, overexpressing c-Jun can antagonize the expression of depletion-related genes, enhancing cell viability and proliferation. However, long-term overexpression may interfere with the normal differentiation process of cells and lead to cell dysfunction.

[0044] (4) Epigenetic regulation

[0045] a. DNA methylation: DNA demethylation agents can reduce the methylation levels of some exhaustion-related genes and restore CAR-T cell function. However, this approach involves nonspecific regulation, and demethylation agents are likely to affect global gene expression.

[0046] b. Histone modification regulation: Using histone deacetylase inhibitors to increase chromatin access in memory T cell-related genes and restore CAR-T cell function. This approach also has the problem of nonspecific regulation, making it more difficult to regulate.

[0047] (5) CAR-T cell subtype optimization

[0048] a. Using Tscm or Tcm to create CAR-T cells: Tscm and Tcm cell subtypes have high proliferation and differentiation potential, and CAR-T cells created using them as starting cells are more resistant to exhaustion. However, this method is more difficult to isolate and expand, and is more expensive.

[0049] b. Cytokine secretion modification: Genetically engineering CAR-T cells to secrete IL-7 and IL-15 helps maintain CAR-T cell memory and cell survival. This approach may lead to an imbalance in cellular self-regulation, and excessive cytokine secretion may cause local immune disorders.

[0050] (6) Combination therapy

[0051] Combination strategies: For example, CAR-T cells can be combined with immune checkpoint inhibitors and metabolic regulators. This approach integrates multiple mechanisms to synergistically enhance CAR-T cell resistance to exhaustion and restore anti-tumor activity. However, the downside of this combination strategy is that it can increase the complexity of clinical trials, requiring thorough verification of treatment dosing and safety. Furthermore, interactions between the combination regimens may lead to unforeseen adverse reactions.

[0052] (8) Limitations in clinical practice

[0053] Existing technologies mostly focus on blood tumors and have limited effects on improving the problem of CAR-T cell exhaustion in solid tumors.

[0054] Long-term follow-up data show that more than 50% of patients who relapse after CAR-T treatment are associated with CAR-T cell exhaustion, highlighting the shortcomings of existing regulatory strategies.

[0055] In summary, existing methods have developed a variety of strategies to reduce CAR-T cell exhaustion, mainly from the aspects of optimizing CAR design, improving metabolic environment, regulating transcription factors and epigenetic status, optimizing CAR-T cell subtypes to relieve tumor microenvironment inhibition, etc. However, they generally have shortcomings such as difficulty in balancing efficacy and safety, insufficient dynamic regulation, and entity.

[0056] Based on this, the present invention proposes an innovative strategy to inhibit CAR-T exhaustion by targeted regulation of the endoplasmic reticulum stress pathway (ER stress). The IRE1-XBP1 pathway is a signaling pathway in cells. Under endoplasmic reticulum stress conditions, inositol-requiring enzyme 1 (IRE1) is activated and produces active XBP1 protein (XBP1s) by splicing the mRNA of X-box binding protein 1 (XBP1), thereby regulating gene expression in cells to respond to endoplasmic reticulum stress. Through research, the present invention found that the IRE1-XBP1 pathway is the core signaling axis of ER stress, and its excessive activation will drive CAR-T cell exhaustion-related phenotypes (such as PD-1 / LAG-3 upregulation and reduced cytokine secretion). Compared with directly inhibiting IRE1, the core innovation of the present invention is to indirectly block the activity of the IRE1-XBP1 pathway by inhibiting its upstream regulatory factor GADD34 (growth arrest and DNA damage-inducible protein 34). Screening experiments showed that inhibition of GADD34 not only effectively reduced the IRE1-XBP1 signal intensity, but also synergistically alleviated endoplasmic reticulum stress by regulating protein translation recovery (PPP1R15A / B complex function), thereby more comprehensively maintaining CAR-T cell homeostasis.

[0057] In view of this, the present invention proposes for the first time the application of GADD34 inhibitors in CAR-T therapy.

[0058] The present invention provides an embodiment of the present invention that provides the use of a GADD34 inhibitor in the preparation of a drug for enhancing CAR-T cell tumor therapy.

[0059] In one embodiment, the drug inhibits CAR-T cell exhaustion by inhibiting the IRE1-XBP1 pathway, thereby enhancing the anti-tumor efficacy of CAR-T cells. Specifically, inhibition of the IRE1-XBP1 pathway is demonstrated by inhibition of XBP1 mRNA splicing, IRE1 phosphorylation, and XBP1s expression.

[0060] In one embodiment, the GADD34 inhibitor is selected from one or more of a small molecule compound that inhibits GADD34, a nucleic acid drug that targets GADD34, and a gene editing tool that knocks out or knocks down the gene encoding GADD34; the GADD34 inhibitor inhibits the activity of GADD34 or reduces the expression of GADD34. Specifically, the small molecule compound that inhibits GADD34 can be Sephin1, and the structure of Sephin1 is: However, it is not limited thereto and may also be other structures that specifically inhibit GADD34. The nucleic acid drug targeting GADD34 may be a small interfering RNA (siRNA) or antisense oligonucleotide targeting GADD34. The gene editing tool for knocking out or knocking down the GADD34 encoding gene may be CRISPR-Cas9 and an sgRNA targeting the GADD34 encoding gene; the primer pair sequence of the sgRNA targeting the GADD34 encoding gene may be one or more of SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

[0061] In one embodiment, the tumor is a solid tumor; preferably, the solid tumor is pancreatic cancer; more preferably, the pancreatic cancer is pancreatic adenocarcinoma.

[0062] In one embodiment, the CAR-T cell is a B7-H3 CAR-T cell. B7-H3 CAR-T cells refer to CAR-T cells that target B7 homolog 3 protein (B7-H3).

[0063] Specifically, the present invention experimentally verified that a GADD34 inhibitor (Sephin 1) can promote the killing effect of B7-H3 CAR-T cells on human orthotopic pancreatic adenocarcinoma cells (BXPC3). Therefore, a GADD34 inhibitor (Sephin 1) can be used to prepare a drug that enhances the therapeutic effect of B7-H3 CAR-T cells on pancreatic adenocarcinoma.

[0064] An embodiment of the present invention also provides an in vitro method for producing CAR-T cells with improved tumor treatment effects, wherein the CAR-T cells are treated with a GADD34 inhibitor.

[0065] In one embodiment, the GADD34 inhibitor is Sephin 1; the concentration of Sephin 1 is 0.5 to 50 μM.

[0066] In a third aspect, a composition for tumor treatment is provided, comprising: a GADD34 inhibitor and CAR-T cells.

[0067] In one embodiment, the composition further comprises an immune checkpoint inhibitor; the immune checkpoint inhibitor can be a PD-1 antibody.

[0068] In one embodiment, the composition further comprises a pharmaceutically acceptable excipient.

[0069] The present invention will be further described below with reference to specific examples.

[0070] Example 1

[0071] The gene encoding GADD34 is PPP1R15A. To endogenously inhibit the expression of GADD34, this example constructed a PPP1R15A gene knockdown cell line, as follows:

[0072] Based on the PPP1R15A gene sequence, three sgRNAs for knocking down the PPP1R15A gene (respectively designated as sgRNA1, sgRNA2, and sgRNA3) and one sgRNA for negative control (respectively designated as CTRL sgRNA) were designed. The primer sequences for sgRNA1, sgRNA2, sgRNA3, and CTRL sgRNA are as follows:

[0073] CTRL sgRNA forward primer (SEQ ID NO.1):

[0074] 5'-TTGGACCAGGATGGGCACCACCCGTTTAAGAGC-3'.

[0075] CTRL sgRNA reverse primer (SEQ ID NO. 2):

[0076] 5'-TTAGCTCTTAAACGGGTGGTGCCCATCCTGGTCCAACAAG-3'.

[0077] sgRNA1 forward primer (SEQ ID NO.3):

[0078] 5'-TTGGCGTGGCGACAAGCGGGCGCGTTTAAGAGC-3'.

[0079] sgRNA1 reverse primer (SEQ ID NO.4):

[0080] 5'-TTAGCTCTTAAACGCGCCCGCTTGTCGCCACGCCAACAAG-3'.

[0081] sgRNA2 forward primer (SEQ ID NO.5):

[0082] 5'-TTGGGTTCCGACCCCTTACTCACGTTTAAGAGC-3'.

[0083] sgRNA2 reverse primer (SEQ ID NO.6):

[0084] 5'-TTAGCTCTTAAACGTGAGTAAGGGGTCGGAACCCAACAAG-3'.

[0085] sgRNA3 forward primer (SEQ ID NO.7):

[0086] 5'-TTGGCAGCCGGTGAGTAAGGGGTGTTTAAGAGC-3'.

[0087] sgRNA3 reverse primer (SEQ ID NO.8):

[0088] 5'-TTAGCTCTTAAACACCCCTTACTCACCGGCTGCCAACAAG-3'.

[0089] Dissolve the CTRL sgRNA forward primer and CTRL sgRNA reverse primer in ddH2O to a final concentration of 100 μM. Add 1 μl of each to 48 μl of 1× annealing buffer and anneal as follows: 95°C for 5 minutes, followed by a cooling rate of 0.8°C / s to 25°C. Ligate 1 μl of the annealed product into the CRISPRi / a V2 vector (Addgene, Plasmid #84832, restriction sites BstXⅠ and BlpⅠ) to generate a plasmid carrying the CTRL sgRNA.

[0090] The sgRNA1 forward primer and sgRNA1 reverse primer were dissolved in ddH2O to a final concentration of 100 μM, and the plasmid carrying PPP1R15AsgRNA1 was obtained by referring to the method for synthesizing a plasmid carrying CTRL sgRNA.

[0091] The sgRNA2 forward primer and sgRNA2 reverse primer were dissolved in ddH2O to a final concentration of 100 μM, and the plasmid carrying PPP1R15AsgRNA2 was obtained by referring to the method for synthesizing a plasmid carrying CTRL sgRNA.

[0092] The sgRNA3 forward primer and sgRNA3 reverse primer were dissolved in ddH2O to a final concentration of 100 μM, and the plasmid carrying PPP1R15AsgRNA3 was obtained by referring to the method for synthesizing a plasmid carrying CTRL sgRNA.

[0093] The plasmid carrying PPP1R15AsgRNA1, the plasmid carrying PPP1R15AsgRNA2, the plasmid carrying PPP1R15AsgRNA3 and the plasmid carrying CTRL sgRNA were respectively transferred into the HEK293T-CRISPRi cell line (abbreviated as 293Ci) by lentivirus. After 48 hours, puromycin was added to screen and obtain three PPP1R15A gene knockdown cell lines (respectively marked as 293Ci-sgPPP1R15A#1, 293Ci-sgPPP1R15A#2, 293Ci-sgPPP1R15A#3) and a negative control cell line (respectively marked as 293Ci-sgCTRL).

[0094] Example 2

[0095] In this example, RT-qPCR was used to detect the expression of the PPP1R15A gene in the three PPP1R15A gene knockdown cell lines and the negative control cell line in Example 1 to verify whether the PPP1R15A gene was successfully knocked down, as follows:

[0096] 293Ci-sgPPP1R15A#1, 293Ci-sgPPP1R15A#2, 293Ci-sgPPP1R15A#3 and 293Ci-sgCTRL cell lines were collected and used The RNA samples were extracted using the Cell RNA Kit (Yeasen, Cat. No. 19231ES50). The specific steps are detailed in the instructions. The concentration and purity of the extracted RNA were tested. 1 μg of RNA was taken for reverse transcription. cDNA was obtained by reverse transcription using the One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen, Cat. No. AT311-03). Primers were designed based on the PPP1R15A gene sequence and synthesized by Shanghai Bioengineering. The designed primer sequences are as follows:

[0097] PPP1R15A forward primer (SEQ ID NO.9):

[0098] 5'-ATGATGGCATGTATGGTGAGC-3'.

[0099] PPP1R15A reverse primer (SEQ ID NO.10):

[0100] 5'-AACCTTGCAGTGTCCTTATCAG-3'.

[0101] A 20 μl reaction system was prepared according to the instructions of AceQ qPCR SYBR Green Master Mix (Vazyme, product number CQ111-02). All operations were performed on ice. Three parallel tubes were set up for each sample, and all amplification reactions were repeated more than three times to ensure the reproducibility of the results. Amplification program: 95°C 60s, (95°C 15s, 60°C 60s) × 45 cycles. SYBRGreen was used as a fluorescent marker, and the qPCR reaction was performed on a fluorescent real-time quantitative PCR instrument. The △△CT method was used for relative quantification of genes. All experiments were repeated three times, and the data were expressed as mean ± variance. GraphPad Prism was used for statistical plotting. The results are shown in the figure. Figure 1 As shown. Figure 1 It can be seen that compared with the 293Ci-sgCTRL cell line, the expression of PPP1R15A gene was downregulated in 293Ci-sgPPP1R15A#1, 293Ci-sgPPP1R15A#2, and 293Ci-sgPPP1R15A#3 cell lines.

[0102] Example 3

[0103] In this example, under the condition of endoplasmic reticulum stress, the splicing of XBP1 mRNA in the three PPP1R15A gene knockdown cell lines and the negative control cell line of Example 1 was detected, as follows:

[0104] 293Ci-sgPPP1R15A#1, 293Ci-sgPPP1R15A#2, and 293Ci-sgCTRL cell lines were treated with 500 nM thapsigargin (Tg) and 2.5 μM tunicamycin (Tm), respectively, for 24 h to induce endoplasmic reticulum stress. RNA was extracted, and RT-PCR was performed. Agarose gel electrophoresis was performed, and the grayscale values of the upper and lower bands were analyzed using ImageJ. The ratio was used to reflect the degree of splicing of XBP1 mRNA. The results are shown in Figure 2. Figure 2 As shown. Figure 2 It can be seen that under endoplasmic reticulum stress, the splicing of XBP1 mRNA in the PPP1R15A gene knockdown cell line was inhibited compared with the negative control cell line.

[0105] 293Ci-sgPPP1R15A#1, 293Ci-sgPPP1R15A#2, 293Ci-sgPPP1R15A#3, and 293Ci-sgCTRL cell lines were treated with 500 nM Tg for 6 h and 24 h, respectively. Figure 3 As shown. Figure 3 It can be seen that after 24 h of Tg treatment, the phosphorylation level of IRE1 and the expression level of XBP1s in the PPP1R15A gene knockdown cell line were inhibited compared with the negative control cell line.

[0106] During RT-PCR, the primer sequences for XBP1 mRNA were designed as follows:

[0107] XBP1 forward primer (SEQ ID NO.11):

[0108] 5'-CCTTGTAGTTGAGAACCAGGAG-3'.

[0109] XBP1 reverse primer (SEQ ID NO.12):

[0110] 5'-GGTCCAAGTTGTCCAGAATGC-3'.

[0111] according to Prepare a 20 µl reaction using Master Mix (With Dye) (yeasen, Cat. No. 10102ES03) according to the instructions. Amplification protocol: 98°C for 3 min, followed by 25 cycles of (98°C for 10 s, 62°C for 20 s, 72°C for 15 s).

[0112] Example 4

[0113] This example examines the effects of GADD34 inhibitors (such as Sephin1) on XBP1 mRNA splicing, IRE1 phosphorylation, and XBP1s expression, as follows:

[0114] HEK293T cells were treated with Sephin1 (MCE, Cat. No. HY-111022) at different concentrations (0, 0.8 μM, 3.75 μM) for 24 h in the presence or absence of ER stress. The cells were then harvested, RNA was extracted, and RT-PCR and agarose gel electrophoresis were performed. The results are shown in Figure 2. Figure 4 As shown. Figure 4 It can be seen that under the condition of endoplasmic reticulum stress, after the addition of Sephin1, the splicing of XBP1 mRNA is inhibited, and within a certain range of Sephin1 drug concentration, the degree of inhibition of XBP1 mRNA splicing is positively correlated with the concentration of Sephin1.

[0115] HEK293T cells were treated with Sephin1 at different concentrations (0, 1 μM, 5 μM) for 24 h with or without ER stress. The cells were then harvested, proteins were extracted, and Western blotting was performed to observe changes in the expression levels of proteins associated with ER stress. The results are as follows: Figure 5 As shown. Figure 5 It can be seen that under the condition of endoplasmic reticulum stress, after the addition of Sephin1, the phosphorylation level of IRE1 and the expression level of XBP1s were inhibited, and the inhibitory effect was positively correlated with the concentration of Sephin1; however, no significant changes in the phosphorylation level of eIF2α were observed.

[0116] Example 5

[0117] This example examines the effect of a GADD34 inhibitor (such as Sephin1) on XBP1 mRNA splicing in B7-H3 CAR-T cells during co-culture of B7-H3 CAR-T and BXPC3, as follows:

[0118] First, peripheral blood mononuclear cells (PBMC, STEMCELL Technologies) were activated for 2 days on NTC 24-well plates pre-coated with CD3 (1 μg / ml, Biolegend) and CD28 (1 μg / ml, BD Biosciences). Then, the activated T cells were transfected with B7-H3 CAR retrovirus on NTC 24-well plates pre-coated with reverse transcriptase (Retronectin). After 3 days, the transfected CAR-T cells were removed and cultured with IL-7 (10 ng / ml, PeproTech) and IL-15 (5 ng / ml, PeproTech). After 7 to 9 days of culture, B7-H3 CAR-T cells were used for co-culture with tumor cells (BXPC3).

[0119] Tumor cells (BXPC3) were plated at 0.2×10 6 The cells were seeded in a 12-well plate at a ratio of 1:1 and B7-H3 CAR-T cells were added in triplicate at an effector-target ratio of 1:1 in the presence of DMSO or 30 μM Sephin1. After 3 days of co-culture, the cells in the 3 parallel wells were combined, washed and resuspended in 3 mL of fresh culture medium. 1 mL of cells was used for cell counting and intracellular cytokine staining, and 2 mL of cells were added to the tumor cells inoculated the day before for subsequent rounds of co-culture. B7-H3CAR-T cells in co-culture on days 0, 3, 6, and 9 were taken, RNA was extracted, RT-PCR was performed, and agarose gel electrophoresis was performed. The data were expressed as mean ± variance, and statistical plots were performed using GraphPad Prism. The results are shown in Figure 5. Figure 6 As shown. Figure 6It can be seen that the degree of XBP1 splicing in the Sephin1-treated group on day 9 was significantly lower than that in the DMSO-treated group. Sephin1 can inhibit the splicing of XBP1 mRNA in B7-H3 CAR-T cells during co-culture of B7-H3 CAR-T and BXPC3.

[0120] Example 6

[0121] This example examines the effects of GADD34 inhibitors (such as Sephin 1) on CAR-T cell depletion, INF-γ and TNF-α secretion, and tumor killing, as follows:

[0122] The B7-H3 CAR-T cells in the co-culture of Example 5 were collected, washed and resuspended, and then treated with 50 μg / mL phorbol ester (PMA, Beyotime, Catalog No.: S1819), 1 mM ionomycin (Ionomycin, Beyotime, Catalog No.: S1672) and 5 μg / mL brefeldin A (Biolegend, Catalog No.: 420601) for 4 hours to induce cytokine production. The B7-H3 CAR-T cells were then stained for CD8 and live / dead cells (Zombie Aqua, Biolegend, Catalog No.: 423102), and then stained with BD Cytofix / Cytoperm TM Fixation / permeabilization kit (BD Bioscience, catalog number: 554714) was used for fixation and permeabilization overnight at 4°C, and then intracellular IFN-γ and TNF-α were stained. Flow cytometry antibodies were purchased from Biolegend, including: AF700-conjugated anti-CD8 (clone SK-1), AF647-conjugated anti-IFN-γ (clone 4S.B3), and PE-conjugated anti-TNF-α (clone MAB11). Flow cytometry data were collected on a NovoCyteQuanteon flow cytometer (Agilent) using NovoExpress software and analyzed by FlowJo software (v10.7, Tree Star). The results are shown in Figure 2. Figure 7 and Figure 8 As shown. Figure 7 It can be seen that on the 9th day (statistics show only D9), the secretion levels of interferon gamma (INF-γ) and tumor necrosis factor alpha (TNF-α) in the Sephin1-treated group were significantly higher than those in the DMSO-treated group. Figure 8It can be seen that Sephin1 treatment inhibited the exhaustion process of CAR-T cells and had better proliferation and tumor killing activity than the DMSO-treated group. This was demonstrated by the increase in the number of CAR-T cells and the decrease in residual tumor cells in the Sephin1-treated group after each round of co-culture. Figure 7 and Figure 8 It can be seen that Sephin1 can inhibit the exhaustion of CAR-T cells and promote the secretion of INF-γ and TNF-α and tumor killing effect.

[0123] In summary, the present invention established a stable PPP1R15A gene knockdown cell line in the HEK293-CRISPRi cell line through Examples 1 and 2; Example 3 demonstrated that endogenous inhibition of GADD34 expression can inhibit XBP1 mRNA splicing, IRE1 phosphorylation, and XBP1s expression; Example 4 demonstrated that the use of a GADD34 inhibitor (Sephin1) can also inhibit XBP1 mRNA splicing, IRE1 phosphorylation, and XBP1s expression; and Examples 5 and 6 demonstrated that the use of Sephin1 during co-culture of B7-H3CAR-T and BXPC3 can inhibit XBP1 mRNA splicing, inhibit CAR-T cell exhaustion, promote INF-γ and TNF-α secretion, and tumor killing. Therefore, GADD34 inhibitors (Sephin1) can provide a new approach for enhancing CAR-T tumor therapy.

[0124] Compared with the prior art, the core innovation of this invention lies in the following differentiated designs:

[0125] (1) Breakthrough in target selection: Existing technologies for regulating T cell exhaustion mostly focus on immune checkpoints (such as PD-1) or metabolic pathways, while directly inhibiting IRE1, a key node in endoplasmic reticulum stress (ERS), may interfere with its physiological functions (such as RIDD activity) and lead to unexpected toxicity (such as RNA degradation or pro-apoptosis risk). This invention reveals for the first time that GADD34 is a regulatory hub upstream of the IRE1-XBP1 pathway. By inhibiting GADD34 (rather than IRE1 itself) to regulate the translation repair link, it selectively and indirectly blocks the pro-exhaustion signal of IRE1-XBP1, while retaining other adaptive functions of IRE1 (such as other branches of the unfolded protein response), achieving precise regulation and reducing off-target toxicity.

[0126] (2) Synergistic mechanism of action: The present invention's scheme for inhibiting GADD34 can not only downregulate IRE1-XBP1 signaling, but also restore the PPP1R15A / B complex by regulating the translation of proteins in which it participates, thereby reducing the abnormal accumulation of misfolded proteins, thereby delaying exhaustion from the dual perspectives of "signaling pathway inhibition" and "protein homeostasis restoration" and enhancing the metabolic adaptability of CAR-T cells.

[0127] (3) Drugability advantage: The GADD34 inhibitor of the present invention is preferably a small molecule compound such as Sephin1. Compared with gene editing or antibody drugs, small molecule compounds are easier to integrate with existing CAR-T preparation processes (such as in vitro pretreatment or in vivo combined administration), and their blood-brain barrier penetrability and other characteristics provide additional advantages for the treatment of solid tumors.

[0128] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

[0129] In the present invention, the amino acid sequence of GADD34 encoded by the PPP1R15A gene is as follows:

[0130] Amino acid sequence of GADD34 (SEQ ID NO.13):

[0131] MAPGQAPHQATPWRDAHPFFLLSPVMGLLSRAWSRLRGLGPLEPWLVEAVKGAALVEAGLEGEARTPLAIPHTPWGRRPEEEAEDSGGPGEDRETLGLKTSSSLPEAWGLLDDDDGMYGEREATSVPRGQGSQFADGQRAPLSPSLLIRTLQGSDKNPGEEKAEEEGVAEEEGVNKFSYPPSHRECCPAVEEEDDEEAVKKEAHRTSTSALSPGSKPSTWVSCPGEEENQATEDKRTERSKGARKTSVSPRSSGSDPRSWEYRSGEASEEKEEKAHKETGKGEAAPGPQSSAPAQRPQLKSWWCQPSDEEEGEVKALGAAEKDGEAECPPCIPPPSAFLKAWVYWPGEDTEEEEDEEEDEDSDSGSDEEEGEAEASSSTPATGVFLKSWVYQPGEDTEEEEDEDSDTGSAEDEREAETSASTPPASAFLKAWVYRPGEDTEEEEDEDVDSEDKEDDSEAALGEAESDPHPSHPDQRAHFRGWGYRPGKETEEEEAAEDWGEAEPCPFRVAIYVPGEKPPPPWAPPRLPLRLQRRLKRPETPTHDPDPETPLKARKVRFSEKVTVHFLAVWAGPAQAARQGPWEQLARDRSRFARRITQAQEELSPCLTPAARARAWARLRNPPLAPIPALTQTLPSSSVPSSPVQTTPLSQAVATPSRSSAAAAAALDLSGRRG。

Claims

1. Application of GADD34 inhibitors in the preparation of drugs to enhance CAR-T cell tumor therapy.

2. The use according to claim 1, characterized in that The drug inhibits the exhaustion of CAR-T cells by inhibiting the IRE1-XBP1 pathway, thereby enhancing the tumor-killing effect of CAR-T cells.

3. The use according to claim 1, characterized in that The GADD34 inhibitor is selected from one or more of a small molecule compound that inhibits GADD34, a nucleic acid drug that targets GADD34, and a gene editing tool that knocks out or knocks down the GADD34 encoding gene.

4. The use according to claim 1, characterized in that The GADD34 inhibitor is Sephin1.

5. The use according to claim 1, characterized in that The tumor is a solid tumor.

6. The use according to claim 5, characterized in that The solid tumor is pancreatic cancer.

7. The use according to claim 1, characterized in that The CAR-T cells are B7-H3CAR-T cells.

8. An in vitro method for producing CAR-T cells with improved tumor treatment effects, characterized in that: CAR-T cells were treated with GADD34 inhibitor.

9. A composition for treating tumors, characterized in that: include: GADD34 inhibitors and CAR-T cells.

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