Klotho-modified CAR-T structures and their applications

Abstract

The present invention belongs to the technical field of tumor cell immunotherapy, and specifically relates to a Klotho-modified CAR-T structure and its application. The Klotho-modified CAR-T structure provided by the present invention is: CD8a-TIGIT-41BB-CD3Z-P2A-Klothoβ; the amino acid sequence of the Klothoβ is shown in SEQ ID NO.1. The present invention solves the technical defects of rapid exhaustion and apoptosis of T cells in the application of existing CAR-T cells in the treatment of solid tumors. The provided CAR-T structure effectively improves the survival cycle of CAR-T cells in tumor tissues, lays a good technical foundation for improving the application effect of CAR-T cells, and shows good application potential and practical value.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor cell immunotherapy, and specifically relates to a Klotho-modified CAR-T structure and an application thereof. Background Art

[0002] In the prevention and treatment of tumors, the immune system-based CAR-T (Chimeric Antigen Receptor T-Cell) treatment has been widely studied and developed in recent years due to its technical advantages such as few side effects and a wide range of indications. In clinical practice, CAR-T cells are used to eliminate CD19 by targeting CD19. + Tumor cells have shown good preventive and therapeutic effects in the treatment of acute lymphoblastic leukemia. However, the application of this technology in solid tumors is not satisfactory at present, and there are still many problems to be solved. One of the important problems is the problem of CAR-T cells dying, exhausting and apoptosis in tumor tissues.

[0003] In the tumor microenvironment, the function of T cells is regulated by multiple metabolic pathways, including aerobic glycolysis, glutamine catabolism, and fatty acid oxidation. However, in some tumors, despite the use of T cell adoptive therapy or PD-1 monoclonal antibody treatment, the mitochondrial activity of CD8+ T cells decreases, which directly affects the proliferation and function of cells. The decrease in mitochondrial activity interferes with the normal metabolic activity of T cells and is considered to be one of the main reasons for the ineffectiveness of T cell adoptive therapy. Further observations have found that in the differentiation process of T cells, mitochondrial metabolism is not only a key factor in the differentiation of memory CD8 + The typical energy metabolism pattern of T cells is also the memory CD8 + Abnormal mitochondrial metabolism regulates CD8 + Activation and differentiation of T cells into CD8 + Key regulators of T cell responses. Tumor-infiltrating T memory cells are the main effector components of anti-tumor immunity in most tumor types, but they are often rapidly exhausted due to the limitations of the tumor microenvironment. Similarly, CAR-T cells face similar dilemmas in the treatment of solid tumors due to the tumor microenvironment.

[0004] Klotho is a β-glucuronidase associated with the aging mechanism, and its most prominent function is to regulate phosphate homeostasis. However, Klotho has multiple pleiotropic effects, including inhibiting major signaling pathways, reducing oxidative stress, and inhibiting inflammation. Studies by Kaszubowska L et al. found that with the onset of human aging, the concentration of Klotho in serum decreases, and the concentration of Klotho in serum is significantly positively correlated with the secretion of IFN-γ by T cells (reference [Anti-Inflammatory Klotho Protein Serum Concentration Correlates with InterferonGamma Expression Related to the Cellular Activity of Both NKT-like and TCells in the Process of Human Aging. L. Kaszubowska, J. Foerster, JJKaczor, MJ Karnia and Z. Kmieć. International Journal of Molecular Sciences 2023]). The study by Yating Q et al. showed that serum Klotho was negatively correlated with pan-cancer risk, and this negative correlation was dependent on serum Klotho concentration (reference [Association of serum Klotho levels with cancer and cancer mortality: Evidence from National Health and Nutrition Examination Survey. Y. Qiao, F. Liu, Y. Peng, P. Wang, B. Ma, L. Li, et al. Cancer Medicine 2022]). The study by Shiyi Kan et al. found that mitochondrial biogenesis and fusion depend on Klotho expression (reference [FGF19 increases mitochondrial biogenesis and fusion inchondrocytes via the AMPKα-p38 / MAPK pathway. S. Kan, C. Pi, L. Zhang, D. Guo, Z. Niu, Y. Liu, et al. Cell Communication and Signaling 2023]).At the same time, Klotho is also involved in the regulation of tumor cells, tumor-associated macrophages and fibroblasts. This shows that Klotho plays an important role in T cell survival.

[0005] Therefore, based on the important role of Klotho in T cell survival, designing related CARs based on Klotho and thereby improving the persistence of CAR-T cell survival in tumor tissues is of great technical significance for improving the application of CAR-T technology in solid tumors and further improving the treatment effect of tumors. Summary of the invention

[0006] The present invention aims to solve the technical defects of rapid exhaustion and apoptosis of T cells in the application of existing CAR-T cells in the treatment of solid tumors, and provides a Klotho-modified CAR-T structure and its application. Based on the role of Klothoβ in T cell energy metabolism, the present invention uses it as a "target", combines the application characteristics of CAR-T technology, and designs targeted CAR to improve the survival cycle of T cells in tumor tissues. It has very important technical significance for improving the application of CAR-T technology in solid tumors and further improving the treatment effect of tumors.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The first object of the present invention is to provide a Klotho-modified CAR-T structure, wherein the Klotho-modified CAR-T structure is: a human CD8a molecule signal peptide, a human TIGIT extracellular region, a human CD8 molecule transmembrane region, a 41BB molecule intracellular region, a human CD3z molecule intracellular region, a P2A connection sequence and a Klothoβ coding sequence connected in sequence, namely: CD8a—TIGIT—41BB—CD3Z—P2A—Klothoβ; the amino acid sequence of the Klothoβ is shown in SEQ ID NO.1.

[0009]

[0010] Preferably, the amino acid sequence of the transmembrane region of the human CD8 molecule is shown in SEQ ID NO.2;

[0011] MALPVTALLLPLALLLHAARP (SEQ ID NO.2)

[0012] The amino acid sequence of the human TIGIT extracellular region is shown in SEQ ID NO.3;

[0013] MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP (SEQ ID NO.3)

[0014] The amino acid sequence of the human CD8a molecule signal peptide is shown in SEQ ID NO.4;

[0015] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO.4)

[0016] The amino acid sequence of the intracellular region of the 41BB molecule is shown in SEQ ID NO.5;

[0017] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO.5)

[0018] The amino acid sequence of the intracellular region of the human CD3z molecule is shown in SEQ ID NO.6;

[0019] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO.6)

[0020] The amino acid sequence of the P2A linker sequence is shown in SEQ ID NO.7.

[0021] GSGATNFSLLKQAGDVEENPGP (SEQ ID NO.7)

[0022] Preferably, the amino acid sequence of the Klotho-modified CAR-T structure is shown in SEQ ID NO.8.

[0023]

[0024] The second object of the present invention is to provide a method for preparing the above-mentioned Klotho-modified CAR-T structure lentiviral vector, comprising the following steps:

[0025] (1) Synthesize the gene sequence of the Klotho-modified CAR-T structure described in claim 1: the specific structure of the CAR-T structure is: a human CD8a molecule signal peptide, a human TIGIT extracellular region, a human CD8 molecule transmembrane region, a 41BB molecule intracellular region, a human CD3z molecule intracellular region, a P2A linker sequence and a Klothoβ coding sequence connected in sequence, namely: CD8a-TIGIT-41BB-CD3Z-P2A-Klothoβ; the amino acid sequence of Klothoβ is shown in SEQ ID NO.1;

[0026] (2) Using pCDH-EF1-conGFP plasmid as an expression vector, digesting it with EcoR I, and then integrating and recombining the gene sequence in step (1) into the pCDH-EF1-conGFP plasmid; the manufacturer of the In-Fusion HD Cloning Kits is Clontech, and the model number is 639649.

[0027] (3) The ligation product in step (2) was transformed into STABL3 competent cells, and the plasmid was further extracted after screening and expansion culture to construct a lentiviral vector pCDH-EF1-CD8a-TIGIT-41BB-CD3Z-P2A-Klothoβ expressing the Klotho-modified CAR-T structure.

[0028] Preferably, the step (2) comprises using In-Fusion HD Cloning Kits, Clontech, model 639649, to integrate and recombinantly integrate the gene sequence in step (1) into the pCDH-EF1-conGFP plasmid.

[0029] Another object of the present invention is to provide a lentiviral vector prepared by the above preparation method.

[0030] Another object of the present invention is to provide T cells infected by the above-mentioned lentiviral vector.

[0031] Another object of the present invention is to provide the use of the above-mentioned T cells in the preparation of anti-tumor drugs, wherein the tumor includes cervical cancer.

[0032] Preferably, the nucleotide sequence of CD8a-TIGIT-41BB-CD3z-Klothoβ in step (1) is as shown in SEQ ID NO.9.

[0033]

[0034] Another object of the present invention is to provide an application of the amino acid sequence of Klothoβ in the preparation of an anti-tumor drug, wherein the amino acid sequence of Klothoβ is shown in SEQ ID NO.1.

[0035] Preferably, the amino acid sequence of Klothoβ is used in the preparation of a vector for resisting T cell aging.

[0036] The inventive concept and principle of the present invention:

[0037] In the existing technology, the use of lentiviral vectors as "vector tools" to introduce exogenous genes so that the exogenous genes can play a targeted role in the corresponding "preventive and therapeutic" functions is an ideal "targeted" prevention and treatment strategy that has been widely studied and applied. However, in practical applications, the "targeting" of exogenous genes plays a fundamental role in determining the relevant prevention and treatment results. Therefore, selecting appropriate "targeting" sites and designing reasonable CARs based on them are the basic prerequisites for the application of CAR-T (chimeric antigen receptor T cells) technology.

[0038] In the present invention, the inventors take Klothoβ as a "key target" based on its role in T cells, and design and develop a restrictive CAR structure that resists T cell aging by deeply integrating the unique advantages of CAR-T technology, aiming to optimize the survival persistence of T cells in the tumor microenvironment, thereby effectively addressing the technical bottlenecks of rapid exhaustion and apoptosis of T cells faced by current CAR-T therapy in the field of solid tumor treatment. The present invention is further verified by experiments, and the results show that the CAR design based on the present invention can significantly improve the survival time of CAR-T cells in tumor tissues, laying a solid technical foundation for improving the effect of CAR-T cell therapy in clinical applications, and predicting its broad application prospects and important practical value.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] First, based on the role of Klothoβ in T cell energy metabolism, the present invention uses it as a "target" and designs a Klotho-modified CAR-T structure, which can improve the survival cycle of T cells in tumor tissues. This has very important technical significance for improving the application of CAR-T technology in solid tumors and further improving the treatment effect of tumors.

[0041] Secondly, the present invention solves the technical defects of rapid exhaustion and apoptosis of T cells in the application of existing CAR-T cells in the treatment of solid tumors. The preliminary test results of the present invention show that the CAR-T structure provided by the present invention effectively improves the survival cycle of CAR-T cells in tumor tissues, lays a good technical foundation for improving the application effect of CAR-T cells, and shows good application potential and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 CAR structure diagram. (A) is a schematic diagram of the structure of the recombinant expression plasmid vector pCDH-EF1-CD8a-TIGIT-41BB-CD3Z-P2A-Klothoβ of the present invention. (B) is a diagram of CAR transfection efficiency detected by GFP;

[0043] Figure 2 Klotho promotes memory differentiation of T cells; in the figure, the specific cell types are: naïve T cells (TN), central memory T cells (TCM), effector T cells (Recently activated effector memory or effector T cells, TEM), exhausted T cells (exhausted T cells, TEX);

[0044] Figure 3 Klotho supports T cell metabolic adaptation by enhancing mitochondrial metabolism. (A) T cells were collected 48 hours after lentiviral infection and single-cell suspensions were prepared. Mitochondrial mass, ΔΨm, and ROS were detected by flow cytometry. (B) T cells were labeled with CFSE, and the proliferation differences of Isotype or Klotho-overexpressing T cells were detected by flow cytometry after 24 hours of culture. *P<0.05, **P<0.01;

[0045] Figure 4 CAR-T cells targeting CD155 can effectively and specifically eliminate target cells. (A) Flow cytometry was used to detect the expression of CD155 on the surface of cervical cancer cell lines (HCA1, CAC1C) and human cervical epithelial immortalized cells (H8); (B) After different groups of T cells were co-incubated with target cells at an effector-target ratio of 5:1 for 8 hours, the fluorescence intensity of surviving tumor cells was detected using a biological imaging system and the specific killing effect of different groups of T cells on target cells was analyzed. ****P<0.0001;

[0046] Figure 5Improve the AICD of CAR-T cells by regulating the expression of Klotho. (A) Flow chart of the detection of AICD of CAR-T cells; (B) Collect the incubated CAR-T cells and prepare single cell suspension, label the CAR-T cells with PI and Annexin V, and detect the apoptosis of CAR-T cells;

[0047] Figure 6 The expression of Klotho improves the ability of CAR-T cells to cope with tumor recurrence. (A) Mouse experimental model diagram; (B) Mouse tumor bioluminescence detection diagram; (C) Mouse tumor bioluminescence fluorescence statistics diagram; (D) Mouse survival curve. DETAILED DESCRIPTION

[0048] The above contents of the present invention are further described in detail below by means of specific implementation methods in the form of examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples.

[0049] Biology and Materials:

[0050] The non-obese diabetic / severe combined immunodeficiency (NOD / SCID) mice, 6-8 weeks old female mice, were purchased from Beijing Weitong Lihua Co., Ltd. (Beijing, China) and raised in a sterile environment;

[0051] The human cervical cancer-related cell lines: HCA1, CAC1C, purchased from ATCC;

[0052] The human cervical immortalized cell line: H8, purchased from ATCC;

[0053] During the experiments, all cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS, HyClone, Chicago, IL, USA) and 100 U / mL penicillin and 100 μg / mL streptomycin (Invitrogen, Carlsbad, CA, USA);

[0054] The fluorescent cell line was obtained as follows: before the experiment, tumor cells were transfected with a viral supernatant containing luciferase-GFP, and the GFP channel was sorted using a FACS AriaTM cell sorter (BD Biosciences, San Jose, CA, USA), and finally a cell line stably expressing luciferase-green fluorescent protein (GFP) was obtained.

[0055] The technologies involved in the present invention are all conventional molecular cloning techniques. The designed enzymes, primers, reagents and reaction conditions can be reasonably selected based on the experience of those skilled in the art unless otherwise specified. The designed reagents and consumables are common products on the market, and the detection methods and instruments involved are also well known and proficiently mastered by those skilled in the art.

[0056] Example 1 Construction of Klotho-modified CAR-T structure of the present invention

[0057] The present invention uses Klotho to modify and transform CAR-T cells. First, a specific CAR structure is designed, and then the T cells are labeled. The specific process is as follows:

[0058] 1. Design of CAR structure

[0059] The Klotho-modified CAR-T structure provided by the present invention is an amino acid sequence connected by several protein fragments, named: CD8a-TIGIT-41BB-CD3z-Klothoβ, and the specific structure is: a human CD8a molecule signal peptide, a human TIGIT extracellular region, a human CD8 molecule transmembrane region, a 41BB molecule intracellular region, a human CD3z molecule intracellular region, a P2A connection sequence and a Klothoβ coding sequence connected in sequence; that is: CD8a-TIGIT-41BB-CD3Z-P2A-Klothoβ. Among them: the amino acid sequence of Klothoβ is shown in SEQ ID NO.1; the amino acid sequence of the human CD8a molecule signal peptide is shown in SEQ ID NO.2; the amino acid sequence of the human TIGIT extracellular region is shown in SEQ ID NO.3; the amino acid sequence of the human CD8 molecule transmembrane region is shown in SEQ ID NO.4; the amino acid sequence of the 41BB molecule intracellular region is shown in SEQ ID NO.5; the amino acid sequence of the human CD3z molecule intracellular region is shown in SEQ ID NO.6; the amino acid sequence of the P2A connecting sequence is shown in SEQ ID NO.7.

[0060] Based on the design of the above CAR structure, it was commissioned to be synthesized by Sangon Biotech Co., Ltd., wherein the amino acid sequence of the Klotho-modified CAR-T structure is shown in SEQ ID NO.8, and the nucleotide sequence of the Klotho-modified CAR-T structure is shown in SEQ ID NO.9.

[0061] 2. Construction of recombinant lentiviral expression plasmid vector

[0062] Based on the characteristics of CAR-T technology, the present invention uses pCDH-EF1-conGFP plasmid as an expression vector to further construct a recombinant lentiviral expression plasmid vector, so as to facilitate subsequent use in infecting T cells to prepare CAR-T cells. The specific construction process of the lentiviral expression vector is described as follows:

[0063] (1) Entrust Sangon Biotech Co., Ltd. to synthesize the coding sequence DNA of the Klotho-modified CAR-T structure, as shown in the above SEQ ID NO.9, for future use.

[0064] (2) Using pCDH-EF1-conGFP plasmid (purchased from Novopro, model number V007087) as an expression vector, it was digested with restriction endonuclease EcoR I (purchased from New England Biolabs, model number R0101V) at 37°C; then, referring to the instruction manual, the coding sequence DNA in step (1) was integrated and recombined into the pCDH-EF1-conGFP plasmid using In-Fusion HD Cloning Kits, purchased from Clontech, model number 639649.

[0065] (3) The ligation product in step (2) was transformed into STABL3 competent cells (purchased from weidibio, catalog number Stbl3). Before transformation, the competent cells were taken out from -80°C and placed on ice. The entire ligation system was added to the competent cells and allowed to stand on ice for 2 hours. After standing, the competent cells were quickly moved to a 42°C water bath, heat-shocked for 45 seconds, and then quickly returned to ice for 2 minutes. 500µl LB culture medium was added and shaken at 37°C for 1 hour. A suitable volume of cells was aspirated and applied to an LB gel culture plate containing antibiotics. The plate was inverted and cultured at 37°C for 12 hours for screening. After screening and expansion culture, the plasmid was further extracted to prepare the lentiviral expression plasmid pCDH-EF1-CD8a-TIGIT-41BB-CD3 expressing Klothoβ recombinant. Z -P2A-Klothoβ.

[0066] The constructed lentiviral expression plasmid pCDH-EF1-CD8a-TIGIT-41BB-CD3 Z -P2A-Klothoβ structure diagram Figure 1 As shown in (A), the related operations in the construction process can refer to the conventional operations in the prior art and will not be repeated here.

[0067] As a control, the present invention prepared the control recombinant plasmid pCDH-EF1-CD8a-TIGIT-41BB-CD3 by the above method. Z , used for subsequent experimental control.

[0068] Example 2 Preparation of CAR-T cells

[0069] 1. Lentiviral packaging

[0070] 293T cells were used as the target cells to be initially transfected, as follows:

[0071] First, 293T cells (purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences, model SCSP-502) were plated in a six-well plate and incubated for 24 h (37°C, DMEM complete medium (purchased from Thermo Fisher Scientific, model 12491015);

[0072] Subsequently, the culture medium was replaced with OPTI-MEM culture medium (purchased from Thermo Fisher Scientific, model 31985070), 1.5 g of the lentiviral expression plasmid constructed in Example 1, 1.5 g of the packaging plasmid psPAX2, and Pmd2.G1 g, and 8 μl of liposome transfection reagent were added, and mixed evenly to transfect the target cell 293T;

[0073] Twelve hours after transfection, the medium was replaced with normal DMEM;

[0074] Finally, after continuing to culture for 48 hours, the virus supernatant (i.e., the packaged virus-like particles) was collected and centrifuged at 1500 rpm for 10 minutes. The supernatant was stored at -80°C for subsequent use in infecting T cells.

[0075] 2. Preparation of purified CD3 + T cells

[0076] First, mononuclear cells were isolated from human peripheral blood using density gradient centrifugation;

[0077] Then, T cell separation magnetic beads were used for separation and purification to obtain purified CD3 + T cells;

[0078] Afterwards, the purified T cells were cultured in 24-well cell culture plates with RPMI-1640 containing IL-2 (100 IU / mL, PeproTech, Suzhou, Jiangsu, China) and L-glutamine (2 mM, Gibco / Life Technologies / Thermo Fisher Scientific, Waltham, MA, USA);

[0079] Finally, before use, T cells were activated with CD3 / CD28 antibodies (1 μl / 10 7 Cells were stimulated and activated for 2 days.

[0080] The specific operation method can be referred to the existing technology (Zhang Kai. (2024). IL-24 improves efficacy of CAR-T cell therapy by targeting stemness of tumor cells. British Journal of Cancer.), which will not be repeated here.

[0081] 3. T cell infection

[0082] Use a 24-well cell culture plate and add T cells (10 6 / well), add 1mL of the virus supernatant collected in step (1) and polybrene (8μg / mL) to each well and incubate overnight for infection;

[0083] After 24 hours, the medium was replaced with normal RPMI-1640 containing IL-2 (100 IU / mL) and L-glutamine (2 mM).

[0084] 4. T cell expansion

[0085] To facilitate subsequent experimental applications, the infected T cells in step (iii) were incubated and washed three times by centrifugation, and then RPMI1640 medium containing 1000 U IL-2 and 5% fetal bovine serum was added (the medium was changed every 2-3 days) to further expand the T cells. The prepared cells were recorded as:

[0086] TIGIT.CAR-T (containing the control lentiviral vector plasmid pCDH-EF1-CD8a-TIGIT-41BB-CD3 Z )、TIGIT.CAR.KL-T (containing the lentiviral vector plasmid pCDH-EF1-CD8a-TIGIT-41BB-CD3 Z -P2A-Klothoβ).

[0087] In addition, as a blank control, referring to the above operation, the present invention packages the pCDH-EF1 empty plasmid containing GFP into lentivirus and directly infects T cells, and the prepared cells are recorded as: GFP-T (equivalent to the blank control T cell group expressing only GFP).

[0088] Example 3 Cell experiment and animal experiment

[0089] Based on Example 2, the present invention prepared CAR-T cells by transfection of the constructed lentiviral expression plasmid, and further conducted preliminary cell experiments. The specific experimental process is as follows:

[0090] (1) Virus packaging detection

[0091] For T cells infected 5 days after the infection in step (iv) of the above Example 2, the expression of CAR on the surface of T cells was detected by flow cytometry.

[0092] The experimental results are as follows Figure 1 As shown in (B), it can be seen that the CAR expression positive rate reaches 60%~70%. This result shows that the CAR expression plasmid constructed by the present invention is successfully infected with T cells after being packaged into lentiviral particles, and is well expressed, and can be used for subsequent experiments.

[0093] (2) Detection of differentiation of different T cells

[0094] During the above-mentioned evaluation of T cell CAR expression, the differentiation of T cells was further detected and analyzed.

[0095] T cells infected 5 days after the infection in step (iv) of Example 2 above were collected, the cell pellet was resuspended in PBS, and then 1µl CD3 antibody, 1µl CD45 antibody, and 1µl CD62L antibody were added, and incubated at 4°C in a light-proof environment for 15 minutes; the data were analyzed using a C6 flow cytometer (Becton Dickinson), and the data were analyzed using FlowJo software (FlowJo, LLC, Ashland, Covington, KY, USA).

[0096] The experimental results are as follows Figure 2 As shown by Figure 2 It can be seen that compared with the TIGIT.CAR group cells, in the presence of Klothoβ, the proportion of different types of cells in the TIGIT.CAR.KL group cells changed significantly, and the proportion of central memory T cells (TCM) was significantly higher than that of other control groups. In other words, Klothoβ promotes the further differentiation of T cells into central memory subtype TCM, and the high proportion of central memory subtype TCM can make T cells survive better in the tumor microenvironment, so that CAR-T cells can better exert their anti-tumor function, thereby improving the therapeutic effect.

[0097] (3) Detection of mitochondrial metabolism and proliferation of different T cells

[0098] During the above-mentioned evaluation of T cell CAR expression, the mitochondrial metabolism and proliferation of T cells were further detected and analyzed.

[0099] T cells infected 5 days after the above Example 2 step (iv) were collected, and the cell pellets were resuspended in PBS, followed by adding 1µl Mito Tracker, 1µl TMRM, and 1µl MitoSOX to detect mitochondrial mass, ΔΨm, and mitochondrial reactive oxygen species (ROS). The cells were incubated at 4°C in a dark environment for 15 minutes; the cells were detected and analyzed using a C6 flow cytometer (Becton Dickinson), and the data were analyzed using FlowJo software (FlowJo, LLC, Ashland, Covington, KY, USA).

[0100] The experimental results are as follows Figure 3 As shown in (A), the analysis showed that the mitochondrial mass, ΔΨm and mean fluorescence intensity (MFI) of mitochondrial ROS in CAR-T cells overexpressing Klothoβ were significantly increased compared with the control group.

[0101] T cells were collected one day after infection in step (iv) above, and the cell pellet was resuspended in PBS, followed by the addition of 1 µl CFSE to detect T cell proliferation. The data were analyzed on the first and second days using a C6 flow cytometer (Becton Dickinson), and the data were analyzed using FlowJo software (FlowJo, LLC, Ashland, Covington, KY, USA).

[0102] The experimental results are as follows Figure 3 As shown in (B), the analysis shows that the CFSE fluorescence of Klotho-overexpressing CAR-T cells is more significantly weakened, which means that Klothoβ overexpression significantly enhances the proliferation ability of CAR-T cells. These results show that Klothoβ plays a key role in regulating mitochondrial potential by regulating mitochondrial biogenesis-related genes, thereby promoting CAR-T cell proliferation and meeting the bioenergy requirements of T cells.

[0103] (4) Detection of apoptosis in different tumor cells

[0104] Tumor cells with high expression of CD155 were used as experimental subjects, CAR-T cells were used as experimental "agents", and tumor cell apoptosis was used to detect and evaluate the technical effect of the constructed CAR. The specific experimental process is briefly described as follows:

[0105] Human cervical cancer cell lines HCA1-fluc, CAC1C-fluc with high expression of CD155 and human cervical immortalized cell line H8-fluc with negative CD155 were used as target cells, and T cells treated with different methods were used as effector cells (Mock-T, TIGIT.CAR-T, TIGIT.CAR.KL-T). T cells and target cells were co-incubated in 96-well plates for 6 hours at an effector-target ratio of 5:1, with three replicate wells in each group.

[0106] After the incubation, a small animal in vivo imaging device (purchased from Caliper Life Science, model IVIS Lumina, Series Ⅲ spectrometer) was used to detect the fluorescence changes expressed by the tumor and calculate the fluorescence values.

[0107] The experimental results are as follows Figure 4 As shown. It can be seen that compared with the control group, the TIGIT.CAR-T group can specifically kill HCA1 and CAC1C tumor cells with positive CD155 expression, while TIGIT.CAR.KL-T cells can also effectively kill CD155-positive tumor cells at the same ratio. This result shows that the presence of Klothoβ does not affect the killing effect of CAR-T cells on CD155 antigen-positive tumor cells.

[0108] (5) Detection of activation-induced apoptosis (AICD) of different T cells

[0109] In the above-mentioned evaluation process of CAR-T cells killing tumor cells, the present invention further detected and analyzed the AICD of T cells.

[0110] Soluble CD155 was used in the culture system of CAR-T cells. After 12 hours of culture, the medium without CD155 was replaced. After 3 days of culture, the medium containing soluble CD155 was replaced again and cultured for 12 hours to detect CAR-T cell apoptosis. After the incubation, the cell supernatant was collected, and the cell pellet was resuspended in Annexin V-binding buffer (BioLegend, San Diego, CA, USA), and then 1µl Annexin V antibody (BioLegend) was added and incubated at 4℃ in a dark environment for 15 minutes; Propidium (propidium iodide, Sigma) was added before loading. The C6 flow cytometer (Becton Dickinson) was used for detection and analysis, and the data were analyzed using FlowJo software (FlowJo, LLC, Ashland, Covington, KY, USA).

[0111] The experimental results are as follows Figure 5 As shown. It can be seen that compared with the TIGIT.CAR-T group, fewer CAR-T cells in the TIGIT.CAR.KL-T group underwent apoptosis, i.e., AICD, after multiple long-term antigen stimulation. This result preliminarily suggests that the expression of Klothoβ can improve apoptosis caused by activation of CAR-T cells due to long-term antigen stimulation in the tumor microenvironment.

[0112] (6) Detection of tumor recurrence in mice

[0113] Based on the above experiments, further experiments were conducted on mice. The specific process is briefly described as follows:

[0114] Mouse experimental model Figure 6 As shown in (A), the present invention uses NOD / SCID immunodeficient mice purchased from Vital River Company, and subcutaneously inoculates 3×10 5 HCA1-fluc cells (100ul) were used to construct a mouse subcutaneous transplant tumor model. When the mouse tumor fluorescence signal appeared, the model was successfully established. After 7 days, 5×10 6 Different groups of T cells (100ul) (TIGIT.CAR-T, TIGIT.CAR.KL-T) were used for treatment (3 mice in each group). 14 days after the injection of T cells via tail vein, the mouse tumor fluorescence signal was detected to have completely disappeared and then the mouse was subcutaneously inoculated with 3×10 resuspended in PBS. 5 HCA1-fluc cells (100ul) were used to construct a mouse tumor recurrence model.

[0115] During the experiment, small animal in vivo imaging equipment was used to detect fluorescence changes expressed by the tumor (bioluminescence photos were taken every 7 days).

[0116] When using small animal live imaging equipment to detect fluorescence changes expressed in tumors, mice were first anesthetized with 3% isoflurane (RWD Life Science, Shenzhen, China) in an induction chamber; then each mouse was intraperitoneally injected with 100 ul of d-luciferin solution (0.15 mg / ml, Yeasen Biotech Co., Ltd., Shanghai, China) using a syringe. After 10 minutes, fluorescence was detected using the animal live imaging equipment IVIS Lumina, Series Ⅲ spectrometer (Caliper Life Science), and finally analyzed using live image 4.3.1 software (PerkinElmer, Waltham, MA, USA).

[0117] The experimental results are as follows Figure 6 As shown. The statistical results and analysis show that:

[0118] CAR-T cells co-expressing Klotho were better able to cope with secondary tumors, with a secondary tumor formation rate of 1 / 3, compared with 2 / 3 in the control group, and the survival of mice was prolonged. This result preliminarily confirmed that the expression of Klothoβ can enable CAR-T cells to better cope with tumor recurrence.

[0119] From the above experimental results, it can be seen that the present invention further improves the prevention and treatment effects of related tumors by further modifying the existing CAR structure targeting CD155 and utilizing Klothoβ to further promote T cell subtype differentiation, thereby improving T cell exhaustion.

[0120] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. Klotho-modified CAR-T, characterized in that: The Klotho-modified CAR-T is: a human CD8a molecule signal peptide, a human TIGIT extracellular region, a human CD8 molecule transmembrane region, a 41BB molecule intracellular region, a human CD3ζ molecule intracellular region, a P2A connection sequence and a Klothoβ coding sequence connected in sequence, that is: CD8a—TIGIT—41BB—CD3ζ—P2A—Klothoβ; the amino acid sequence of Klothoβ is shown in SEQ ID NO.1; The amino acid sequence of the human TIGIT extracellular region is shown in SEQ ID NO.

3.

2. The Klotho-modified CAR-T according to claim 1, characterized in that The amino acid sequence of the transmembrane region of the human CD8 molecule is shown in SEQ ID NO.2; The amino acid sequence of the human CD8a molecule signal peptide is shown in SEQ ID NO.4; The amino acid sequence of the intracellular region of the 41BB molecule is shown in SEQ ID NO.5; The amino acid sequence of the intracellular region of the human CD3ζ molecule is shown in SEQ ID NO.6; The amino acid sequence of the P2A linker sequence is shown in SEQ ID NO.

7.

3. The Klotho-modified CAR-T according to claim 1, characterized in that The amino acid sequence of the Klotho-modified CAR-T is shown in SEQ ID NO.

8.

4. The method for preparing a Klotho-modified CAR-T lentiviral vector according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Synthesize the gene sequence of the Klotho-modified CAR-T described in claim 1: the specific structure of the CAR-T is: a sequence of human CD8a molecule signal peptide, human TIGIT extracellular region, human CD8 molecule transmembrane region, 41BB molecule intracellular region, human CD3ζ molecule intracellular region, P2A linker sequence and Klothoβ, namely: CD8a-TIGIT-41BB-CD3ζ-P2A-Klothoβ; the amino acid sequence of Klothoβ is shown in SEQ ID NO.1; (2) Using pCDH-EF1-conGFP plasmid as an expression vector, digesting it with EcoR I, and then integrating and recombining the gene sequence in step (1) into the pCDH-EF1-conGFP plasmid; (3) The ligation product in step (2) was transformed into STABL3 competent cells, and the plasmid was further extracted after screening and expansion culture to construct a lentiviral vector pCDH-EF1-CD8a-TIGIT-41BB-CD3ζ-P2A-Klothoβ expressing Klotho-modified CAR-T.

5. The preparation method according to claim 4, characterized in that: The step (2) includes using In-Fusion HD Cloning Kits to integrate and recombine the gene sequence in step (1) into the pCDH-EF1-conGFP plasmid; the manufacturer of the In-Fusion HD Cloning Kits is Clontech, and the model number is 639649.

6. A lentiviral vector prepared by the preparation method according to any one of claims 4 to 5.

7. T cells infected with the lentiviral vector according to claim 6.

8. The use of the T cells according to claim 7 in the preparation of anti-tumor drugs, characterized in that: The tumor is cervical cancer.

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