Chimeric antigen receptor targeting human alpha-enolase, CAR-T cell and application

Abstract

The invention discloses a chimeric antigen receptor targeting human alpha-enolase, a CAR-T cell and application of the chimeric antigen receptor. The invention relates to a chimeric antigen receptor targeting human alpha-enolase, the chimeric antigen receptor comprises an scFv structural domain aiming at the human alpha-enolase, a transmembrane region and an intracellular signal transduction region, and the amino acid sequence of the chimeric antigen receptor is SEQUENCE No.1. The chimeric antigen receptor aiming at the tumor metabolic protein ENO1 is constructed, on one hand, tumor cells are recognized and killed through the highly expressed ENO1, and on the other hand, the chimeric antigen receptor has the effect of inhibiting invasion and metastasis of the tumor cells. Compared with ENO1 monoclonal antibody targeted therapy, the ENO1CAR therapy can directly kill tumor cells, and the effect is stronger; compared with other CAR treatment targets, the ENO1 has more adaptations and can play a role in inhibiting invasion and metastasis of tumors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology engineering, and in particular to a chimeric antigen receptor targeting human α-enolase, a CAR-T cell and uses thereof. Background Art

[0002] Abnormal cell energy metabolism is one of the basic characteristics of tumor cells. Normal cells mainly meet metabolic requirements through the tricarboxylic acid cycle, fatty acid oxidation or glutamine oxidation. Only under hypoxia and stress conditions will they perform anaerobic glycolysis, while tumor cells perform glycolysis regardless of aerobic or hypoxic conditions, which is the Warburg effect. The abnormally high expression of key glycolytic enzymes in tumor cells is highly correlated with the enhanced proliferation, invasion and metastasis of tumor cells. Targeting key glycolytic enzymes is a new strategy for anti-tumor treatment in recent years.

[0003] Human α-enolase (ENO1) is one of the key enzymes of glycolysis. It is highly expressed in a variety of tumor tissues, and the expression level is highly correlated with the malignancy of the tumor. In recent years, there have been a number of tumor vaccines and monoclonal antibody therapies targeting ENO1, which is a potential target for tumor treatment. ENO1 is involved in the glycolysis pathway and is mainly located in the cytoplasm. ENO1 located on the cell membrane surface is also called plasminogen binding protein, which is highly related to tumor cell invasion and can be used as an ideal target for chimeric antigen receptors (CAR). Bioinformatics analysis and our previous immunohistochemistry results showed that ENO1 membrane expression is mainly in tumor tissues, and is generally not expressed or lowly expressed in normal tissues.

[0004] CAR therapy has been a research hotspot in recent years. Its specific recognition domain is tumor-associated antigen (TAA). Kymriah (autologous CD19 CAR-T) is the first approved CAR-T therapy. Its efficacy in treating relapsed or refractory acute lymphoblastic leukemia is as high as 83%. There are also CARs targeting other TAAs. According to data from Clinical Trials (Clinical Trial.gov), as of February 7, 2023, there are 1,186 registered CAR-T clinical trials worldwide, of which 612 are in China, ranking first, and 399 are in the United States, which is still growing rapidly. However, no CAR targeting tumor energy metabolism has been seen. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide a chimeric antigen receptor, CAR-T cell and use targeting human α-enolase. The present invention constructs a chimeric antigen receptor (ENO1CAR) targeting the tumor metabolic protein ENO1, which recognizes and kills tumor cells through highly expressed ENO1 on the one hand, and inhibits the invasion and metastasis of tumor cells on the other hand.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0007] In the first aspect, a chimeric antigen receptor targeting human α-enolase comprises a scFv domain targeting human α-enolase, a transmembrane region and an intracellular signal transduction region, and the amino acid sequence of the chimeric antigen receptor is SEQUENCE No. 1. Furthermore, the scFv domain targeting human α-enolase comprises a heavy chain variable region with an amino acid sequence as shown in SEQUENCE No. 2, a connecting peptide with an amino acid sequence as shown in SEQUENCE No. 3 and a light chain variable region with an amino acid sequence as shown in SEQUENCE No. 4.

[0008] The second aspect is a nucleotide sequence encoding the chimeric antigen receptor as described above.

[0009] The third aspect provides an expression vector comprising the nucleotide described above.

[0010] In a fourth aspect, a CAR-T cell targeting human α-enolase is transduced with the vector as described above.

[0011] In a fifth aspect, a method for constructing a CAR-T cell targeting human α-enolase comprises the following steps:

[0012] (1) Construction of a chimeric antigen receptor targeting human α-enolase;

[0013] (2) Chimeric antigen receptor transduction of NK cells.

[0014] In a sixth aspect, a composition comprises the CAR-T cells as described above or the CAR-T cells constructed by the above construction method, and a pharmaceutically acceptable carrier.

[0015] A seventh aspect is the use of the above cells in the preparation of drugs for treating tumor diseases.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention constructs a chimeric antigen receptor targeting the tumor metabolic protein ENO1, which can recognize and kill tumor cells through highly expressed ENO1 on the one hand, and inhibit the invasion and metastasis of tumor cells on the other hand. Compared with ENO1 monoclonal antibody targeted therapy, ENO1 CAR therapy can directly kill tumor cells and has a stronger effect; ENO1 has more indications than other CAR therapeutic targets and can inhibit the invasion and metastasis of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A The structure diagram of the ENO1 CAR of the present invention; wherein the core structure of CAR includes CD8α signal peptide, ENO1 single-chain antibody, CD8 transmembrane region and intracellular signal transduction region (4-1BB and CD3ζ), 5'LTR / 3'LTR, Moloney murine leukemia virus long terminal repeat series; MMLVΨ, Moloney murine leukemia virus packaging signal; pol region, reverse transcriptase binding site, MMLV reverse transcriptase site contains splice acceptor site; P2A, protein 2A peptide derived from porcine Teschovirus type 1;

[0019] Figure 1B This is the recombinant plasmid map of SFG.CNb30_opt.ENO1 CAR, in which ENO1 CAR (including SS signal peptide, ENO1-scFv and CD8 hinge-TM-41BB-CD3Z and other core structures) is recombined into SFG.CNb30_opt retroviral vector through Ncol and MluI restriction sites;

[0020] Figure 1C The electrophoresis identification results are shown in Figure 2. From left to right, they are DL10,000 DNA marker (M), undigested plasmid (1, 7868 bp), and target bands double-cut by Ncol and MluI (2, 1458 bp and 6410 bp);

[0021] Figure 1D It is a schematic diagram of the domain structure of ENO1 CAR, which shows that the sequencing identification is correct, and the dotted box shows the recombinant target sequence ENO1 CAR;

[0022] Figure 2 The expression of ENO1 in normal tissues and tumor cells. In normal tissues, ENO1 is not expressed in the cell membrane of lung (A), liver (B), kidney (C), and heart (D). In liver, kidney, and heart, it is only expressed in the cytoplasm and cannot be recognized by ENO1 CAR. In histological sections of patients with lung cancer (E) and glioma (F), the capsule expression is strongly positive.

[0023] Figure 3Flow cytometry was used to detect ENO1 CAR expression, where A. NT Fab had no expression; B. ENO1 CAR-T had high expression;

[0024] Figure 4 Flow cytometry was used to detect the specific activation phenotype (CD69) of ENO1 CAR-T and NT when co-cultured with target cells. The target cells used included immortalized microglial cells HCM3, human glioma cells LN299, monocytic leukemia cells THP-1, and human lung cancer cells H460. When ENO1 CAR-T was co-cultured with various target cells, the expression of CD69 was significantly higher than that of the NT group (P < 0.05).

[0025] Figure 5 Flow cytometry was used to detect the specific activation phenotype (CD25) of ENO1 CAR-T and NT when they were co-cultured with target cells. When ENO1 CAR-T was co-cultured with various target cells, the expression of CD25 was significantly higher than that of the NT group (P < 0.05).

[0026] Figure 6 Flow cytometry was used to detect the killing effect (live / dead) of ENO1 CAR-T and NT on target cells when they were co-cultured with target cells. ENO1 CAR-T was co-cultured with immortalized microglia HCM3, human glioma cells LN299, monocytic leukemia cells THP-1 and human lung cancer cells H460, and the killing ratio (Dead%) was significantly higher than that of the NT group.

[0027] Figure 7 Hematoxylin staining was used to observe the killing effect. After NT and ENO1 CAR-T were co-cultured with HCM3, the supernatant was removed and hematoxylin staining was performed. A large amount of HCM3 adherent to the wall was still observed in the HCM3 ONLY and NT groups, while the HCM3 retention in the ENO1CAR-T killing group was significantly reduced.

[0028] Figure 8 LEGENDplex multifactor staining was used to detect the secretion levels of 13 cytokines when ENO1 CAR-T and NT were co-cultured with target cells. Among them, the secretion of IL-4, IL-6, IL-17A, sFas, sFasL, IFN-γ and Granzyme B by ENO1 CAR-T was significantly increased, which was related to its immune specific response, killing and safety mechanism. DETAILED DESCRIPTION

[0029] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0030] Example 1 Construction and identification of ENO1 CAR retroviral vector

[0031] The whole gene synthesis ENO1 CAR sequence (amino acid sequence No. 1) has a structure as shown in Figure 1A As shown, the core elements include a leader sequence, Anti-ENO1 scFv, a hinge region and a transmembrane domain, and an intracellular signal transduction domain; Anti-ENO1 scFv includes a heavy chain variable region (V H , amino acid sequence No.2), linker (Linker, amino acid sequence No.3) and light chain variable region (V L , amino acid sequence No.4).

[0032] The ENO1 CAR gene was cloned into the SFG.CNb30_opt retroviral vector by molecular cloning. The steps included extraction of template plasmid and vector plasmid; PCR amplification of ENO1 CAR target fragment; restriction enzyme digestion of SFG.CNb30_opt vector to linearize it; gel recovery of target fragment and vector, ligation and recombination, and transformation of DH5α competent cells, identification of positive clones by PCR, and restriction enzyme digestion identification after plasmid extraction by shaking bacteria ( Figure 1B ) and sequencing identification ( Figure 1C ), and finally construct the vector plasmid SFG.CNb30_opt.ENO1 CAR ( Figure 1D ).

[0033] Example 2 Expression of ENO1 in normal tissues and tumor cells

[0034] Immunohistochemistry was used to identify the expression in normal human tissues and tumor cells. Normal human lung, liver, kidney, and heart histological sections as well as lung cancer and glioma histological sections from tumor patients were obtained (approved by the Ethics Committee of Southern Medical University and with informed consent from the patients). Immunohistochemistry was used to detect the expression of ENO1. The steps were as follows: 2um slices were baked at 65°C for 60 minutes; dewaxing to water: three cylinders of xylene, 6 minutes each; two cylinders of 100% alcohol, 3 minutes each; one cylinder of 95% alcohol, 3 minutes; washing with distilled water for 3 minutes, high-pressure repair with citrate buffer for 3 minutes, and cooling naturally to room temperature; 3% H202 inhibited endogenous peroxidase: room temperature for 10 minutes; goat serum blocked for 30 minutes, antibody ENO1 (ab227978) diluted 1:100 with antibody diluent, incubated at 37℃ for 1 hour; PBS rinsed for 2 minutes×5 times; secondary antibody: enzyme-labeled goat anti-rabbit IgG polymer, incubated at 37℃ for 30 minutes; PBS rinsed for 2 minutes×5 times; DAB color development (Shanghai Gene): 2 minutes; hematoxylin counterstaining for 1 minute, hydrochloric acid alcohol differentiation for 5 seconds, tap water blueing for 2 minutes; gradient alcohol dehydration, xylene transparent, neutral gum sealing, observation under ordinary optical microscope ( Figure 2 ).

[0035] Example 3 ENO1 CAR-T preparation

[0036] ①Retroviral packaging

[0037] The SFG-ENO1 CAR target plasmid was co-transfected with pRDF (envelope plasmid, env) and pEQ-PAM3(-E) (packaging plasmid, gag-pol) into 293T / 17 cells to package retrovirus, and the supernatant was collected and frozen at -80%;

[0038] ②T cell activation and CAR gene transduction

[0039] Day 0T cell activation

[0040] Isolation of PBMC: Take concentrated buffy coat from healthy blood donors, disinfect the blood bag with 75% alcohol, cut off the sample braid in the blood bag, and carefully squeeze out the blood; isolate PBMC according to the instructions of Tianjin Haoyang Company's human peripheral blood lymphocyte separation solution (Ficoll), take a 50ml centrifuge tube, first add the same amount of separation solution as the blood sample, about 20mL, carefully draw the blood sample with a pipette and slowly spread it on the surface of the separation solution; horizontal centrifuge, 1000g×30min, 9 up and 2 down; after centrifugation, the centrifuge tube is divided into four layers from top to bottom. The first layer is the plasma layer. The second layer is the ring-shaped milky white lymphocyte layer. The third layer is the transparent separation solution layer. The fourth layer is the red blood cell layer; carefully draw the second ring-shaped milky white lymphocyte layer into another 50mL centrifuge tube with a pipette, add 50mL PBS, centrifuge and wash at 400g×5min, resuspend the cells with 50mL PBS, and count them for subsequent experiments.

[0041] T cell activation: According to the instructions of the T Cell TransAct kit from Miltenyi Biotec, magnetic beads-coupled Anti-CD3 / CD28 was added to activate PBMCs, and the cells were cultured with OptiVitro T cell serum-free medium from Excell (Shanghai Jitai Yikesai Biotechnology Co., Ltd.) supplemented with 100 IU / mL IL-2.

[0042] Day 1 Coated with RetroNectin (recombinant human fibronectin)

[0043] CAR gene transduction can be performed 48 hours after T cells are activated. RetroNectin can be coated at 4°C one day in advance, or at 37°C for 2 hours. The corresponding volume of RetroNectin solution is prepared with PBS to coat a 24-well plate. The appropriate volume and concentration are prepared according to the instructions. The edge of the well plate is sealed with Parafilm (sealing film) and coated overnight at 4°C. The RetroNectin solution is recovered and frozen at -20°C and can be used twice.

[0044] Day 2CAR gene transduction

[0045] The virus solution was taken out half an hour in advance, thawed at room temperature, and the 48-hour and 72-hour virus solutions were mixed in a ratio of 1:1; the RetroNectin solution was recovered, 2 mL of virus solution was added to each well, and it was gently added to the wall, the edge was sealed with Parafilm, and centrifuged at 2000g×90 minutes, RT; the activated T cells were collected, counted, and centrifuged at 400g×5 minutes, RT; resuspended with T cell culture medium (containing 100 IU / mL IL-2) to 0.1×106 / mL; after the centrifugation, the virus solution was aspirated and 2 mL of T cells were gently added to each well; centrifuged at 400g×5 minutes, and cultured in a 37°C 5% CO2 incubator.

[0046] Day 5 (Day 3 after transduction) Transduction efficiency detection

[0047] After 72 hours of virus infection, the cells were counted, half of the medium was replaced, and a small amount of cells were taken to detect CAR expression by flow cytometry (Fab antibody to detect scFv, Figure 3 ).

[0048] Example 4 Specific activation and killing of ENO1 CAR-T

[0049] The specific activation of ENO1 CAR-T was detected in cell models, including immortalized microglial cells HCM3, human glioma cells LN299, monocytic leukemia cells THP-1 and human lung cancer cells H460. Flow cytometry was used to detect the expression of ENO1 in the cell models ( Figure 4 ).

[0050] On Day 13 after gene transduction, specific activation and killing detection were performed. Non-Transduced CAR-T (NT, non-transduced group) and ENO1 CAR-T were co-cultured with the above cell models at a ratio of 2:1 for 12 hours, and flow cytometry was used to detect the specific activation phenotype ( Figure 5 ) and killing effect ( Figure 6 ).

[0051] Day 13 after gene transduction, NT and ENO1 CAR-T were co-cultured with HCM3 for 12 hours (48-well plate, E:T = 5 × 10 5 :1×10 5 ), remove the supernatant and perform hematoxylin staining to observe the retention of HCM3 (adherent cells) ( Figure 7 )

[0052] Example 5 ENO1 CAR-T specific cytokine release

[0053] On Day 13 after gene transduction, Non-Transduced CAR-T and ENO1 CAR-T were co-cultured with HCM3 for 12 hours (48-well plate, E:T = 5 × 10 5 :1×10 5) The supernatant was collected and the release of cytokines was detected using Biolegend's LEGENDplex multifactor detection kit Human CD8 / NK Panel (13-plex) with V-bottom Plate (V-bottom). The detected cytokines included 13 types (IL-2, IL-4, IL-10, IL-6, IL-17A, TNF-α, sFas, sFasL, IFN-γ, Granzyme A, Granzyme B, Perforin and Granulysin). Figure 8 ).

[0054] The above-mentioned embodiments merely express the implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. Any technical solution obtained in the form of equivalent replacement or equivalent transformation shall fall within the protection scope of the present invention.

Claims

1. A chimeric antigen receptor targeting human α-enolase, characterized in that: It comprises a scFv domain targeting human α-enolase, a transmembrane region and an intracellular signal transduction region, and the amino acid sequence of the chimeric antigen receptor is SEQUENCE No.

1.

2. The chimeric antigen receptor targeting human α-enolase according to claim 1, characterized in that: The scFv domain against human α-enolase comprises a heavy chain variable region with an amino acid sequence as shown in SEQUENCE No.2, a connecting peptide with an amino acid sequence as shown in SEQUENCE No.3, and a light chain variable region with an amino acid sequence as shown in SEQUENCE No.

4.

3. A nucleotide, characterized in that It encodes the chimeric antigen receptor according to claim 1 or 2.

4. An expression vector, characterized in that: It comprises the nucleotide according to claim 3.

5. A CAR-T cell targeting human α-enolase, characterized in that: It is transduced with the vector according to claim 4.

6. A method for constructing a CAR-T cell targeting human α-enolase, characterized in that: It includes the following steps: (1) constructing a chimeric antigen receptor targeting human α-enolase as described in claim 1; (2) Chimeric antigen receptor transduction of T cells.

7. A composition comprising the CAR-T cell of claim 5 or the CAR-T cell constructed by the construction method of claim 6, and a pharmaceutically acceptable carrier.

8. Use of the cell according to claim 5 in the preparation of a drug for treating a tumor disease.

Images