CCR9-targeted chimeric antigen receptor and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a CCR9-targeted chimeric antigen receptor and application thereof, the chimeric antigen receptor comprises an antigen binding structural domain, a hinge region, a transmembrane structural domain, a co-stimulation structural domain and a signal transduction structural domain which are connected in sequence from N terminal to C terminal; wherein the C end of the signal transduction structural domain is further connected with a CXCR4 chemotactic factor receptor structural domain, and the amino acid sequence of the CXCR4 chemotactic factor receptor structural domain is as shown in SEQ ID NO. 2; the antigen binding structural domain is an anti-CCR9 single-chain variable fragment, and the amino acid sequence of the anti-CCR9 single-chain variable fragment is as shown in SEQ ID NO. 1. The CAR enables immune cells expressing the CAR to obtain stronger tissue chemotactic ability and in-vivo distribution breadth through the energization of CXCR4, and particularly, the ability of the CAR to migrate to bone marrow is remarkably improved, so that the CAR is more beneficial to treatment of acute T lymphocytic leukemia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a chimeric antigen receptor targeting CCR9 and its application. Background Technology

[0002] Acute T-cell lymphoblastic leukemia (T-ALL) is an aggressive leukemia caused by the malignant transformation of immature T cells. Chimeric antigen receptor T-cell (CAR-T) cell therapy has made breakthrough progress in relapsed / refractory acute B-cell lymphoblastic leukemia (R / R B-ALL) and has become a guideline-recommended salvage therapy for R / R B-ALL. However, the application of CAR-T cell therapy in T-ALL still faces many challenges, mainly due to the lack of specific targets on the surface of T-ALL cells. Currently used pan-T cell antigens such as CD5 and CD7 have an expression rate of over 90% in normal T cells and natural killer cells (NK cells), leading to "cannibalism" of CAR-T cells during preparation and after infusion, poor expansion capacity, depletion of normal T cells / NK cells, and consequently, fatal opportunistic infections, seriously threatening patient survival. Therefore, developing novel CAR-T cell products that are highly specific, easy to prepare, and have low toxicity is of great significance for the treatment of T-ALL. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a chimeric antigen receptor targeting CCR9 and its application, overcoming the clinical bottleneck of insufficient bone marrow infiltration in traditional CAR-T therapy.

[0004] The technical solution provided by this invention is as follows:

[0005] This invention provides a chimeric antigen receptor targeting CCR9, wherein the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, a sequentially connected antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signal transduction domain. The signal transduction domain is further connected to a CXCR4 chemokine receptor domain at its C-terminus, and the amino acid sequence of the CXCR4 chemokine receptor domain is shown in SEQ ID NO.2. The antigen-binding domain is an anti-CCR9 single-chain variable fragment, and the amino acid sequence of the anti-CCR9 single-chain variable fragment is shown in SEQ ID NO.1.

[0006] Furthermore, the hinge region is the CD8α hinge region with an amino acid sequence as shown in SEQ ID NO.4; And / or, the transmembrane domain is a CD8α transmembrane domain with an amino acid sequence as shown in SEQ ID NO.5; And / or, the co-stimulatory domain is a 4-1BB co-stimulatory domain with an amino acid sequence as shown in SEQ ID NO.6; And / or, the signal transduction domain is the CD3ζ intracellular signal domain with an amino acid sequence as shown in SEQ ID NO.7.

[0007] Furthermore, the chimeric antigen receptor also includes a CD8α signal peptide and a T2A self-cleaving peptide. The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.3, and the amino acid sequence of the T2A self-cleaving peptide is shown in SEQ ID NO.8. The CXCR4 chemokine receptor domain is linked to the C-terminus of the signal transduction domain via the T2A self-cleaving peptide.

[0008] Furthermore, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.10.

[0009] The present invention also provides a nucleic acid encoding the chimeric antigen receptor targeting CCR9 described above, the nucleotide sequence of which is shown in SEQ ID NO.20.

[0010] The present invention also provides a recombinant expression vector comprising the nucleic acid as described above.

[0011] The present invention also provides an engineered immune effector cell comprising the chimeric antigen receptor described above, the nucleic acid described above, or the recombinant expression vector described above.

[0012] Furthermore, the immune effector cells are selected from T cells, B cells, NK cells, macrophages, dendritic cells, immune effector cells differentiated from induced pluripotent stem cells, or any combination thereof.

[0013] The present invention also provides a pharmaceutical composition comprising the chimeric antigen receptor described above or the engineered immune effector cells described above, and one or more pharmaceutically acceptable excipients and / or carriers.

[0014] The present invention also provides the use of the chimeric antigen receptor described above, the engineered immune effector cells described above, or the pharmaceutical composition described above in the preparation of a medicament for the diagnosis, prevention, and / or treatment of diseases or conditions related to CCR9 expression, wherein the disease or condition related to CCR9 expression is acute T-lymphoblastic leukemia. Beneficial effects

[0015] This invention discloses for the first time a chimeric antigen receptor targeting CCR9. This CAR, through CXCR4 activation, enhances the tissue chemotaxis and in vivo distribution of immune cells expressing this CAR, particularly significantly improving their ability to migrate to the bone marrow, thus making it more beneficial for the treatment of acute T-lymphoblastic leukemia. CC motif chemokine receptor 9 (CCR9) is highly expressed on T-ALL but extremely poorly expressed on normal T cells, possessing the potential as a target for CAR-T therapy. The signaling axis formed by chemokine receptor 4 (CXCR4) and its ligand CXCL12 maintains hematopoietic homeostasis under physiological conditions by precisely regulating the homing process of hematopoietic stem cells. As a G protein-coupled receptor, CXCR4, upon binding to CXCL12, activates multiple downstream signaling pathways such as PI3K / Akt and MAPK / ERK, mediating the directed migration and retention of hematopoietic stem cells in the bone marrow microenvironment. This mechanism not only provides a theoretical basis for hematopoietic stem cell transplantation, but also opens up new directions for the innovation of CAR-T cell therapy strategies. By engineering CAR-T cells to highly express CXCR4, their chemotaxis to bone marrow lesions can be significantly enhanced, thereby overcoming the clinical bottleneck of insufficient bone marrow infiltration in traditional CAR-T therapy.

[0016] In in vitro experiments, CXCR4-empowered anti-CCR9 CAR-T cells exhibited similar immunomodulatory activities to anti-CCR9 CAR-T cells, including in vitro tumor killing and CAR-T activation levels. In in vivo experiments, CXCR4-empowered anti-CCR9 CAR-T cells showed increased chemotaxis towards bone marrow. Through CXCR4 empowerment, anti-CCR9 CAR-T cells gained stronger tissue chemotaxis and wider in vivo distribution, particularly a significantly enhanced ability to migrate to the bone marrow. This indicates that CXCR4-empowered anti-CCR9 CAR-T cells have a more significant therapeutic advantage in treating T-ALL patients and demonstrate stronger clinical translational potential. Attached Figure Description

[0017] Figure 1 These are the expression characteristics of CCR9 on leukocytes in T-ALL patients; among them, A represents the expression characteristics of CCR9 in newly diagnosed and relapsed T-ALL; B and C represent the expression characteristics of CCR9 in one case of T-ALL in newly diagnosed and relapsed T-ALL after induction chemotherapy.

[0018] Figure 2 This describes the expression characteristics of CCR9 in normal lymphocytes and hematopoietic stem and progenitor cells; where A represents the expression of CD4 by flow cytometry. + The expression characteristics of CCR9 on T lymphocytes; B is CD8. +The expression characteristics of CCR9 on T lymphocytes; C stands for CD56. + Expression characteristics of CCR9 on NK cells; D represents CD19. + The expression characteristics of CCR9 on B lymphocytes; E represents CD34. + Expression characteristics of CCR9 on hematopoietic stem and progenitor cells; F represents T-ALL leukemia cells and CD4+. + T, CD8 + T, CD56 + NK cells, B cells, and CD34 + Expression characteristics of CCR9 antigen in hematopoietic stem and progenitor cells.

[0019] Figure 3 This is a characteristic of the expression level of the chemokine CXCL12 protein.

[0020] Figure 4 These are the expression characteristics of CXCR4 in primary leukemia cells and cell lines; where A represents the expression characteristics of CXCR4 in primary leukemia cells from a T-ALL patient; and B to D represent the expression characteristics of CXCR4 in cell lines Jurkat, MOLT-4, and K562.

[0021] Figure 5 These are structural design diagrams of CXCR4, αCCR9 CAR, and CXCR4-enabled αCCR9 CAR.

[0022] Figure 6 These are schematic diagrams of the structures of αCCR9 CAR-T, CXCR4 αCCR9 CAR-T, and CXCR4-T.

[0023] Figure 7 This is a diagram of HEK293T cells 24 hours after plasmid transfection; where A is αCCR9 CAR-T; B is CXCR4 αCCR9 CAR-T; and C is CXCR4-T (Note: Cell status under 100x microscope).

[0024] Figure 8 This is a graph showing the expression of CAR and CXCR4; where A represents the expression ratio of CXCR4 and CAR on day 7 of culture; and B represents the characteristics of CXCR4 expression changes at different time points.

[0025] Figure 9 These are the cell states of CAR-T cells during in vitro culture; A and D show the cell morphology of αCCR9 CAR-T cells under 100x and 200x magnification on day 6 of culture; B and E show the cell morphology of CXCR4 αCCR9 CAR-T cells under 100x and 200x magnification on day 6 of culture; C and F show the cell morphology of CXCR4-T cells under 100x and 200x magnification on day 6 of culture.

[0026] Figure 10 This is an analysis of the killing results of CXCR4 and αCCR9 CAR-T cells.

[0027] Figure 11 These are the activation characteristics of CXCR4 αCCR9 CAR-T cells by target cells; where A represents the CD25 expression characteristics of CXCR4 αCCR9 CAR-T and αCCR9 CAR-T cells after co-incubation with target cells; and B represents the CD69 expression characteristics of CAR-T cells after co-incubation with MOLT-4 target cells.

[0028] Figure 12 The images show the chemotaxis of CXCR4-enabled αCCR9 CAR-T cells observed under a fluorescence microscope (×400x). A represents the chemotaxis of αCCR9 CAR-T cells to CXCL12-free medium, CXCL12-containing medium, MOLT-4 cells, and mesenchymal stem cells; B represents the chemotaxis of CXCR4 αCCR9 CAR-T cells to CXCL12-free medium, CXCL12-containing medium, MOLT-4 cells, and mesenchymal stem cells.

[0029] Figure 13 This is a diagram showing the efficacy of CXCR4 αCCR9 CAR-T in a hematologic malignancy model. A represents the in vivo animal experiment flowchart; B represents the fluorescence in vivo imaging results; C represents the fluorescence intensity comparison curves for each group; and D represents the mouse body weight change curve.

[0030] Figure 14 This is a fluorescence in vivo imaging of CAR-T cells in tumor-free mice; where A is the flowchart of the in vivo animal experiment; B is the mouse in vivo imaging; and C is the percentage of fluorescence intensity in the bilateral thighs of the mouse relative to the total fluorescence intensity of the whole body. Detailed Implementation

[0031] Specific embodiments of the present invention are illustrated in the following examples. It should be understood that these examples are merely illustrative, and the scope of the present invention is not limited thereto. Methods without specific conditions can generally be performed with reference to known methods in the art (e.g., the relevant descriptions in *Molecular Cloning: A Laboratory Manual*) or the conditions recommended by the reagent supplier. Unless otherwise specified, all reagents used in the experiments are conventional reagents in the art and can be purchased commercially.

[0032] Example 1: Feasibility Analysis of Applying CCR9 Target and CXCR4-CXCL12 Pathway to the Treatment of T-ALL

[0033] 1. Detection of CCR9 expression levels in different T-ALL cell lines

[0034] Leukemia cell samples were collected from T-ALL patients. The samples were diluted with 100 μL of PBS and mixed thoroughly. Then, 2 μL of CCR9-APC antibody was added, gently mixed, and stained at 4°C in the dark for 30 minutes. After staining, 1.5 mL of physiological saline was added and mixed thoroughly, then centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in 1.5 mL of physiological saline and centrifuged again (1500 rpm, 5 minutes). Finally, the cell pellet was resuspended in 0.4 mL of PBS and transferred to flow cytometry tubes. CCR9 expression was detected using a Beckman Coulter flow cytometer.

[0035] The study used flow cytometry to detect the expression level of CCR9 in T-ALL primary leukemia cells. Figure 1 The results showed that CCR9 was highly expressed in T-ALL leukemia cells. Figure 1 (A). In newly diagnosed T-ALL patients, the expression level of CCR9 was 48.52%±14.67%. As the disease progressed to the relapsed / refractory stage, the expression level of CCR9 antigen significantly increased to 88.57%±3.5%, which was significantly higher than that of newly diagnosed patients (48.52%±14.67%, P=0.022). This suggests that CCR9 expression is upregulated as the disease progresses. Furthermore, in newly diagnosed T-ALL patients, the proportion of CCR9 expression on residual leukemia cells was significantly higher than before chemotherapy, increasing from 28.48% to 58.6% (A). Figure 1 (B to C). This phenomenon suggests that CCR9 may exist as a marker for T-ALL leukemia stem cells.

[0036] 2. Expression characteristics of CCR9 in normal lymphocytes and hematopoietic stem and progenitor cells

[0037] To further validate the potential of CCR9 as a relatively specific target for T-ALL, this study examined the activity of CCR9 antigen in peripheral blood T lymphocytes, B lymphocytes, NK cells, and CD34 cells from healthy volunteers. + Expression characteristics on hematopoietic stem and progenitor cells. This detection is of great significance for early detection and avoidance of side effects such as cannibalism, severe immunosuppression, and severe myelosuppression that may occur in αCCR9 CAR-T cell therapy.

[0038] Flow cytometry results showed that CCR9 expression levels were extremely low in peripheral blood lymphocytes from healthy individuals, specifically as follows: CD4 + T cells: CCR9 expression rate was 2.73% ± 1.45% ( Figure 2 (A) CD8 + T cells: CCR9 expression rate was 2.87% ± 2.31% ( Figure 2(Chinese B); CD56 + NK cells: CCR9 expression rate was 1.30% ± 0.69% ( Figure 2 (C) CD19 + B lymphocytes: CCR9 expression rate was 0.85% ± 0.44% ( Figure 2 (D); CD34 + Hematopoietic stem and progenitor cells: CCR9 expression rate was only 0.03% ± 0.05% ( Figure 2 (E). Statistical analysis showed that, compared with normal T lymphocytes and CD56... + Compared with NK cells, B lymphocytes, NK lymphocytes, and hematopoietic stem and progenitor cells, the expression level of CCR9 on primary T-ALL leukemia cells was significantly higher (P < 0.001). Figure 2 (F). This result clearly indicates that CCR9 plays a role in normal CD4. + T cells and CD8 + The extremely low expression of CCR9 on T cells means that αCCR9CAR-T cells are highly unlikely to self-kill during treatment, and also have a very low chance of killing normal T lymphocytes. Furthermore, CCR9 is expressed at CD34... + The expression level on hematopoietic stem and progenitor cells is extremely low, indicating that αCCR9 CAR-T cells have no direct killing effect on hematopoietic stem and progenitor cells.

[0039] In summary, the low expression of CCR9 in normal lymphocytes and hematopoietic stem and progenitor cells further confirms its potential as a relatively specific target antigen on the surface of T-ALL leukemia cells. This finding provides important evidence for the safety and efficacy of αCCR9 CAR-T cell therapy and lays the foundation for subsequent clinical applications.

[0040] 3. Detection of CXCL12 expression characteristics in primary T-ALL specimens, target cells, mesenchymal stem cells, and umbilical vein endothelial cells.

[0041] A series of samples were tested using the LEGENDplex™ Human CXCL12 kit. The samples included 72-hour supernatant from in vitro cultured cells (NCT, MOLT-4, hMSC), as well as bone marrow and peripheral blood supernatant from mice and T-ALL patients. After pretreatment by centrifugation at 1000 rpm for 5 minutes, the experimental procedure strictly followed the kit instructions: a standard concentration gradient was prepared, 50 μL of sample or standard was sequentially mixed with capture microspheres and detection antibody, incubated, and washed. Finally, fluorescence intensity was analyzed by flow cytometry, and the absolute concentration of CXCL12 was calculated based on a standard curve.

[0042] Quantitative results such as Figure 3The results showed that, among cell lines, hMSCs secreted the highest concentration of CXCL12 (3289.33±478.40 pg / mL), significantly higher than NCT cells (223.50±19.09 pg / mL, P=0.0414); MOLT-4 cells were in the middle range (1181.79±2.41 pg / mL). In clinical samples, the concentration of CXCL12 in the bone marrow supernatant of T-ALL patients (4717.50±702.12 pg / mL) was significantly higher than that in normal human bone marrow (1999.29±556.87 pg / mL, P=0.0385). Furthermore, the CXCL12 level in mouse bone marrow (2821.83±729.74 pg / mL) was also significantly higher than that in their peripheral blood (650.67±13.27 pg / mL, P=0.0087). These data confirm at the protein level that CXCL12 is highly expressed in the T-ALL microenvironment, providing direct evidence for subsequent CAR-T cell therapy targeting CXCR4 and related preclinical mouse model studies.

[0043] 4. Detection of CXCR4 expression characteristics in T-ALL primary cells and target cells.

[0044] (1) Take 500 μL of bone marrow specimen and mix thoroughly. Take target cells (MOLT-4, Jurkat, K562 cells) and add the following antibodies to the specimen: CD45-FITC 8 μL, CD4-FITC 8 μL, CD8-PE 8 μL, CD3-ECD 8 μL, CD7-A700 3 μL, CXCR4-APC 2 μL. Mix gently and stain at 4°C in the dark for 30 minutes (for target cell lines, only add CXCR4 antibody).

[0045] (2) Add 200 μL of red blood cell lysis buffer, mix gently, and place in a 40°C water bath for 10 minutes to lyse (except for target cell lines).

[0046] (3) Add the stop solution, mix well and let stand at room temperature for 15 minutes.

[0047] (4) Add 1.5 mL of physiological saline, mix well and centrifuge at 1500 rpm for 5 minutes.

[0048] (5) After removing the supernatant, add 1.5 mL of physiological saline, mix well, and centrifuge again at 1500 rpm for 5 minutes.

[0049] (6) Add 0.4 mL of PBS to resuspend the cells and transfer them into a flow cytometer.

[0050] (7) The labeled samples were analyzed by Beckman Coulometer flow cytometry to detect the expression characteristics of CXCR4.

[0051] Results: The mean expression level of CXCR4 in primary T-ALL cells from T-ALL patients was 85.23% ± 17.8%. The expression characteristics of CXCR4 in one T-ALL patient were... Figure 4 (A), expression in Jurkat cells was 25% ( Figure 4 The expression level of B in MOLT-4 cells was 99%. Figure 4 The expression level in C cells was 21.8% in K562 cells. Figure 4 (D).

[0052] In summary, the experimental results show that primary T-ALL patients' leukemia cells and the T-ALL cell line MOLT-4 not only secrete increased levels of CXCL12, but also highly express its receptor CXCR4. This is related to the strong adhesion and chemotaxis of T-ALL cells, which result in high invasiveness (Teicher BA, Fricker SP. CXCL12 (SDF-1) / CXCR4 pathway in cancer. Clin Cancer Res. 2010;16(11):2927-31.). Based on this, CAR-T cells can utilize the properties of chemokines to enhance CAR-T cell migration to the bone marrow, thereby increasing the depth of myeloremission in T-ALL.

[0053] Example 2: Construction of CXCR4-empowered anti-CCR9 CAR-T cells

[0054] 1. Preparation of materials and reagents

[0055] 1.1 Materials

[0056] Cell lines: MOLT-4, Jurkat, Raji-B, and HUVEC were obtained from ATCC, USA; NCG mice were obtained from Shanghai Southern Model Biotechnology.

[0057] 1.2 Reagent Preparation

[0058] (1) Polyethyleneimine (PEI) stock solution (1 mg / mL): prepared using PEI 40000.

[0059] (2) Prepare 50×TAE Buffer, pH 8.3.

[0060] (3) DMEM complete medium: Add 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture to DMEM medium and mix the three thoroughly.

[0061] (4) Target cell 1640 complete medium: Add 10% FBS and 1% penicillin-streptomycin to 1640 basal medium, which is suitable for the culture of MOLT-4, Jurkat, human chronic myeloid leukemia cell line K562 and MOLT-4 luciferase cells.

[0062] (5) Complete CAR-T cell culture medium: serum-free medium (GT-T551 H3)-TAKARA.

[0063] (6) Mesenchymal stem cell culture medium: a mixture of platelet lysis buffer and 10% FBS, suitable for in vitro culture of mesenchymal stem cells.

[0064] (7) CAR-T cell cryopreservation solution: FBS and DMSO are added in a ratio of 9:1 and mixed thoroughly. The prepared cell cryopreservation solution is stored in a 4°C freezer for later use.

[0065] 2. Experimental Procedure

[0066] 2.1 Construction of anti-CCR9 CAR target plasmid and CXCR4-enhanced anti-CCR9 CAR target plasmid

[0067] (1) Design of target gene CAR

[0068] like Figure 5 In this study, a CAR structure targeting the CCR9 antigen was designed. The αCCR9 CAR backbone, from the N-terminus to the C-terminus, sequentially includes a CD8α signal peptide (CD8α Leader), an αCCR9 scFv (anti-CCR9 scFv, a single-chain variable fragment), a CD8α hinge region and transmembrane domains (CD8α Hinge and CD8α TM), a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain. This structure was named αCCR9 4-1BBζ CAR (i.e., αCCR9 CAR, also known as anti-CCR9 CAR). The designed sequence was synthesized, and subsequently, the target plasmid for the anti-CCR9 CAR was constructed.

[0069] The CXCR4 sequence was determined by consulting literature and databases. A single CXCR4 target gene structure was designed and tandemly linked with an αCCR9 CAR. From the amino terminus to the carboxyl terminus, the structure sequentially includes a CD8α signal peptide, an αCCR9 scFv, a CD8α hinge region and transmembrane domain, a 4-1BB co-stimulatory domain, a CD3ζ intracellular signal domain, a T2A self-cleaving peptide, and the CXCR4 sequence. This structure was named CXCR4 αCCR9 CAR (also known as CXCR4-enabled anti-CCR9 CAR). The designed sequence was synthesized to construct the CXCR4 αCCR9 CAR target plasmid.

[0070] (2) Vector digestion

[0071] Prepare the reagents, mix them according to the system in the table below, and incubate in a 37°C water bath until the next day.

[0072] Table 1 Enzyme digestion system

[0073]

[0074] (3) PCR amplification of the target gene and gel electrophoresis recovery of the target DNA fragment

[0075] (4) Transformation of competent cells

[0076] Prepare a reaction solution by mixing 1 μL of the linearized plasmid expression vector (digested by enzymes), 10 μL of the target DNA fragment recovered by gel electrophoresis, and 15 μL of seamless cloning reaction solution at a ratio of 2:1. Incubate the reaction solution in a 40°C water bath for 30 minutes, then transfer it to ice at 4°C for later use.

[0077] Remove competent HEK293T cells and place them on ice at 4°C. After complete thawing, add 10 μL of reaction solution containing the target gene and vector, and mix gently. Continue to incubate the mixture on ice at 4°C for 30 minutes. After the ice bath, heat shock the mixture at 42°C for 90 seconds to promote plasmid transformation, then immediately return it to the ice bath at 4°C for 30 minutes to stabilize the membrane structure.

[0078] (5) Screening and identification of positive clones

[0079] Observe the growth of colonies on the plate, select round, well-defined, medium-sized independent colonies, and inoculate them into 10 μL of bacterial culture medium. Use 1 μL of the mixture as a PCR template to amplify positive clones. After the PCR reaction, add 2 μL of loading buffer to the reaction solution, and load 10 μL of the product into the corresponding wells of a 1.5% agarose gel for electrophoresis. Extract the bands corresponding to the expected positions. Collect the clones corresponding to the band positions and continue culturing and amplifying. Extract plasmids and sequence the DNA. Import the sequenced base sequences into SnapGene software and use its alignment function to compare and analyze them with the target sequence to determine if the inserted fragment is correct and if there are any mutations or deletions, thus confirming the accuracy of the clone construction.

[0080] Results: αCCR9 CAR-T was constructed, as shown in the schematic diagram. Figure 6 As shown in Figure A, an αCCR9 CAR-T cell receptor mediated by the chemokine receptor CXCR4 (i.e., CXCR4 αCCR9 CAR-T) was constructed to enhance the migration ability of CAR-T cells. A schematic diagram of the structure of the CXCR4-receptor αCCR9 CAR is shown below. Figure 6As shown in B.

[0081] Simultaneously, CXCR4-T cells (chemokine receptor CXCR4-T) were constructed, increasing the migration and chemotactic abilities of T cells. This served as a control for CXCR4 αCCR9 CAR-T cells, demonstrating whether the addition of CXCR4 affected the cytotoxic effects of T cells. (Structural diagram follows) Figure 6 (C)

[0082] 2.2 Packaging of Lentiviral Vectors

[0083] (1) Small-scale plasmid extraction

[0084] Follow the instructions in the plasmid extraction kit manual.

[0085] (2) HEK293T cell resuscitation and passage

[0086] Remove one vial of cryopreserved HEK293T cells from the liquid nitrogen container and place it in a 37°C water bath to thaw the cells. Use a pipette to transfer the liquid from the cryopreservation tube to a 15 mL sterile centrifuge tube containing 10 mL of PBS, mix well by pipetting, and centrifuge at 1000 rpm for 5 minutes, discarding the supernatant. Add an appropriate amount of DMEM complete medium to the centrifuge tube to adjust the cell concentration, and seed the cells in 10 cm culture dishes. Observe the HEK293T cell status under a microscope to ensure uniform cell distribution. Incubate the cells in a 37°C, 5% CO2 incubator.

[0087] (3) Transfection of HEK293T packaging cells

[0088] When HEK293T cells reached 80% confluence, they were passaged. HEK293T cells in logarithmic growth phase were harvested, centrifuged to remove the supernatant, and resuspended in fresh DMEM complete medium. Simultaneously, 1 µg / μL of PEI transfection reagent, packaging plasmid, and target plasmid were prepared. The target plasmid, pPac-GP, pPac-R, and pEnv-G plasmids were mixed thoroughly at a ratio of 20:10:8:6. Then, PEI was slowly added dropwise to the plasmid mixture at a 1:3 mass ratio, gently shaking the EP tube while adding the PEI to ensure thorough mixing. The mixture was incubated at room temperature for 20 minutes to form the PEI-DNA complex.

[0089] The complex was evenly added dropwise to fresh culture medium containing HEK293T cells for transfection. Approximately 5 hours later, the original culture medium was discarded and replaced with fresh DMEM complete medium. Culture was continued to collect the viral supernatant. One day post-transfection, cell status and density were observed under a microscope, and appropriate fluid replenishment was performed. The cells were then placed in a cell culture incubator for further culture.

[0090] (4) Collection and concentration of lentiviral vectors

[0091] Two days after HEK293T cell transfection, all culture supernatant was collected and centrifuged at 500 rpm for 10 minutes at 4°C. The resulting lentiviral vector harvest solutions were filtered through a 0.22 μM PES syringe filter and centrifuged at 25,000 rpm for 2 hours at 4°C. The supernatant was discarded, and 200 μL of serum-free Opti-MEM was added to the centrifuge tube. After mixing by pipetting, the mixture was incubated at 4°C for 2 hours to dissolve. The concentrated viral supernatant was then aliquoted and stored at -80°C for later use.

[0092] (5) Virus titer determination

[0093] Activation time was 24 to 48 hours. The T cell culture flasks were removed from the incubator, and 0.5 mL of cells were collected and counted using trypan blue staining. Based on the count results, the activated T cells were divided into three equal parts. One part served as a negative control T cell, i.e., untransfected T cells (NCT), for further expansion and culture. The other two parts were used for lentiviral infection and gene transduction. The required viral load was calculated based on the total cell count. The viral titer (Integration Units per mL, IU / mL) was calculated using the following formula based on the PCR results:

[0094] Note: : The average number of viral copies integrated into the genome of each cell; Virus dilution factor; The total number of cells at the time of infection; : Volume of virus added (μL).

[0095] result:

[0096] The target plasmid was co-transfected into HEK293T cells with packaging plasmids pPac-GP and pPac-R, and envelope plasmid pEnv-G, respectively, for lentivirus production. The status of HEK293T cells was observed 24 hours after transfection as follows: Figure 7 Figures A through C show shortened cell processes, rounded cell shape, and some cells showing fusion, indicating successful transfection and entry into the viral shedding phase. The final viral vector concentrate was quantified using a functional titer method, and the relevant titer results are shown in Table 2.

[0097] Table 2 Lentiviral titers

[0098]

[0099] 2.3 Preparation and Identification of CXCR4-Enabled Anti-CCR9 CAR-T Cells

[0100] (1) Isolation of peripheral blood mononuclear cells (PBMCs) from healthy donors

[0101] This study was approved through ethical review, and informed consent was obtained from three healthy volunteers before peripheral blood samples (50 mL each) were collected. Peripheral blood cells (PBMCs) were isolated using density gradient centrifugation: EDTA-anticoagulated whole blood was first mixed with an equal volume of PBS, then slowly added to Ficoll separation buffer, and centrifuged at 800g for 30 minutes. Interfacial white membrane cells were collected, washed twice with physiological saline (450 rpm × 5 minutes), resuspended, and counted after trypan blue staining, ultimately yielding approximately 2 × 10⁶ cells. 8 One PBMC is available for backup.

[0102] (2) CD3 + T cell sorting and activation

[0103] CD3 was isolated from PBMCs using an immunomagnetic bead positive sorting method. + T cells. The cell suspension was incubated with CD3 magnetic beads at 4°C in the dark for 15 minutes, followed by collection of CD3 using a magnetic sorting column. + T cells. The sorted cells were resuspended in T cell-specific culture medium and the density was adjusted to 3 × 10⁶ cells / day. 6 / mL, inoculated into culture flasks pre-coated with anti-CD3 / CD28 antibody, and IL-2 (final concentration 1000 IU / mL) was added. The culture was activated at 37℃ and 5% CO2 for 48 hours.

[0104] (3) Lentiviral transduction and CAR-T cell expansion

[0105] Forty-eight hours after T cell activation, T cells were transduced with CXCR4-empowered anti-CCR9 CAR lentivirus, anti-CCR9 CAR lentivirus, and CXCR4 lentivirus at different multiplicity of infection (MOIs) (50, 80, 120). Untransduced T cells (NCT) served as a control. After transduction, the cell density was adjusted to 5 × 10⁶ cells / year. 5 / mL, and cultured continuously for 10-12 days, during which the culture medium is replenished regularly and the cell status and proliferation are monitored.

[0106] (4) Harvesting and preservation of CAR-T cells

[0107] On day 12 of culture, CAR-T cells were collected, washed by centrifugation at 1500 rpm for 5 minutes, resuspended in cryopreservation buffer, and cultured at 1×10⁻⁶ cells / day. 7 Cells are aliquoted and stored in liquid nitrogen for long-term preservation.

[0108] result:

[0109] Flow cytometry results showed that CXCR4 αCCR9 CAR-T cells, αCCR9 CAR-T cells, and CXCR4-T cells were successfully prepared in this study. Statistical analysis showed that the average infection efficiency of αCCR9 CAR-T cells (45.31% ± 8.43%) was not significantly different from that of CXCR4 αCCR9 CAR-T cells (38.15% ± 1.24%) (P = 0.312), nor was it significantly different from that of CXCR4-T cells (46.93% ± 6.93%) (P = 0.179). Figure 8 (A). Compared with the CXCR4-T cell control group, CXCR4 αCCR9 CAR-T cells maintained stable CXCR4 levels during expansion culture. Figure 8 (B) CXCR4 expression remained at 42.45% ± 5.44% without downregulation as CXCR4 αCCR9 CAR-T cells expanded.

[0110] Example 3: In vitro functional validation of CXCR4-enhanced anti-CCR9 CAR-T cells.

[0111] 1. Evaluation of LDH killing effect in CXCR4-enhanced anti-CCR9 CAR-T cells

[0112] Take 5×10 6 Effector cells NCT, anti-CCR9 CAR-T cells, CXCR4-empowered anti-CCR9 CAR-T cells, and CXCR4-T cells (overexpressing CXCR4 protein on a T cell basis, (CXCR4 NCBI Gene ID: 7852)). Approximately 3 × 10⁻⁶ cells were collected from each cell type. 5 MOLT-4 cells (strongly CCR9 positive), Jurkat cells (weakly CCR9 positive), and K562 cells (CCR9 negative) were used as target cells. Effector cells from different treatment groups were loaded into labeled EP tubes and washed twice. Cells were stained with trypan blue and then counted. Effector cells were adjusted to the desired density and added to labeled 96-well plates.

[0113] Effector cells were divided into different effector-to-target ratio groups (E:T = 10:1, 5:1, 2.5:1, 1:1), with positive control, negative control, and experimental groups in each group. Target cells were divided into control group, maximum lysis control group, and experimental group. Each condition was configured in triplicate. 1640 complete medium containing 4% fetal bovine serum was added to each well to a total volume of 100 μL. The cells were incubated at 37°C with 5% CO2 for 24 hours. Cell lysis buffer was then added to each well of the maximum lysis group, and incubation continued for 1 hour to obtain maximum LDH release. After incubation, the 96-well plate was centrifuged at 250g for 5 minutes at room temperature, and 50 μL of supernatant from each well was collected and transferred to a new ELISA plate. An equal volume of LDH substrate was added, and the plate was incubated at room temperature in the dark for 30 minutes.

[0114] The OD value of each well was measured using a full-wavelength microplate reader, and the average value was taken. The killing efficiency of CAR-T cells in each group was calculated according to the formula to evaluate the killing ability.

[0115] result:

[0116] LDH release experiment results showed that ( Figure 10Both CXCR4 and αCCR9 CAR-T cells exhibited significant killing activity against CCR9-positive target cells. Specifically, the killing efficiencies of the two CAR-T cell types against Jurkat and MOLT-4 cells were as follows: the killing efficiencies of αCCR9 CAR-T cells against Jurkat cells at effector-to-target ratios of 5:1, 2.5:1, and 1:1 were 35.94%±1.79%, 40.56%±3.64%, and 32.88%±8.94%, respectively. The killing efficiencies of αCCR9 CAR-T cells against MOLT-4 cells at effector-to-target ratios of 5:1, 2.5:1, and 1:1 were 22.65%±3.64%, 24.84%±8.94%, and 32.52%±1.93%, respectively. The killing efficiencies of CXCR4 αCCR9 CAR-T cells against Jurkat cells at effector-to-target ratios of 5:1, 2.5:1, and 1:1 were 29.83%±7.70%, 41.42%±1.82%, and 35.83%±9.24%, respectively. The killing efficiencies of CXCR4 αCCR9 CAR-T cells against MOLT-4 cells at the same ratios were 43.92%±1.82%, 41.43%±9.24%, and 33.38%±2.19%, respectively. LDH analysis indicated no significant difference in the killing efficacy of the two CAR-T cell types against Jurkat and MOLT-4 cells (P>0.05), suggesting that the introduction of CXCR4 did not weaken the killing ability of αCCR9 CAR-T cells against CCR9-positive target cells. It is noteworthy that even under the low target-to-cell ratio of 1:1, both types of CAR-T cells maintained high killing activity and showed no significant killing effect on CCR9-negative K562 cells, confirming the specific recognition ability of CAR-T cells for the CCR9 antigen.

[0117] 2. Activation Capability Assessment

[0118] Sufficient effector cells were collected: NCT cells, anti-CCR9 CAR-T cells, CXCR4-empowered anti-CCR9 CAR-T cells, and CXCR4-T cells; target cells: MOLT-4, Jurkat, and K562. Cells were co-cultured at a 5:1 effector-target ratio for 24 hours. Flow cytometry was used to detect the expression levels of CD25 and CD69 in effector cells before and after co-incubation. Blank represents baseline data. Cells were collected 24 hours after co-incubation to detect changes in the expression of the above activation markers. By comparing the expression levels of activation markers in effector cells at different time points, the immune activation capacity of CAR-T cells and their recognition and response intensity to different target cells were evaluated.

[0119] Collect cells from each group into centrifuge tubes. Centrifuge at 1500 rpm for 5 minutes and discard the supernatant. Wash twice. Resuspend the cell pellet in 100 μL PBS in each tube, then add 1 μL each of CD3, CD25, CD69, and CCR9-CAR antibodies, and incubate at room temperature in the dark for 30 minutes. Wash the cells twice with an appropriate amount of PBS and centrifuge. Resuspend the cell pellet in 200 μL PBS, gently pipette to mix, and then detect the expression of CD25 and CD69 in CD3+CAR+ cells using flow cytometry to characterize the activation effect of target cells on CAR-T cells.

[0120] result:

[0121] This study aimed to investigate the activation status of CXCR4 αCCR9 CAR-T cells after co-culturing with target cells. Flow cytometry was used to detect the expression levels of CD25 and CD69 in CAR-T cells 24 hours after co-culturing with target cells to assess whether T cells were activated by the target cells.

[0122] The results show that ( Figure 11 After co-culturing with different target cells, the expression levels of CD25 and CD69 in αCCR9 CAR-T cells showed significant differences. Specifically, in the αCCR9 CAR-T cell activation assay, the CD25 positivity rate was 25.57%±8.52% in the Blank group (blank group), 27.82%±9.13% in the K562 group, 27.75%±6.54% in the Jurkat group, and 43.63%±8.51% in the MOLT-4 group. Statistical analysis showed that compared with the Jurkat and K562 groups, the CD25 expression level of αCCR9 CAR-T cells activated by MOLT-4 cells was significantly upregulated (P=0.039 and P=0.002), and compared with the Blank group (blank group), P<0.001, indicating that MOLT-4 cells have a significant activating effect on αCCR9 CAR-T cells.

[0123] Further analysis of the activation characteristics of CXCR4 αCCR9 CAR-T cells revealed that after co-culturing with MOLT-4 cells, CD25 expression was upregulated by 41.60% ± 7.89%. Compared with the Jurkat group, K562 group, and Blank group (blank group), the P values ​​were 0.002, 0.004, and <0.001, respectively, indicating that CXCR4 αCCR9 CAR-T cells can be significantly activated by MOLT-4 cells. Similarly, when αCCR9 CAR-T cells and CXCR4 αCCR9 CAR-T cells were activated by MOLT-4 cells, the CD69 positivity rates were 23.64% ± 5.47% and 22.42% ± 3.12%, respectively, showing a significant upward trend compared with the Blank group (blank group) (P < 0.001). =0.047, P = 0.048).

[0124] In summary, the experimental results indicate that CXCR4 αCCR9 CAR-T cells and αCCR9 CAR-T cells have similar activation characteristics and can both be significantly activated by the target cell MOLT-4.

[0125] Example 4: Detection of CXCR4-enhanced anti-CCR9 CAR-T chemotactic migration ability

[0126] First, CXCL12 was diluted to a concentration of 200 ng / mL for later use, and 2% FBS TAKARA medium was prepared for diluting the effector cells.

[0127] 1. Divide the effector cells into two groups: all effector cells were pre-stained with CFSE.

[0128] 2. Count the effector cells (αCCR9 CAR-T and CXCR4 αCCR9 CAR-T) and adjust the concentration to 5 × 10⁻⁶. 6 / mL.

[0129] 3. Add 600 μL of blank culture medium, 600 μL of culture medium containing CXCL12, hMSC culture supernatant, normal human bone marrow supernatant, T-ALL patient bone marrow supernatant and mouse bone marrow supernatant to the lower chamber respectively.

[0130] 4. Effector cells are added to the upper chamber.

[0131] 5. Incubate in an incubator (37℃, 5% CO2) for 4 hours.

[0132] 6. Take photos using a fluorescence microscope, capturing images under both white and green light.

[0133] result:

[0134] This study used Transwell assays to evaluate the chemotactic capacity of CXCR4 αCCR9 CAR-T cells and αCCR9 CAR-T cells. In the experiment, the two types of CAR-T cells were co-incubated with CXCL12, MOLT-4, and hMSC cells, respectively, and cell migration was observed under a light microscope to analyze the chemotactic capacity.

[0135] Fluorescence microscopy revealed that CXCR4 αCCR9 CAR-T cells exhibited significantly higher chemotactic activity towards CXCL12, MOLT-4, and mesenchymal stem cells compared to αCCR9 CAR-T cells. Figure 13 ).

[0136] Example 5: Validation of in vivo anti-leukemia activity in a T-ALL mouse model

[0137] 1. Efficacy evaluation of CXCR4 αCCR9 CAR-T cell anti-T-ALL hematologic malignancy model

[0138] 1.1 Twenty female SPF-grade NCG mice aged 6-8 weeks were used. All mice underwent acclimatization for 3-5 days before the experiment, and their numbers and weights were recorded.

[0139] 1.2 Modeling: Mice were fixed in a mouse restraint device, and their tails were disinfected with 75% alcohol. A thoroughly mixed MOLT-4 luciferase cell suspension (concentration 3×10⁻⁶) was drawn. 7 100 μL ( / mL) was slowly injected via the tail vein at the distal 1 / 3 of the tail.

[0140] After successful tumor implantation was confirmed by fluorescence imaging, patients were randomly assigned to groups based on the imaging results to ensure a balanced tumor burden in each group. Treatment was then administered via tail vein injection of different types of effector cells (NCT, αCCR9 CAR-T, CXCR4 αCCR9 CAR-T, and CXCR4-T) according to the group (ensuring consistent procedure).

[0141] 1.3 Treatment Plan

[0142] Mice were divided into four groups: NCT cell group; αCCR9 CAR-T group; CXCR4 αCCR9 CAR-T group; and CXCR4-T group. Five mice were in each group, and each mouse received 100 μL of effector cells, for a total of 1 × 10⁻⁶ cells. 7 One effector cell.

[0143] Each group returns 1×10 7 Effector T cells (cell concentration 1×10⁻⁶) 8 ( / mL) CAR-T cells were injected into each group of effector cells via the tail vein. The day of CAR-T cell infusion was defined as D0. The mice's mental state, body temperature, weight changes, and food intake were observed daily. A comfortable and clean housing environment was provided to avoid unexpected deaths.

[0144] 1.4 Monitoring and Detection

[0145] 1) Weight monitoring: The first time on the day of reinfusion and once a week thereafter;

[0146] 2) In vivo imaging: Reinfusion twice in the first week and once a week thereafter;

[0147] If mice exhibit abnormal behavior during the experiment, such as significant weight loss or lethargy, this study will follow animal welfare principles and implement humane treatment (euthanasia) as appropriate to minimize animal suffering.

[0148] result:

[0149] To further investigate the therapeutic effect of CXCR4 αCCR9 CAR-T cells in the bone marrow of a T-ALL mouse model, this study established a T-ALL leukemia model via tail vein injection. On day 3, each mouse was injected via tail vein with 3 × 10⁻⁶ CAR-T cells. 6 One MOLT-4-Luciferase cell was used, and in vivo imaging indicated successful modeling.

[0150] The experimental procedure is as follows Figure 13 As shown in Figure A. Experimental results showed that the fluorescence signal in mice in the CXCR4-T and NCT groups continuously increased over time, indicating a continuous increase in leukemia burden. From day 15 onwards, mice in both groups died successively, suggesting that these two groups of cells did not produce a significant therapeutic effect. Figure 13 (B) In contrast, the fluorescence signals of mice in both the CXCR4 αCCR9 CAR-T group and the αCCR9 CAR-T group showed a decreasing trend from day 11, and this decreasing trend remained stable, indicating a significant in vivo anti-leukemia effect. However, there was no significant difference in fluorescence intensity between the two CAR-T cell treatment groups (P=0.394), indicating that the two types of CAR-T cells were comparable in their anti-leukemia efficacy. Figure 13 (C). By day 15, the fluorescent signals in the mice of the two CAR-T treatment groups had basically disappeared, indicating that the leukemia burden was significantly reduced, or even possibly completely eliminated.

[0151] In addition, the body weight of mice in each group was monitored in this study. The results showed that the body weight of mice in all four groups did not decrease significantly, and there was no statistically significant difference between the groups (P=0.227). Figure 13 (D). During the treatment, no significant changes were observed in physiological indicators such as body temperature and food intake in the mice of each group, indicating that CXCR4 αCCR9 CAR-T cells have good safety and tolerability in vivo.

[0152] In conclusion, both CXCR4 αCCR9 CAR-T cells and αCCR9 CAR-T cells demonstrated significant and rapid anti-leukemia efficacy in a mouse model of T-ALL hematologic malignancies with tail vein tumor formation, and no significant adverse reactions occurred during treatment, suggesting that they have good safety profiles.

[0153] Example 6: Dynamic distribution of CAR-T cells in vivo

[0154] Staining CAR-T cell membranes with near-infrared fluorescent probes (DIR)

[0155] 1. Preparation of staining solution

[0156] (1) Preparation of storage solution: Dissolve 5 mg DIR in 1 mL of anhydrous DMSO to prepare a 5 mM storage solution.

[0157] (2) Preparation of working solution: 40 μL of stock solution is diluted with 40 mL of PBS to make working solution.

[0158] 2. CAR-T cell staining

[0159] (1) 3×10 of αCCR9 CAR-T and CXCR4 αCCR9 CAR-T were taken. 7 Cells, centrifuged, supernatant discarded.

[0160] (2) Take 30 mL of working solution to resuspend the cells 3×10 7 Each cell.

[0161] (3) Incubate the cells at 37°C in a 5% CO2 incubator for 20 minutes.

[0162] (4) Place 60 mL of complete cell culture medium TAKARA in an incubator for 20 minutes to wash the DIR cells that have not bound to CAR-T cells.

[0163] (5) After incubating the cells for 20 minutes, centrifuge at 1000-1500 rpm for 5 minutes. Discard the supernatant and resuspend the cells in 37°C TAKAR.

[0164] (6) Repeat step (3) more than twice (you can wash it once).

[0165] (7) Resuspend the cells in 600 μL PBS. For later use.

[0166] 3. DIR staining detection

[0167] CAR-T cells can be imaged using fluorescence microscopy or analyzed by flow cytometry to verify whether DIR has successfully stained CAR-T cells.

[0168] 4. Dynamic detection of CAR-T cells in tumor-free mouse models

[0169] 1) Grouping: 3 mice in the NCT group; 5 mice in the αCCR9 CAR-T group; 5 mice in the CXCR4 αCCR9 CAR-T group; 5 mice in the CXCR4-T group.

[0170] 2) Monitoring and Detection

[0171] (1) Weight monitoring: once on the day of reinfusion and once a week thereafter;

[0172] (2) Blood sampling from the inner canthus: the first time on the day of infusion and twice a week thereafter; after the experiment, the mice were sacrificed and organs were taken for flow cytometry analysis of CAR-T cell distribution.

[0173] (3) Near-infrared in vivo imaging: The distribution of CAR-T cells in vivo was observed during the first infusion on the day of infusion and twice a week thereafter.

[0174] Results: To investigate the migration and homing ability of CAR-T cells, CAR-T cells labeled with the near-infrared fluorescent dye DIR were injected into tumor-free NCG mice via the tail vein, and the dynamic distribution of cells in different organs was tracked in real time using an in vivo fluorescence imaging system. Figure 14 The test procedure is as follows: Figure 14 As shown, in vivo imaging results revealed that, compared with the NCT, CXCR4-T, and αCCR9CAR-T groups, CXCR4 and αCCR9 CAR-T cells had a significantly wider distribution range in tumor-free mice. Figure 14 (B). Further comparison of fluorescence intensity in the mouse trunk, brain, and legs revealed that the fluorescence intensity ratio in the bilateral thighs of the CXCR4 αCCR9 CAR-T cell group was significantly higher than that in the NCT and αCCR9 CAR-T groups. Figure 14 (C). Four hours after CAR-T infusion and at D1, D3, D6, and D9, the proportion of fluorescence intensity in both thighs of the CXCR4 αCCR9 CAR-T cell group was significantly higher than that of the NCT group, with P values ​​of 0.0315, <0.0001, <0.0001, <0.0001, and 0.0365, respectively. At D1, D3, and D6 after CAR-T infusion, the αCCR9 CAR-T cell group showed a significantly higher fluorescence intensity than the NCT group, with P values ​​of 0.0054, 0.0140, and 0.0015, respectively. Furthermore, at D3 and D6 after CAR-T infusion, the proportion of fluorescence intensity in both thighs of the CXCR4 αCCR9 CAR-T cell group was higher than that of the αCCR9 CAR-T cell group, with P values ​​of 0.0406 and 0.0439, respectively. In summary, this suggests that the distribution of CXCR4 αCCR9 CAR-T cells and αCCR9 CAR-T cells in the bone marrow is increased, and the chemotactic ability of CXCR4 αCCR9 CAR-T cells is more significant.

[0175] The embodiments described herein are intended to illustrate the technical solutions of the present invention through specific examples, and their purpose is to assist in understanding the core concept of the present invention, rather than to exhaustively describe all implementation methods. Those skilled in the art, after grasping the essence of the present invention, can deduce various variations in its specific applications, and these variations should all be included within the necessary scope defined by the claims. Therefore, the content of the embodiments in this specification should not be construed as a limitation of the present invention.

Claims

1. A chimeric antigen receptor targeting CCR9, characterized in that, The chimeric antigen receptor comprises, from the N-terminus to the C-terminus, an antigen-binding domain, a hinge region, a transmembrane domain, a co-stimulatory domain, and a signal transduction domain connected in sequence. The signal transduction domain is further connected to a CXCR4 chemokine receptor domain at its C-terminus, and the amino acid sequence of the CXCR4 chemokine receptor domain is shown in SEQ ID NO.

2. The antigen-binding domain is an anti-CCR9 single-chain variable fragment, and the amino acid sequence of the anti-CCR9 single-chain variable fragment is shown in SEQ ID NO.

1.

2. The chimeric antigen receptor targeting CCR9 according to claim 1, characterized in that, The hinge region is the CD8α hinge region with an amino acid sequence as shown in SEQ ID NO.4; And / or, the transmembrane domain is a CD8α transmembrane domain with an amino acid sequence as shown in SEQ ID NO.5; And / or, the co-stimulatory domain is a 4-1BB co-stimulatory domain with an amino acid sequence as shown in SEQ ID NO.6; And / or, the signal transduction domain is the CD3ζ intracellular signal domain with an amino acid sequence as shown in SEQ ID NO.

7.

3. The chimeric antigen receptor targeting CCR9 according to claim 1, characterized in that, The chimeric antigen receptor further includes a CD8α signal peptide and a T2A self-cleaving peptide. The amino acid sequence of the CD8α signal peptide is shown in SEQ ID NO.3, and the amino acid sequence of the T2A self-cleaving peptide is shown in SEQ ID NO.

8. The CXCR4 chemokine receptor domain is linked to the C-terminus of the signal transduction domain via the T2A self-cleaving peptide.

4. The chimeric antigen receptor targeting CCR9 according to claim 1, characterized in that, The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.

10.

5. A nucleic acid encoding a chimeric antigen receptor targeting CCR9 as described in claim 1, wherein the nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

20.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 5.

7. An engineered immune effector cell, characterized in that, It comprises the chimeric antigen receptor as described in any one of claims 1-4, the nucleic acid as described in claim 5, or the recombinant expression vector as described in claim 6.

8. The engineered immune effector cells according to claim 7, characterized in that, The immune effector cells are selected from T cells, B cells, NK cells, macrophages, dendritic cells, immune effector cells differentiated from induced pluripotent stem cells, or any combination thereof.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a chimeric antigen receptor as described in any one of claims 1-4 or an engineered immune effector cell as described in any one of claims 7-8, and one or more pharmaceutically acceptable excipients and / or carriers.

10. The use of a chimeric antigen receptor as described in any one of claims 1-4, or an engineered immune effector cell as described in any one of claims 7-8, or a pharmaceutical composition as described in claim 9, in the preparation of a medicament for the diagnosis, prevention, and / or treatment of diseases or conditions associated with CCR9 expression, characterized in that, The disease or condition associated with CCR9 expression is acute T-lymphoblastic leukemia.

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