Novel anti-CCR9 CAR, immune cell and preparation method and application of novel anti-CCR9 CAR

Abstract

The invention discloses a novel anti-CCR9 CAR. A nucleotide sequence of the novel anti-CCR9 CAR sequentially comprises nucleotide sequences of a coding anti-CCR9 scFv, a hinge and transmembrane domain, a costimulatory domain, a CD3 zeta intracellular signal domain and CXCR4 from a 5'end to a 3 'end. The invention also relates to an immune cell with the CAR and related application. The CAR is energized by CXCR4, so that immune cells with the CAR have stronger tissue chemotactic ability and in-vivo distribution range, and particularly, the ability of migration to marrow and extramedullary leukemia lesions is remarkably improved, thereby being more beneficial to treatment of acute T lymphocytic leukemia combined with extramedullary diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to a chimeric antigen receptor (CAR), in particular to a new anti-CCR9 CAR, immune cells and preparation method and application thereof. BACKGROUND

[0002] Acute T lymphoblastic leukemia (T-ALL) is derived from the malignant transformation of early T lineage progenitor cells, accounting for about 25% of adult acute lymphoblastic leukemia. Although the remission rate can reach 70%-90%, about 50% of adult patients eventually develop primary drug resistance or hematological relapse, and progress to relapsed / refractory T-ALL (R / R T-ALL). The complete remission (CR) rate of R / R T-ALL re-induction is only 20%-30%, and the five-year overall survival rate is less than 20%, with a very poor prognosis. Chimeric antigen receptor T-cell (CAR-T) therapy has achieved a breakthrough effect in R / R B-ALL and has been listed as a standard salvage treatment in international guidelines; however, the clinical application of CAR-T in T-ALL still faces significant obstacles. The main bottleneck is the lack of tumor-restricted surface antigens: the commonly used target points such as CD5 and CD7 are pan-T antigens, which are synchronously highly expressed in normal T cells and >90% of natural killer (NK) cells. Off-target toxicity causes long-term depletion of T / NK cells, induces fatal opportunistic infections, and significantly increases the treatment-related mortality rate. Therefore, it is urgent to develop a new type of CAR-T product with relative specificity, simple preparation and controllable toxicity.

[0003] C-C motif chemokine receptor 9 (CCR9) is universally highly expressed in T-ALL blast cells, while almost absent in mature peripheral T cells and NK cells, with an ideal tumor-normal distinction. Taking advantage of its differential expression profile, it avoids fratricide and off-target toxicity caused by pan-T antigens, and provides a precise and easily clinically transformed immunotherapy strategy for T-ALL.

[0004] In addition, more than 50% of T-ALL patients have extramedullary disease (EMD) in the central nervous system, mediastinum or liver and spleen at the time of initial diagnosis. Patients with EMD have high tumor burden and strong invasiveness, and have a significantly increased risk of early relapse. Although the CR rate of CD7-CAR-T in treating bone marrow lesions can reach more than 90%, the CR rate of EMD lesions is only about 50%, and more than half of the patients relapse locally within 6-12 months, indicating that CAR-T has insufficient efficacy in clearing residual leukemia in the extramedullary space. Chemokine receptor 4 (C-X-C motif chemokine receptor 4, CXCR4) and its ligand CXCL12 not only regulate the homing of hematopoietic stem cells, but also mediate the extramedullary infiltration of T-ALL cells. Traditional CAR-T lacks CXCR4, has low bone marrow niche retention rate, and limited ability to migrate to EMD lesions, resulting in short treatment response. SUMMARY

[0005] In a first aspect, in order to solve or improve the problem of short EMD treatment effect, low CR rate and high relapse rate, the present application provides a new anti-CCR9 CAR. The technical scheme is that the nucleic acid sequence of the new anti-CCR9 CAR comprises, from 5' end to 3' end, nucleic acid sequences encoding anti-CCR9 scFv, hinge and transmembrane domain, costimulatory domain, CD3ζ intracellular signaling domain and CXCR4.

[0006] In some embodiments, the nucleic acid sequence of the new anti-CCR9 CAR further comprises nucleic acid sequences encoding 5'LTR, EF1α, CD8a leader peptide and 3'LTR, and from 5' end to 3' end, the nucleic acid sequences encoding 5'LTR, EF1α, CD8a leader peptide, anti-CCR9 scFv, CD8a hinge and transmembrane domain, costimulatory domain, CD3ζ intracellular signaling domain, CXCR4 and 3'LTR.

[0007] In some embodiments, the amino acid sequences of the light chain variable region (VL) and the heavy chain variable region (VH) of the anti-CCR9 scFv are respectively shown in SEQ ID NO: 1 and 2 (corresponding to the VL and VH of AFA64), or respectively shown in SEQ ID NO: 3 and 4 (corresponding to the VL and VH of AFF84); the amino acid sequence of CXCR4 is shown in SEQ ID NO: 5.

[0008] In some embodiments, the hinge is a CD8a hinge; the CXCR4 is connected by T2A.

[0009] In some embodiments, the amino acid sequence of the new anti-CCR9 CAR is shown in SEQ ID NO: 6 or 7.

[0010] In some implementations, the nucleic acid sequence of the novel anti-CCR9 CAR is shown in SEQ ID NO: 8 or 9.

[0011] Secondly, the present invention also discloses a vector having a nucleic acid sequence encoding a novel anti-CCR9CAR as described in the first aspect (e.g., a vector for gene delivery / cloning), or having a novel anti-CCR9CAR amino acid sequence as described in the first aspect (e.g., a vector for targeted drug delivery / antibody conjugation).

[0012] Thirdly, the present invention also discloses a host cell comprising one or more of the following: a nucleic acid sequence encoding the novel anti-CCR9 CAR as described in the first aspect, an amino acid sequence of the novel anti-CCR9 CAR as described in the first aspect, and a vector as described in the second aspect.

[0013] Fourthly, the present invention also discloses an immune cell carrying the novel anti-CCR9 CAR as described in the first aspect.

[0014] In some embodiments, the immune cell is one or more of CAR-T cells, CAR-NK cells, CAR-DC cells, CAR-B cells, CAR-monocytes, and CAR-M cells. CAR-T cells are preferred.

[0015] In some implementations, the immune cells encompass those generated in vivo or in vitro.

[0016] In some implementations, the immune cells are CXCR4-empowered anti-CCR9 CAR-T.

[0017] Fifthly, the present invention also discloses a method for preparing immune cells as described in the fourth aspect, which involves transducing immune cells with a lentiviral vector carrying the novel anti-CCR9 CAR.

[0018] In a sixth aspect, the present invention also discloses the use of novel anti-CCR9 CARs as described in the first aspect or immune cells as described in the fourth aspect in the preparation of medicaments for treating tumors with high CCR9 expression complicated by extramedullary diseases.

[0019] In some implementations, the tumor with high CCR9 expression and concurrent extramedullary disease is acute T-lymphoblastic leukemia with concurrent extramedullary disease.

[0020] This study co-expressed CXCR4 on the αCCR9 CAR-T backbone, constructing a "CXCR4-enabled αCCR9 CAR-T" (CXCR4-αCCR9 CAR-T). Utilizing the adhesion-chemotaxis mechanism of the CXCR4 / CXCL12 axis, the study enhanced CAR-T's dual homing and deep infiltration into both bone marrow and extramedullary lesions, thereby improving the clearance efficiency of residual leukemia lesions, prolonging the duration of remission in patients with EMD-T-ALL, and reducing the relapse rate.

[0021] This invention discloses for the first time a novel anti-CCR9 CAR, which, through CXCR4 activation, enables immune cells carrying the CAR to have stronger tissue chemotaxis and wider distribution in vivo, especially significantly enhanced migration to bone marrow and extramedullary leukemia lesions, thus making it more beneficial for the treatment of acute T-lymphocytic leukemia complicated with extramedullary diseases.

[0022] In in vitro experiments, CXCR4-empowered anti-CCR9 CAR-T cells exhibited similar cytotoxic activity to anti-CCR9 CAR-T cells, including cytokine secretion and CD107a expression. However, in in vivo experiments, CXCR4-empowered anti-CCR9 CAR-T cells showed superior efficacy against extramedullary lesions compared to anti-CCR9 CAR-T cells, with increased chemotaxis towards both bone marrow and extramedullary lesions. CXCR4 empowerment resulted in enhanced tissue chemotaxis and wider in vivo distribution of anti-CCR9 CAR-T cells, particularly a significantly improved migration to bone marrow and extramedullary leukemia lesions. This indicates that CXCR4-empowered anti-CCR9 CAR-T cells offer a more significant therapeutic advantage in treating T-ALL patients with concurrent extramedullary disease, demonstrating greater clinical translational potential.

[0023] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The T-ALL cell line highly expressed the antigen CCR9. A. CCR9 IgG blank control; B. Target cells were K562, Jurkat, and MOLT-4 cell lines, respectively.

[0026] Figure 2 Binding curves of AFA64 scfv and AFF84 scfv with Molt 4 cell line.

[0027] Figure 3 CAR structure diagram.

[0028] Figure 4 The CAR positivity rate of AFA64 CAR-T and AFF84 CAR-T transduction.

[0029] Figure 5 CAR-T surface CD107A expression was observed after co-incubation with target cells.

[0030] Figure 6 Expression characteristics of CXCL12 protein. Note: The expression level of CXCL12 was higher in bone marrow (T-ALL BM) and peripheral blood (T-ALL PB) of T-ALL patients. The expression level of CXCL12 in bone marrow of T-ALL patients was higher than that in normal human bone marrow (Normal BM), P < 0.05 (n=6); the expression level of CXCL12 in mouse bone marrow (NCG BM) was higher than that in peripheral blood (NCG PB), P < 0.01 (n=6).

[0031] Figure 7 The expression characteristics of CXCR4 in primary leukemia cells and cell lines. Among them, A. CXCR4 is highly expressed in primary leukemia cells from a T-ALL patient; BD. CXCR4 is expressed in cell lines MOLT-4, Jurkat, and K562.

[0032] Figure 8 Schematic diagrams of the structures of αCCR9 CAR-T, CXCR4 CAR-T, and CXCR4-T. From left to right in the diagram, they represent αCCR9 CAR-T, CXCR4 CAR-T, and CXCR4-T.

[0033] Figure 9 Immunofluorescence expression intensity of CCR9 CAR and CXCR4.

[0034] Figure 10 Comparison of CAR-T cell proliferation capacity. Note: CAR-T cell proliferation was detected by co-incubating MOLT-4, Jurkat, and K562 with NCT, αCCR9 CAR-T, and CXCR4 αCCR9 CAR-T cells on D1, D3, and D5. MOLT-4 and Jurkat promoted the proliferation of αCCR9 CAR-T and CXCR4 αCCR9 CAR-T cells.

[0035] Figure 11 ,4 The expression level of CD107a when the cells killed the target cells was determined. Among them, there was no difference in the expression level of CD107a when CXCR4 αCCR9 CAR-T cells and αCCR9 CAR-T cells killed the target cells (P=0.775, n=3).

[0036] Figure 12 The percentage of target cells remaining after CAR-T cell killing is represented by a flow cytometry graph.

[0037] Figure 13 Cytokine secretion characteristics of CAR-T cells after co-incubation with different target cells. Note: A. αCCR9 CAR-T and CXCR4 αCCR9 CAR-T cells co-incubated with MOLT-4 cells showed increased cytokine secretion; B. αCCR9 CAR-T and CXCR4 αCCR9 CAR-T cells co-incubated with Jurkat cells showed increased cytokine secretion.

[0038] Figure 14 Chemotaxis characteristics of CXCR4 and αCCR9 CAR-T cells. A. Number of CAR-T cells chemotactically attracted to NCT, CXCL12-containing medium, MOLT-4 cells, and mesenchymal stem cells; B. Percentage of CAR-T cells chemotactically attracted to NCT, CXCL12-containing medium, MOLT-4 cells, and hMSCs.

[0039] Figure 15 Effects of praxavir on the migration of CXCR4 αCCR9 CAR-T cells. A. With the addition of praxavir, the migration of CXCR4 αCCR9 CAR-T cells to hMSCs decreased, P=0.0009 (n=3); B. With the addition of praxavir, the migration of CXCR4 αCCR9 CAR-T cells to MOLT-4 decreased, P=0.004 (n=3).

[0040] Figure 16 1. Anti-leukemia efficacy of CXCR4 αCCR9 CAR-T extramedullary model. A. Experimental flowchart of efficacy in extramedullary leukemia model. Leukemia burden was assessed by monitoring subcutaneous nodule size; B. Efficacy of subcutaneous tumor in mice. CXCR4 αCCR9 CAR-T cells showed significantly better anti-leukemia efficacy than αCCR9 CAR-T cells in the extramedullary model, with a statistically significant difference (P=0.049); C. There was no significant difference in body weight among the groups of mice (P>0.05), indicating that both CAR-T cell therapies were well tolerable.

[0041] Figure 17 The proportions of CCR9 CAR-T, CXCR4 αCCR9 CAR-T and CXCR4-T cells in mouse bone marrow.

[0042] Figure 18 The efficacy of CXCR4 αCCR9 CAR-T combined extramedullary and intramedullary therapy against leukemia. A. In vivo animal experiment flowchart: MOLT-4 cells were subcutaneously and intravenously injected into the same mouse to construct a combined extramedullary and systemic intramedullary leukemia model; B. In vivo imaging to observe systemic leukemia burden; C. On day 21, the fluorescence of CXCR4 αCCR9 CAR-T and αCCR9 CAR-T cells decreased significantly compared to the NCT group (P < 0.0001), indicating better therapeutic effect; D. Mouse body weight change curve. There was no significant difference in body weight change among the groups (P = 0.091).

[0043] Figure 19 CAR-T cell efficacy against extramedullary leukemia. Note: In the CXCR4 αCCR9 CAR-T cell group, 4 mice achieved complete remission of extramedullary leukemia lesions, and 1 mouse relapsed; in the αCCR9 CAR-T group, 2 mice achieved complete remission, and 2 mice experienced partial remission followed by progression. Each broken line represents the volume of a subcutaneous tumor in one mouse.

[0044] Figure 20 1. In vivo fluorescence imaging of CAR-T in tumor-bearing mice. A. Flowchart of in vivo animal experiments; D-3 subcutaneous and tail vein tumors, in vivo fluorescence imaging and weight measurement every 3 days; B. In vivo imaging of mice to detect changes in DIR intensity at various sites; C. The proportion of DIR fluorescence intensity in the bilateral thighs of mice to the total fluorescence intensity of the whole body. The proportion of fluorescence intensity in the bilateral thighs of mice in the CXCR4 αCCR9 CAR-T group was higher than that in the control group (* and § in the figure indicate significant differences: * indicates significance between the CXCR4 αCCR9 CAR-T group and the NCT group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001; § indicates significance between the αCCR9 CAR-T group and the NCT group, § indicates P < 0.05, §§ indicates P < 0.01; αCCR9 CAR-T group, CXCR4 αCCR9 CAR-T group and CXCR4-T group, n=5; NCT group, n=3)). Detailed Implementation

[0045] To facilitate understanding by those skilled in the art, some terms appearing in this document are explained and clarified.

[0046] As used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include plural references.

[0047] In this document, unless otherwise stated, the terms "comprising," "including," or "containing" mean containing the listed technical features, but do not exclude the inclusion of other technical features. The main embodiments of the present invention and corresponding experimental results are summarized below: Example 1. Construction of CCR9 CAR-T cells: Anti-CCR9 single-chain antibodies were designed and screened, and their binding ability was verified. CCR9 CAR-T cells were constructed based on the single-chain antibodies, characterized, and their anti-tumor function was verified.

[0048] Results: AFA64 scfv and AFF84 scfv had similar binding ability to hCCR9 on the MOLT-4 cell line, and there was no significant difference in CAR expression and anti-T-ALL tumor function between AFA64CAR-T and AFF84 CAR-T.

[0049] Example 2. Construction of CXCR4-empowered anti-CCR9 CAR-T cells: The CXCR4 gene was linked to the anti-CCR9 CAR framework via T2A (2A peptide) to construct a lentiviral vector, which was then transduced into CD3+ cells. + T cells were used to obtain CXCR4-empowered anti-CCR9 CAR-T cells (also referred to as CXCR4 αCCR9 CAR-T cells in this invention).

[0050] Result: CXCR4-enhanced anti-CCR9 CAR-T was successfully constructed.

[0051] Example 3. In vitro functional verification of CXCR4-empowered anti-CCR9 CAR-T cells: The proliferation capacity and killing effect of CXCR4-empowered anti-CCR9 CAR-T cells, anti-CCR9 CAR-T cells (also referred to as αCCR9 CAR-T cells in this invention), CXCR4-overexpressed T cells (CXCR4-T), and untransducted T cells (NCT) were compared using CFSE fluorescence intensity changes and CD107A expression methods.

[0052] Results: CXCR4-empowered anti-CCR9 CAR-T cells effectively killed leukemia cells in vitro: CXCR4-empowered anti-CCR9 CAR-T cells, after co-incubation with target cells, expanded effectively and effectively killed CCR9-positive target cells. There was no statistically significant difference in CD107A expression compared to anti-CCR9 CAR-T cells. P >0.05. It also significantly secretes anti-tumor-related immune cytokines.

[0053] Example 4. Chemotactic migration ability of CXCR4-empowered anti-CCR9 CAR-T cells: The chemotactic migration ability of CXCR4-empowered anti-CCR9 CAR-T cells to CXCL12, human mesenchymal stem cells (hMSC), MOLT-4 cells, and bone marrow supernatant from T-ALL patients was evaluated by Transwell assay.

[0054] Results: CXCR4-empowered anti-CCR9 CAR-T cells showed significantly higher chemotaxis toward CXCL12, MOLT-4, and T-ALL bone marrow supernatant than anti-CCR9 CAR-T cells.

[0055] Example 5. Validation of in vivo anti-leukemia activity in T-ALL mouse model: Extramedullary model and combined intramedullary and extramedullary tumor model were established in NCG mice. The efficacy of intramedullary treatment was evaluated by in vivo fluorescence imaging, and the size of subcutaneous leukemia nodules was measured to evaluate the efficacy of extramedullary treatment.

[0056] Results: In the whole-body intramedullary models of extramedullary and intramedullary-extramedullary tumorigenesis, CXCR4-enhanced anti-CCR9 CAR-T cells showed comparable efficacy to anti-CCR9 CAR-T cells. In the extramedullary models of extramedullary and intramedullary-extramedullary tumorigenesis, the CXCR4-enhanced anti-CCR9 CAR-T group had a higher complete remission (CR) rate and a lower relapse rate in the EMD. The proportion of CAR-T cell migration within the bone marrow and EMD was higher in mice in the CXCR4-enhanced anti-CCR9 CAR-T cell group than in the anti-CCR9 CAR-T cell group.

[0057] Example 6. Dynamic distribution of CAR-T cells in vivo: CAR-T cells stained with near-infrared fluorescent probe (DIR) were injected into tumor-bearing mice via the tail vein. Fluorescence imaging was used to understand the dynamic distribution of CAR-T cells in mice and to observe the distribution of CAR-T cells in different organs in vivo.

[0058] Results: In the tumor-bearing NCG mouse T-ALL model, CXCR4-enabled anti-CCR9 CAR-T cells were found to have a wider distribution in organs, especially a higher proportion of bone marrow and extramedullary lesions.

[0059] The sequences used in this paper are shown in Table 1.

[0060] Table 1. Sequence Reference Table The above embodiments will be described in detail below. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in literature in the art, such as *Molecular Cloning: A Laboratory Manual*. Unless otherwise specified, all reagents used in the experiments can be purchased through conventional channels.

[0061] Example 1. Construction of CCR9 CAR-T cells Material: Cell lines: MOLT-4 and K562, from ATCC, USA.

[0062] 1. Reagent preparation (1) Polyethyleneimine (PEI) stock solution (1 mg / mL): prepared using PEI 40000.

[0063] (2) Prepare 50×TAE Buffer, pH 8.3.

[0064] (3) DMEM complete medium: Add 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin to DMEM medium and mix them thoroughly.

[0065] (4) MOLT-4 cells 1640 complete medium: Add 10% FBS and 1% penicillin-streptomycin to 1640 basal medium.

[0066] CAR-T cell culture medium: serum-free medium (GT-T551 H3) - TAKARA.

[0067] Experimental process 1) Detect the expression level of CCR9 in different T-ALL cell lines.

[0068] Take 50 µL of culture medium containing target cells, dilute with 100 µL of PBS, mix thoroughly, add 2 µL of antibody CCR9-APC to the sample, mix gently, and stain at 4°C in the dark for 30 min. Add 1.5 mL of physiological saline, mix well, and centrifuge at 1500 rpm for 5 minutes. Remove the supernatant, add 1.5 mL of physiological saline, mix well, centrifuge again at 1500 rpm for 5 minutes. Resuspend the cells in 0.4 mL of PBS and transfer to a flow cytometry tube. Analyze the expression characteristics of CCR9 in the cell lines using a Beckman Coulter flow cytometer. Flow cytometry results showed that the expression levels of CCR9 varied among different T-ALL cell lines. Figure 1(AD). The expression level of CCR9 in the Jurkat cell line is low, at approximately 24.9%, and the expression intensity is weak; while the expression level of CCR9 in the MOLT-4 cell line is extremely high, reaching 99.73%. However, the human chronic myeloid leukemia K562 cell line does not express CCR9.

[0069] In summary, CCR9 exhibits high expression in T-ALL leukemia cells, and its expression level further increases with disease progression. Different expression intensities and positive rates were also observed in T-ALL cell lines. These experimental results suggest that CCR9 is a potential therapeutic target for T-ALL and has the potential to serve as a target for CAR-T therapy in T-ALL.

[0070] 2) Detect the binding ability of single-chain antibodies to CCR9 on MOLT-4 cells.

[0071] Seamless cloning kit was purchased from Novizan; PrimeStar DNA polymerase was purchased from TAKARA; PUTAM003 expression vector was purchased from Eucardi Biotechnology Co., Ltd.; TOP10 competent cells were purchased from Tiangen Biotech Co., Ltd. Using seamless cloning, AFA64 scfv and AFF84 scFv fragments were fused with the IgG Fc fragment to obtain AFA64 scfv-hFc and AFF84 scFv-hFc recombinant monoclonal antibodies, respectively. These were confirmed to be correct by sequencing by Sangon Biotech Co., Ltd.

[0072] MOLT-4 cells with high CCR9 expression were co-incubated with AFA64 scfv-hFc and AFF84 scFv-hFc at gradient concentrations of 0.0098, 0.0391, 0.1563, 0.625, 2.5, and 10 μg / mL, respectively, followed by incubation with anti-IgG-APC secondary antibody. The average fluorescence intensity of AFA64 scfv-hFc and AFF84 scFv-hFc binding to CCR9 on MOLT-4 cells was analyzed using Beckman Coulter flow cytometry. Figure 2 Flow cytometry results showed that both AFA64 scfv-hFc and AFF84 scFv-hFc could bind to MOLT-4 CCR9, and the binding ability was similar at different concentrations.

[0073] 3) Characterization of CCR9 CAR-T expression and verification of its anti-tumor effect (1) Construction of anti-CCR9 CAR and packaging of lentivirus The lentiviral backbone plasmid vector was PSB1819, obtained from Eukaryotic; pPac-R, pPac-GP, and pEnv-G were all obtained from Eukaryotic; TOP10 competent cells were purchased from Tiangen Biotech Co., Ltd.

[0074] The AFA64 scfv and AFF84 scFv fragments were seamlessly cloned into the lentiviral backbone plasmid vector PSB1819, and second-generation CARs targeting the CCR9 antigen, with 4-1BB as the co-stimulatory signal and CD3ζ as the activation domain, were designed—AFA64CAR and AFF84CAR. Figure 3 As shown in Table 1, the nucleotide and amino acid sequences of AFA64 CAR and AFF84 CAR were prepared by transfecting 293T cells with lentiviral backbone plasmids and lentiviral packaging plasmids pPac-R, pPac-GP, and pEnv-G.

[0075] (2) Preparation and characterization of CCR9 CAR-T cells 10 mL of peripheral blood was collected from healthy volunteers, and mononuclear cells were obtained by Ficoll separation. CD3+ T cells were positively selected using CD3 magnetic beads. After T cells were activated with CD3 / CD28 antibody for 24 hours, they were transduced with the lentiviral vector obtained in step (1) and cultured for another 7 days. The CAR positive expression rate was characterized by flow cytometry. The results showed that the CAR positive expression rates of the AFA64 CAR-T and AFF84 CAR-T groups were 46.4% and 50.97%, respectively. Figure 4 ).

[0076] (3) CD107A assessment of antitumor function Prepare the reagents required for the CD107a experiment in advance according to the following ratios. In a 200 µL incubation system, according to the groups: Blank (effective cells + Golgistop), Negative Control (effective cells), Positive Control (effective cells + Golgisto + stimulate), K562 (effective cells + Golgisto + K562), and Molt-4 (effective cells + Golgistop + Molt-4), add 0.4 µL of stimulate cocktail (500×) and 0.4 µL of golgistop cocktail (500×) to each well. Place appropriate amounts of CAR-T cells, K562 cells, and MOLT-4 cells into separate centrifuge tubes. Centrifuge at 1500 rpm for 5 minutes. Discard the supernatant and wash the cell pellet twice with PBS. Count the cells using the AO / PI staining method and adjust the cell density to the required level. The CAR-T cell count is 5 × 10⁶. 4Target cells were added at a 1:1 effector-to-target ratio, along with the prepared reagents, in an incubation volume of 200 μL. After 6 hours of co-incubation, suitable cells from each group were removed, washed twice with PBS, and then 1 μL each of CD3 antibody, Protein L protein, and CD107a antibody were added to each group of cells. The cells were incubated at 4°C in the dark for 30 minutes at room temperature. After incubation, the cells were washed twice with PBS, and CD107a expression was detected using flow cytometry.

[0077] The results are as follows Figure 5 Both AFA64 CAR-T and AFF84 CAR-T showed significant and specific killing effects on the CCR9-overexpressing cell line MOLT-4, with no significant difference between the two.

[0078] Example 2. Construction of CXCR4-empowered anti-CCR9 CAR-T cells (anti-CCR9 scFv was selected from AFA64 scFv or AFF84 scFv; Examples 2-6 use AFA64 scFv as an example for illustration) Material: Cell lines: MOLT-4, Jurkat, Raji-B, and HUVEC were obtained from ATCC, USA; NCG mice were derived from the Shanghai Southern Model Organisms.

[0079] 1. Reagent preparation (1) Polyethyleneimine (PEI) stock solution (1 mg / mL): prepared using PEI 40000.

[0080] (2) Prepare 50×TAE Buffer, pH 8.3.

[0081] (3) DMEM complete medium: Add 10% fetal bovine serum to DMEM medium. Mix serum (FBS) and 1% penicillin-streptomycin solution thoroughly.

[0082] (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.

[0083] (5) Complete CAR-T cell culture medium: serum-free medium (GT-T551 H3)-TAKARA.

[0084] (6) Mesenchymal stem cell culture medium: a mixture of platelet lysis buffer and 10% FBS, suitable for in vitro culture of mesenchymal stem cells.

[0085] (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.

[0086] 2. Experimental Procedure 1) Detection of CXCL12 expression characteristics in primary T-ALL specimens, target cells, mesenchymal stem cells, and umbilical vein endothelial cells. The CXCL12 kit (from LEGENDplex™ Human CXCL12 (SDF-1) Capture Bead B2, 13X (Biolegend, 740785)) was used to detect the supernatant of cultured NCT, MOLT-4, and hMSC cells after 72 hours, as well as the supernatant of mouse bone marrow and T-ALL patients' bone marrow. Following the kit instructions, standards and staining specimens were prepared, and CXCL12 concentration was detected by flow cytometry. NCT, MOLT-4, and hMSC cells were seeded in 6-well plates and cultured in complete culture medium for 24 hours. After culture, the supernatant from each group was collected and centrifuged at 1000 rpm for 5 minutes to remove residual cells and debris. The supernatant was then transferred to sterile tubes for later use. Following the CXCL12 kit instructions, serial dilutions were performed using the provided standard stock solutions. The stock solutions were diluted with the dilution buffer provided in the kit to establish a series of concentration gradients and a standard curve was constructed. 50 μL of standard / cell supernatant sample was added to each well of an EP tube. After adding the capture antibody provided in the kit and mixing well, incubate at room temperature for 2 hours to allow CXCL12 to fully bind to the antibody. After incubation, add the antibody labeled with a fluorescent dye and continue incubating in the dark for 1 hour, followed by washing. Transfer the washed samples to the flow cytometer's detection tubes and adjust the instrument parameters to capture fluorescence signals. Record the fluorescence intensity data for each sample according to the set acquisition parameters. Plot a standard curve using the fluorescence signals of the standards, and calculate the corresponding CXCL12 concentration based on the fluorescence values ​​of each sample.

[0087] result: Studies have shown that mouse CXCL12 is highly homologous to human CXCL12 (https: / / www.ncbi.nlm.nih.gov / CCDS), providing a basis for the feasibility of CXCR4-modified CAR-T cell therapy and in vivo mouse experiments. This study examined the secretion levels of the chemokine CXCL12 in bone marrow and peripheral blood of T-ALL patients, bone marrow and peripheral blood of NCG mice, normal human bone marrow, NCT cells, K562 cells, Jurkat cells, MOLT-4 cells, and hMSC cells. The results showed ( Figure 6The concentrations of CXCL12 secreted by NCT ​​cells, MOLT-4 cells, and hMSC cells were 223.50±19.09 pg / mL, 1181.79±2.41 pg / mL, and 3289.33±478.40 pg / mL, respectively. The mean concentrations of CXCL12 in mouse peripheral blood and bone marrow were 650.67±13.27 pg / mL and 2821.83±729.74 pg / mL, respectively. The mean concentration of CXCL12 in normal human bone marrow was 1999.29±556.87 pg / mL. The mean concentrations of CXCL12 in peripheral blood and bone marrow of T-ALL patients were 3251.0±649.12 pg / mL and 4717.50±702.12 pg / mL, respectively. Statistical analysis showed that the CXCL12 secreted by hMSC cells during culture was significantly higher than that of NCT cells (…). P= 0.0414). The concentration of CXCL12 in the bone marrow of T-ALL patients was significantly higher than that in the bone marrow of normal individuals. P= 0.0385). The level of CXCL12 in mouse bone marrow was significantly higher than that in peripheral blood ( P= 0.0087).

[0088] This study revealed the high expression levels of CXCL12 protein in the bone marrow and peripheral blood of T-ALL patients, as well as in healthy human bone marrow and cell lines. These results provide a theoretical basis for the subsequent construction of CXCR4-modified CAR-T cell therapy and lay the foundation for its feasibility in in vivo experiments in mice.

[0089] 2) Detect the expression characteristics of CXCR4 in T-ALL primary cells and target cells. The cell processing and staining steps were as follows: First, 500 μl of bone marrow samples or different target cell lines (MOLT-4, Jurkat, K562) were co-incubated with their respective fluorescently labeled antibodies. Bone marrow samples used a multicolor antibody combination (CD45-FITC, CD4-FITC, CD8-PE, CD3-ECD, CD7-A700, CXCR4-APC), while target cell lines used only the CXCR4-APC antibody. All steps were performed at 4°C in the dark for 30 minutes to reduce non-specific binding. Next, the bone marrow samples underwent erythrocyte lysis (40°C water bath for 10 minutes) and stop solution was added (room temperature for 15 minutes) to terminate the reaction. Subsequently, unbound antibodies and cell debris were removed by washing twice with physiological saline and centrifuging (1500 rpm, 5 minutes). Finally, the cell pellet was resuspended in 0.4 ml PBS and analyzed by Beckman Coulter flow cytometry to detect the expression characteristics of CXCR4.

[0090] result: 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 7 A), expressed at 25% on Jurkat cells ( Figure 7 B), with an expression level of 99% in MOLT-4 cells ( Figure 7 C), while the expression level on K562 cells was 21.8% (C). Figure 7 D).

[0091] Both primary T-ALL cells and MOLT-4 cell lines showed high levels of CXCR4 expression. Figure 7 The average expression levels of A and C were 85.23% ± 17.8% and 99%, respectively, while the expression levels of Jurkat and K562 cells were lower. Figure 7 B, D. 25% and 21.8%.

[0092] Based on the previously observed hypersecretion of CXCL12, the CXCR4 / CXCL12 signaling axis may play a crucial role in the pathogenesis of T-ALL, particularly in relation to the strong bone marrow homing ability and invasive phenotype of leukemia cells. Therefore, utilizing this chemokine pathway in CAR-T cell therapy strategies could potentially enhance the migration and infiltration of effector cells into disease target tissues (such as bone marrow and extramedullary lesions of T-ALL), thereby improving the depth of myeloremission and the remission rate of extramedullary lesions in T-ALL.

[0093] 3) Construction of anti-CCR9 CAR target plasmid and CXCR4-enhanced anti-CCR9 CAR target plasmid (1) Design of target gene CAR In this study, a second-generation CAR targeting the CCR9 antigen was designed, using 4-1BB as a co-stimulatory signal and CD3ζ as the activation domain. We named it αCCR9 4-1BBζ CAR (i.e., αCCR9 CAR, also known as anti-CCR9 CAR). The designed sequence was synthesized, and then the target plasmid for the anti-CCR9 CAR was constructed.

[0094] After reviewing literature and databases, a natural CXCR4 sequence was found. A third-generation CAR was designed to tandem the CXCR4 sequence with an αCCR9 CAR, using 4-1BB as a co-stimulatory signal and CD3ζ as the killing domain. We named this CXCR4 αCCR9CAR (also known as CXCR4-enabled anti-CCR9 CAR). After completing the sequence design, the target sequence was first synthesized, and then cloned into an expression vector to construct the CXCR4 αCCR9 CAR target plasmid.

[0095] (2) Vector digestion Prepare the reagents, mix them according to the system in Table 2, and incubate in a 37°C water bath until the next day.

[0096] Table 2. Enzyme digestion system (3) PCR amplification of the target gene and gel electrophoresis recovery of the target DNA fragment (4) Transformation of competent cells Prepare a reaction solution by mixing 1 μL of the linearized plasmid expression vector (digested with 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.

[0097] 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.

[0098] (5) Screening and identification of positive clones 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.

[0099] result: CXCR4 was tandemly linked with CCR9 antibodies VH and VL to form CXCR4-enabled αCCR9 scFv. The CXCR4-enabled αCCR9 CAR backbone, from the 5' amino terminus to the carboxyl terminus, sequentially includes αCCR9 scFv, a CD8a hinge and transmembrane domain, intracellular co-stimulatory signaling molecules 4-1BB, CD3ζ, T2A, and CXCR4, constructing CXCR4-enabled anti-CCR9 CAR-T (i.e., CXCR4 αCCR9 CAR-T), enhancing the migration ability of CAR-T cells. Simultaneously, CXCR4-enabled T cells (CXCR4-T) were constructed, increasing their migration and chemotactic abilities, serving as a control for CXCR4 αCCR9 CAR-T to demonstrate whether the addition of CXCR4 affects the cytotoxicity of T cells. Schematic diagrams of αCCR9 CAR-T, CXCR4 αCCR9 CAR-T, and CXCR4-T are shown below. Figure 8 .

[0100] 4) Packaging of lentiviral vectors (1) Small-scale plasmid extraction Follow the instructions in the plasmid extraction kit manual.

[0101] (2) HEK293T cell resuscitation and passage 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.

[0102] 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.

[0103] The complex was uniformly added dropwise to fresh culture medium containing HEK293T cells for transfection. After about 5 hours, the original culture medium was discarded and replaced with fresh DMEM complete culture medium. The cells were cultured for a longer period to collect the viral supernatant.

[0104] (3) Transfection of HEK293T packaging cells On the second day after transfection with HEK293T cells, the cell confluence and uniformity of distribution were observed under a microscope. One hour before transfection, the culture dish was removed, the original cell culture medium was removed, and 9 mL of fresh Opti-MEM medium was slowly added, taking care to prevent cells from falling off the culture dish. The culture dish was then placed back into the incubator.

[0105] Add CaCl2, the target plasmid, and pre-prepared HBS (250 μL CaCl2 and 44 µg of the target plasmid for CAR-T cells to every 500 μL of HBS) sequentially to a 15 mL centrifuge tube to prepare the transfection reagent. Take HEK293T cells, aspirate 1 mL of the transfection reagent, and drop it into a HEK293T cell culture dish. Return the dish to a 37°C, 5% CO2 cell culture incubator and incubate for 6 hours. Discard the supernatant and add 10 mL of fresh DMEM complete medium. One day after transfection, observe the cell state and density under a microscope and replenish the medium as needed. Continue culturing in a cell culture incubator for 2 days.

[0106] (4) Collection and concentration of lentiviral vectors 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.

[0107] (5) Virus titer determination 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: Note: C: Average number of viral copies integrated into the genome of each cell; D: Virus dilution factor; N: Total number of cells at the time of infection; V: Volume of virus added (μL).

[0108] result: The target plasmid was co-transfected with packaging plasmids pPac-GP and pPac-R, and envelope plasmid pEnv-G into HEK293T cells for lentivirus production.

[0109] The final viral vector concentrate was quantified using the functional titer method, and the relevant titer results are shown in Table 3.

[0110] Table 3. Lentiviral titers 5) Preparation of CXCR4-empowered anti-CCR9 CAR-T cells (1) CAR-T cell source: 50 mL of peripheral blood was collected from each of three healthy volunteers who signed informed consent forms for in vitro experiments and in vivo experiments in mice.

[0111] (2) Isolation of mononuclear cells. Peripheral blood containing EDTA anticoagulant was diluted with an equal volume of sterile PBS and added to Ficoll separation solution (ratio approximately 1:2). The mixture was centrifuged at 800g for 30 minutes at room temperature. After separation, the intermediate white membrane layer, i.e., peripheral blood mononuclear cells (PBMCs), was aspirated and washed twice with 0.9% sodium chloride (1500rpm × 5 minutes). The cells were resuspended, stained with trypan blue, and counted. A final collection of 2 × 10⁶ cells was obtained. 8 One PBMC cell is prepared for use.

[0112] (3) CD3 + T cell sorting Prepare PBMC suspension at 2×10 7 Add CD3 magnetic beads at a ratio of 10 μL / cell, incubate at 4°C in the dark for 15 minutes, then wash and centrifuge. Collect CD3 cells using a magnetic separation column via positive selection. 3+ T lymphocytes.

[0113] (4) CD3 + T cell activation CD3 + Cells were centrifuged at 1500 rpm for 5 minutes and resuspended in T-cell culture medium. Culture flasks pre-coated with anti-CD3 / CD28 antibodies were added, along with IL-2 (final concentration 1000 IU / mL). The cell volume was adjusted to 3 × 10⁶ cells / mL. 6 At a density of / mL, it was activated by incubation in a 37°C, 5% CO2 incubator.

[0114] (5) Lentiviral transduction of cells Forty-eight hours later, remove the T cell culture flask, collect a small number of T cells, stain with trypan blue, add them to a glass slide, and perform cell counting. Based on the cell count results, adjust the cell concentration to 5 × 10⁻⁶ cells / mL. 6 Four vials of CXCR4-enhanced anti-CCR9CAR lentivirus, anti-CCR9CAR lentivirus, and CXCR4 lentivirus were added to three T cell suspensions at MOIs of 50, 80, and 120, respectively. One NCT cell suspension served as a control.

[0115] (6) Expansion of CAR-T cells Observe cell status daily, including density, aggregation, and changes in culture medium, and count cells periodically. Use a cell concentration of 5 × 10⁶ cells / day. 5 Adjust the fluid level by adding fluid at / mL and continue culturing for 10 to 12 days. CAR expression detection, cytotoxic activity assessment, and differentiation subset analysis are performed sequentially after day 7 of lentiviral infection.

[0116] (7) Washing and cryopreserving CAR-T cells On day 12, the remaining expanded CAR-T cells were collected, centrifuged at 1500 rpm for 5 minutes at room temperature, and washed twice. After counting, they were divided into groups of 1×10⁻⁶ cells. 7 Cells / vial, resuspended in cryopreservation solution, and cryopreserved (long-term storage in liquid nitrogen tank).

[0117] result: In this study, the target gene was transduced into T cells using a lentiviral vector, successfully obtaining CXCR4 αCCR9 CAR-T cells, αCCR9 CAR-T cells, and CXCR4-T cells. Flow cytometry was used to detect CAR expression characteristics by detecting CCR9 protein, and CXCR4 antibody was used to detect CXCR4 expression. αCCR9 CAR-T cell culture served as a control. The immunofluorescence intensity of CAR and CXCR4 expression was analyzed. Since normal circulating T cells and NCT cells typically express CXCR4, to verify successful infection of CXCR4 in αCCR9 CAR-T cells and CXCR4-T cells, and successful overexpression in both cell groups, the expression intensity of CXCR4 in the four cell groups was further analyzed using mean fluorescence intensity (MFI). Results showed that CXCR4 expression was not enhanced in αCCR9 CAR-T cells compared to NCT cells, while CXCR4 expression was significantly enhanced in both αCCR9 CAR-T cells and CXCR4-T cells (Figure 9).

[0118] Example 3. In vitro functional validation of CXCR4-enhanced anti-CCR9 CAR-T cells. 1. Assessment of CAR-T cell proliferation capacity NCT cells, anti-CCR9 CAR-T cells, and CXCR4-empowered anti-CCR9 CAR-T cells were stained with CFSE. After staining, they were co-incubated with each target cell at an effector-to-target ratio of 5:1. MOLT-4, Jurkat, and K562 cells were used as target cells. Cells were collected after 24, 72, and 120 hours of incubation, and changes in CFSE fluorescence intensity were detected by flow cytometry.

[0119] As cells proliferate, the CFSE dye is diluted generation by generation during cell division. By monitoring the gradual decrease in fluorescence intensity, the proliferative activity of different CAR-T cells after interaction with target cells can be quantitatively assessed.

[0120] result: Experimental results show that ( Figure 10At different time points, no significant leftward shift of the CFSE fluorescence peak was observed in CAR-T cells after co-incubation with αCCR9 CAR-T cells, CXCR4 αCCR9 CAR-T cells, or CXCR4-T cells co-incubated with K562 cells. This result indicates that K562 cells, as a negative control cell line, failed to effectively activate the proliferation response of CAR-T cells. However, under co-incubation conditions with Jurkat and MOLT-4 cells, the CFSE fluorescence intensity of both CXCR4 αCCR9 CAR-T cells and αCCR9 CAR-T cells decreased significantly, and the fluorescence peak shifted significantly to the left. This phenomenon suggests that these two types of CAR-T cells underwent a significant proliferation response after specific stimulation by the CCR9 antigen. In contrast, CXCR4-T cells, lacking a cytotoxic domain and co-stimulatory signal, could not be activated by Jurkat and MOLT-4 cells, which is consistent with the experimental observation that no decrease in CFSE fluorescence or leftward shift of the peak was observed when CXCR4-T cells were co-incubated with Jurkat and MOLT-4 cells.

[0121] In summary, both αCCR9 CAR-T cells and CXCR4 αCCR9 CAR-T cells exhibited significantly higher proliferation trends than CXCR4-T cells after co-incubation with target cells. Among them, CXCR4 αCCR9 CAR-T cells showed a greater degree of fluorescence attenuation and a more significant leftward shift in the peak value, indicating a stronger proliferative capacity after recognizing the CCR9 target antigen.

[0122] 2. Evaluation of CXCR4-enhanced anti-CCR9 CAR-T toxicity capability (1) CD107a expression level and residual target cell analysis Analysis of target cells with different CCR9 expression levels showed no significant difference in CD107a expression levels between CXCR4 αCCR9 CAR-T cells and αCCR9 CAR-T cells induced by different CCR9 expression intensities (P=0.775). Figure 11When CXCR4-T cells killed Jurkat and MOLT-4 cells, the CD107a expression levels were 2.38%±0.60% and 2.50%±2.24%, respectively. When NCT cells killed Jurkat and MOLT-4 cells, the CD107a expression levels were 1.58%±0.74% and 1.75%±1.01%, respectively. When αCCR9 CAR-T cells killed Jurkat and MOLT-4 cells, the CD107a expression levels were 23.38%±1.79% and 61.50%±3.29%, respectively. When CXCR4 and αCCR9 CAR-T cells killed Jurkat and MOLT-4 cells, the CD107a expression levels were 22.75%±7.55% and 53.89%±3.33%, respectively. Under the same target cell conditions, there was no significant difference in CD107a expression levels between CXCR4 αCCR9 CAR-T cells and αCCR9 CAR-T cells (P > 0.05), further confirming that the introduction of CXCR4 did not have a negative impact on the cytotoxic function of CAR-T cells.

[0123] By detecting CCR9 on target cells to identify residual target cells, it was found that target cells were significantly cleared. Figure 12 A. The residual target cells in the αCCR9 CAR-T group accounted for 1.65%; B. The residual target cell percentage in the CXCR4 αCCR9 CAR-T group accounted for 0.82%; C. The residual target cell percentage in the αCCR9 CAR-T group was the highest at 29.24%.

[0124] (2) Assessment of CAR-T cytokine secretion capacity NCT cells, anti-CCR9 CAR-T cells, and CXCR4-empowered anti-CCR9 CAR-T cells were used as effector cells, while MOLT-4, Jurkat, and K562 cells were used as target cells. The release of cytokines IL-2, IFN-γ, and TNF-α after effector cell killing of target cells was detected using the BD Human Th1 / Th2 / Th17 Cytokine CBA Assay Kit.

[0125] result: To further evaluate the anti-leukemia activity of CXCR4 αCCR9 CAR-T cells, we examined the secretion of cytokines during the killing of target cells.

[0126] The results showed that no significant cytokine secretion was observed in the NCT group and the CXCR4-T control group during co-incubation with target cells. Figure 13In contrast, CXCR4 αCCR9 CAR-T cells exhibited significant cytokine secretion when co-incubated with CCR9-positive target cells. Specific secretion levels were as follows: at a 2.5:1 effector-to-target ratio, when CXCR4 αCCR9 CAR-T cells killed MOLT-4 cells, the secretion levels of IL-2, TNF-α, and IFN-γ reached 424.87 ng / mL, 443.43 ng / mL, and 1883.17 ng / mL, respectively. Under the same conditions, when CXCR4 αCCR9 CAR-T cells killed Jurkat cells, the secretion levels of IL-2, TNF-α, and IFN-γ were 5.92 ng / mL, 13.88 ng / mL, and 535.06 ng / mL, respectively. In addition, both CXCR4 αCCR9 CAR-T cells and αCCR9 CAR-T cells can induce the secretion of a variety of cytokines under stimulation by CCR9-positive target cells (MOLT-4 and Jurkat), among which the secretion level of IFN-γ is significantly increased.

[0127] The introduction of CXCR4 did not weaken the cytokine secretion capacity of CAR-T cells. CXCR4 αCCR9 CAR-T cells exhibited higher cytokine secretion levels under certain conditions; for example, TNF-α secretion was significantly higher in CXCR4 αCCR9 CAR-T cells than in CCR9-positive Jurkat cells. This result further supports the potential application of CXCR4 αCCR9 CAR-T cells in the treatment of T-ALL.

[0128] 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.

[0129] Example 4. Detection of CXCR4-enhanced anti-CCR9 CAR-T chemotactic migration ability 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.

[0130] upper room Effector cells were divided into two groups: all effector cells were pre-stained with CFSE.

[0131] Count the effector cells (αCCR9 CAR-T and CXCR4 αCCR9 CAR-T), adjusting the concentration to 5 × 10⁻⁶. 6 / mL. Plexafor was added to block the binding of CXCR4 to its homologous ligands (stromal cell-derived factor-1α, SDF-1α), verifying that the migration ability of CXCR4 αCCR9 CAR-T cells could be blocked, thus conversely confirming the chemotaxis of CAR-T cells.

[0132] The first group did not add pleruseta, and added 100 μL of effector cells to each well in the upper chamber. Add 100 μL of effector cells (5 × 10⁶ cells) to each well. 5 (cells).

[0133] The second group, treated with plexafor: plexafor concentration was 3.9 μmol / mL, 18 μL per well for 5 × 10⁵ treatments. 5 Calculate the required cell quantity and plexafor dose based on the number of cells and the number of wells.

[0134] Add pleroxafer to effector cells and incubate at 37°C for 30 minutes.

[0135] Downstairs 1. Add 600 μL of culture medium, 600 μL of CXCL12, hMSC culture supernatant, 600 μL of normal human bone marrow supernatant, 600 μL of T-ALL patient bone marrow supernatant and 600 μL of mouse bone marrow supernatant respectively.

[0136] 2. Incubate in an incubator (37℃, 5% CO2) for 4 hours.

[0137] 3. Collect all cells from the lower chamber for flow cytometry analysis, stain with counting beads, and perform flow cytometry counting.

[0138] result: This study used the Transwell assay 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. Cell migration was observed under a light microscope, and absolute counting microspheres were used to count the cells in the lower chamber of the Transwell apparatus to quantitatively analyze their chemotactic capacity.

[0139] Absolute cell counts were performed in the lower chamber of the Transwell device. The proportions of CXCR4 and αCCR9 CAR-T cells chemotactically converting to CXCL12, MOLT-4, and mesenchymal stem cells were 94%, 50%, and 39.8%, respectively, all significantly higher than those of αCCR9 CAR-T cells, and the differences were statistically significant. P All < 0.05 ( Figure 14 ).

[0140] To further verify the chemotactic ability of CXCR4, this study investigated the Transwell chemotactic characteristics after CXCR4 blockade with praxavir. The results showed that praxavir significantly inhibited the chemotactic migration of CAR-T cells to the bone marrow supernatant and target cells of T-ALL patients. Specifically, after the addition of praxavir, the migration rate of CXCR4 αCCR9 CAR-T cells to the bone marrow supernatant of T-ALL patients decreased from 55.09%±8.23% to 15.17%±3.46% (P=0.009). After the addition of praxavir, the migration rate of CXCR4 αCCR9 CAR-T cells to MOLT-4 cells decreased from 37.66%±5.43% to 19.35%±2.37% (P=0.04). Figure 15 ).

[0141] Example 5. Validation of in vivo anti-leukemia activity in a T-ALL mouse model 1. Efficacy evaluation of CXCR4 αCCR9 CAR-T cell anti-extramedullary T-ALL model ① The experimental animals were 12 highly immunodeficient NCG mice (SPF grade), all female and aged 6–8 weeks.

[0142] ② Secure the mice in a mouse restraint device and disinfect their tails with 75% alcohol. Draw pre-mixed MOLT-4 cell suspension into a syringe and insert the needle subcutaneously through the left axilla of the mouse. Inject 100 μL of cell suspension (concentration 5 × 10⁻⁶) subcutaneously into each mouse. 7 / mL). Palpable subcutaneous nodules, larger than 100 mm, formed in mice. 3 After successful tumorigenesis, mice were divided into experimental and control groups. The experimental groups consisted of four subgroups: NCT group, αCCR9 CAR-T group, CXCR4 αCCR9 CAR-T group, and CXCR4-T group. The αCCR9 CAR-T group received 100 μL of effector cells via tail vein injection, totaling 1 × 10⁻⁶ cells. 7 100 μL of αCCR9 CAR-T cells were injected into the CXCR4 αCCR9 CAR-T group via tail vein injection, for a total of 1×10⁶ cells. 7 100 μL of CXCR4 αCCR9 CAR-T cells were injected into the CXCR4-T cell group via tail vein injection, for a total of 1×10⁶ cells. 7 100 μL of CXCR4-T cells. The control group consisted of NCT cells, with 100 μL of effector cells injected via the tail vein, for a total of 1 × 10⁶ cells. 7 One NCT cell. The cells were uniformly grouped according to tumor burden and then infused with effector cells for treatment.

[0143] ③ Treatment options for extramedullary tumors Mice were divided into four groups: αCCR9 CAR-T group; CXCR4 αCCR9 CAR-T group; CXCR4-T group; and NCT group. Each group consisted of three mice, and each mouse received 1×10⁻⁶ CAR-T injections. 7 One effector cell.

[0144] Each group inputs 1 × 10 7 Effector T cells (cell concentration 1×10⁻⁶) 8 Effector cells were administered via tail vein ( / mL). The day of tumor implantation was defined as D0.

[0145] ④ Monitoring and Detection 1) Weight monitoring: The first time on the day of reinfusion and twice a week thereafter; 2) Subcutaneous tumor measurement: reinfusion twice a week; result: To comprehensively evaluate the therapeutic effect of CXCR4 αCCR9 CAR-T cells in an extramedullary T-ALL model, this study used NCG mice to construct an extramedullary leukemia model and conducted an in-depth study on its efficacy.

[0146] Based on the results of previous extramedullary tumorigenesis experiments, this study adopted the subcutaneous tumorigenesis experimental procedure ( Figure 16 (A) Leukemia burden was monitored by measuring subcutaneous tumor volume. At D20, sclerotic nodules appeared at the tumor site. Mice treated with CXCR4 αCCR9 CAR-T cells and αCCR9 CAR-T cells showed a significant slowdown in tumor growth rate in week 1 post-treatment, and tumor volume was unmeasurable after week 2, indicating complete remission of extramedullary leukemia. Notably, the extramedullary lesions shrank faster in the CXCR4 αCCR9 CAR-T cell group than in the αCCR9 CAR-T cell group, and the difference was statistically significant. P= 0.049) Figure 16 B). This study continued observation until day 70 post-treatment. Results showed that no tumor recurrence occurred in either group of mice receiving CAR-T cell therapy. Furthermore, no significant weight loss was observed in the mice receiving CAR-T cell therapy throughout the treatment process. P =0.786), fever, or lethargy, among other adverse reactions, indicate that CXCR4 αCCR9 CAR-T cells and αCCR9 CAR-T cells have good tolerability and few adverse reactions. Figure 16 C).

[0147] CXCR4 αCCR9 CAR-T cells demonstrated superior efficacy in the treatment of extramedullary leukemia, with significantly faster shrinkage of extramedullary lesions compared to αCCR9 CAR-T cells. Furthermore, no relapse was observed during long-term observation, further confirming their good durability. These results indicate that CXCR4 αCCR9 CAR-T cells are a promising candidate for the treatment of extramedullary T-ALL.

[0148] To evaluate the in vivo persistence and tissue homing ability of CXCR4 αCCR9 CAR-T cells, we sacrificed mice at the end of the experiment and measured the proportion of CAR-T cells in their bone marrow. Figure 17 The results showed that the percentage of T cells in the bone marrow of mice in the NCT group was 0.65%±0.41%, in the αCCR9 CAR-T group it was 2.56%±0.39%, and in the CXCR4 αCCR9 CAR-T group it was 1.56%±0.29%. Compared with αCCR9 CAR-T cells, CXCR4 αCCR9 CAR-T cells showed a significantly increased migration to the bone marrow. P= 0.048); compared with CXCR4-T cells, CXCR4 αCCR9 CAR-T cells showed a significantly increased migration to the bone marrow ( ). P= The value was 0.003, which is considered to be related to the expansion of the CAR-T cell killing target cell process. This indicates that the introduction of CXCR4 significantly enhances the chemotaxis and homing ability of CAR-T cells to the bone marrow.

[0149] 2. Efficacy evaluation of CXCR4 αCCR9 CAR-T cell therapy in combined extramedullary and intramedullary T-ALL models Constructing a MOLT-4 luciferase tail vein T-ALL model combined with a MOLT-4 extramedullary model ① Twenty female NCG mice aged 6–8 weeks (SPF grade) were used.

[0150] ② Fix the mouse in the restraint device. After preparing the skin under the left armpit, disinfect the skin with 75% alcohol. Draw a well-mixed suspension of MOLT-4 cells (3×10⁻⁶). 7 100 μL of cell suspension (5 × 10⁹ / mL) was injected subcutaneously into the left axilla of mice (extramedullary tumor model). Simultaneously, 100 μL of a 5 × 10⁹ / mL concentration of [unspecified substance] was slowly injected via the tail vein. 7 100 μL of MOLT-4 luciferase cell suspension (whole-body intramedullary model) was injected to simulate combined extramedullary and intramedullary leukemia. Immediately after injection, pressure was applied to the puncture site for approximately 10 seconds to stop bleeding. Once a clear nodule formed at the subcutaneous injection site and in vivo fluorescence imaging showed a positive result, patients were grouped evenly according to tumor burden, and then CAR-T and other effector cell infusion therapy was initiated.

[0151] ③ Treatment plan Grouping: NCT cell group; αCCR9 CAR-T group; CXCR4 αCCR9 CAR-T group; CXCR4-T group. Five mice were in each group, and each mouse received 1×10⁻⁶ cells. 7 One effector cell.

[0152] The day of CAR-T cell infusion is defined as D0 ( Figure 18 A). Effector cells were injected into each group via the tail vein.

[0153] ④ Monitoring and Detection Weight monitoring: The first time on the day of reinfusion and once a week thereafter; In vivo imaging: twice during the first week and once a week thereafter; Subcutaneous tumor measurement: The size of the subcutaneous tumor is measured twice a week.

[0154] result: To better reflect the clinical characteristics of T-ALL patients with "systemic leukemia combined with extramedullary lesions", this study simultaneously constructed an extramedullary leukemia model and a systemic leukemia model in NCG mice to evaluate the in vivo anti-leukemia activity of CAR-T cells.

[0155] The results show that ( Figure 18 In the CXCR4-T and NCT groups, the fluorescence signal in mice remained continuously enhanced, indicating a progressively worsening leukemia burden, and the mice died successively from day 21 onwards, suggesting no therapeutic effect. In contrast, the fluorescence signal in the CXCR4 αCCR9 CAR-T and αCCR9 CAR-T groups decreased significantly from day 15, showing significant anti-leukemia activity, and the fluorescence intensity decrease was similar in the two groups, suggesting comparable efficacy against systemic leukemia. Figure 18 B). By day 21, almost no fluorescent signal was observed in the two groups of CAR-T treated mice, indicating a significant reduction or even elimination of leukemia burden. Figure 18 C). Furthermore, during treatment, the body weight of mice in both groups remained stable without significant decrease (P=0.091), and no adverse reactions such as lethargy or weight loss were observed, indicating that CXCR4 αCCR9 CAR-T cells showed good tolerance. Figure 18 D).

[0156] Simultaneously, the extramedullary leukemia burden was assessed by measuring the volume of subcutaneous nodules. Results showed ( Figure 19From day 7 after treatment with CXCR4 αCCR9 CAR-T and αCCR9 CAR-T cells, nodule growth slowed and gradually shrank. In the CXCR4 αCCR9 CAR-T group, all four mice achieved complete remission, with only one relapsing on day 50; while in the αCCR9 CAR-T group, two mice achieved complete remission, and the other two showed nodule enlargement after day 40, indicating leukemia progression.

[0157] In conclusion, CXCR4 αCCR9 CAR-T cells demonstrated superior efficacy in treating both systemic and extramedullary leukemia, with a lower relapse rate. This indicates that the introduction of CXCR4 enhances the migration and chemotaxis of CAR-T cells to extramedullary leukemia lesions, significantly improving the treatment outcome for extramedullary leukemia.

[0158] Example 6. In vivo distribution of CAR-T Preparation of DIR dye working solution and CAR-T cell labeling First, dissolve the DIR dye powder in anhydrous DMSO to prepare a 5 mM stock solution. Take 40 μL of this stock solution, dilute it to 40 mL with PBS, and mix well to obtain the DIR working solution.

[0159] 3 × 10⁻⁶ αCCR9 CAR-T cells and CXCR4 αCCR9 CAR-T cells were respectively collected. 7 Centrifuge and discard the supernatant. Resuspend the cell pellet in 30 mL of pre-chilled DIR working solution and incubate at 37°C, 5% CO2, in the dark for 20 minutes. After incubation, centrifuge at 1000-1500 rpm for 5 minutes and discard the supernatant to remove unbound dye. Then, resuspend the cells in pre-warmed complete culture medium and repeat the washing steps twice. Finally, resuspend the cells in 600 μL of PBS for subsequent experiments.

[0160] 2. DIR fluorescence detection CAR-T cells can be imaged and analyzed using fluorescence microscopy and flow cytometry to verify the staining effect.

[0161] 3. Dynamic detection of CAR-T cells in a systemic combined with extramedullary T-ALL mouse model 1) Construct tail vein and subcutaneous T-ALL models.

[0162] Fluorescence in vivo imaging confirmed successful tumor implantation. Mice were then restrained in mouse restraint devices. After disinfecting the tails of each mouse with 75% alcohol, a thoroughly mixed sample of 1×10⁻⁶ alcohol was drawn. 7100 μL of effector cells were injected into mice via the tail vein (D0) after the procedure. Local pressure was applied to stop bleeding after the procedure. The mice were then returned to their cages for further observation and rearing.

[0163] Grouping: NCT group (3 mice); αCCR9 CAR-T group (5 mice); CXCR4 αCCR9 CAR-T group (5 mice); CXCR4-T group (5 mice).

[0164] 2) In vivo dynamic monitoring and endpoint analysis (1) Weight monitoring: The first weight was measured on the day of cell reinfusion (day 0), and then the measurement was repeated once a week at a fixed time until the end of the experiment.

[0165] (2) Hematological parameters: Blood samples were collected from the medial canthal venous plexus on the day of cell infusion and twice a week thereafter. After the mice were sacrificed at the end of the experiment, major organs were collected for subsequent flow cytometry analysis of CAR-T cell infiltration and distribution.

[0166] (3) In vivo imaging tracking: Using near-infrared in vivo imaging technology, mice were imaged on the day of cell infusion and twice a week thereafter to observe the distribution of CAR-T cells in vivo in a non-invasive manner in real time.

[0167] result: To verify the effect of leukemia cells on the distribution of CAR-T cells, this study used tumor-bearing mice with tumors induced subcutaneously and via the tail vein to further verify the in vivo distribution of CAR-T cells. Figure 20 The experimental flowchart is as follows: Figure 20 A. In vivo imaging results showed that, compared with the αCCR9 CAR-T group, the CXCR4 αCCR9 CAR-T cells were more widely distributed in the head and limbs. Figure 20 B). Further analysis of the percentage of fluorescence intensity in the head, trunk, and bilateral thighs relative to the total body fluorescence intensity ( Figure 20 (C) The results showed that 4 hours after CAR-T infusion and at D1, D3, and D6, the fluorescence intensity in the bilateral thighs of mice in the CXCR4 αCCR9 CAR-T cell group was significantly higher than that in the NCT group, with P values ​​of 0.0151, 0.0026, 0.0001, and 0.0006, respectively. At D3 and D6 after CAR-T infusion, the fluorescence intensity in the αCCR9 CAR-T cell group was significantly higher than that in the NCT group, with P values ​​of 0.0170 and 0.0088, respectively.

[0168] The fluorescence intensity percentage in both thighs of the CXCR4 αCCR9 CAR-T cell group and the αCCR9 CAR-T cell group was higher than that of the NCT group, but the increase in the percentage of CXCR4 αCCR9 CAR-T cells was more significant. This suggests that CXCR4 αCCR9 CAR-T cells are more widely distributed in the bone marrow.

[0169] All statistical analyses in Examples 1-6 were performed using GraphPad Prism 9.0. Data are expressed as mean ± standard deviation (SD) or standard error of the mean (SEM). Between-group comparisons were performed using t-tests (paired or unpaired) and one-way and multi-way ANOVA. ns indicates no significance. P <0.05 indicates statistical significance, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001.

[0170] Examples 1-6 demonstrate that CXCR4 empowerment significantly enhances the chemotaxis and distribution of CAR-T cells to bone marrow and extramedullary leukemia lesions, potentially clearing residual leukemia cells in the bone marrow of T-ALL patients and improving the treatment efficacy of extramedullary leukemia. This study suggests that CXCR4-empowered CAR-T cells may represent a key breakthrough in the treatment of complex leukemias and their extramedullary lesions, providing a more reliable treatment option for clinical applications.

[0171] This document uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, based on the ideas of the present invention, there will be changes in the specific implementation methods and application scope. Modifications and improvements to the present invention are possible without exceeding the concept and 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 novel anti-CCR9 CAR, characterized in that, Its nucleic acid sequence contains, from the 5' end to the 3' end, nucleic acid sequences encoding anti-CCR9scFv, hinge and transmembrane domain, co-stimulatory domain, CD3ζ intracellular signaling domain, and CXCR4.

2. The novel anti-CCR9 CAR as described in claim 1, characterized in that, The amino acid sequences of the light chain variable region and the heavy chain variable region of the anti-CCR9 scFv are shown in SEQ ID NO: 1 and 2, or in SEQ ID NO: 3 and 4, respectively; the amino acid sequence of CXCR4 is shown in SEQ ID NO: 5, respectively.

3. The novel anti-CCR9 CAR as described in claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO: 6 or 7.

4. The novel anti-CCR9 CAR as described in claim 1, characterized in that, Its nucleic acid sequence is shown in SEQ ID NO: 8 or 9.

5. A carrier, characterized in that, It has a nucleic acid sequence encoding a novel anti-CCR9 CAR as described in any one of claims 1-4, or has an amino acid sequence of a novel anti-CCR9 CAR as described in any one of claims 1-4.

6. A host cell, characterized in that, It comprises one or more of the following: a nucleic acid sequence encoding the novel anti-CCR9 CAR as described in any one of claims 1-4, an amino acid sequence of the novel anti-CCR9 CAR as described in any one of claims 1-4, and a vector as described in claim 5.

7. An immune cell carrying a novel anti-CCR9 CAR as described in any one of claims 1-4.

8. The immune cells as described in claim 7, characterized in that, It is one or more of CAR-T cells, CAR-NK cells, CAR-DC cells, CAR-B cells, CAR-monocytes, and CAR-M cells.

9. The immune cells as described in claim 8, characterized in that, It covers both in vivo and in vitro generation.

10. The immune cells as described in claim 7, characterized in that, Enabling CXCR4 to fight CCR9 CAR-T.

11. A method for preparing immune cells as described in claim 7, characterized in that, It was obtained by transducing immune cells with a lentiviral vector carrying the novel anti-CCR9 CAR.

12. The use of the novel anti-CCR9 CAR as described in any one of claims 1-4 or the immune cells as described in any one of claims 7-10 in the preparation of a medicament for treating tumors with high CCR9 expression complicated by extramedullary diseases.

13. The application as described in claim 12, characterized in that, The tumor with high CCR9 expression and concurrent extramedullary disease is acute T-lymphoblastic leukemia with concurrent extramedullary disease.

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