Preparation and Application of a Non-Natural Humanized Chimeric Antigen Receptor Against Human CD45RA

Abstract

This invention provides a method for preparing a non-natural humanized chimeric anti-human CD45RA antigen receptor, which specifically binds to the human CD45RA antigen by expressing CAR protein on T cells. Based on a humanized 3A4 antibody, this invention constructs a lentiviral expression vector pLenti / Hu3A4-4-1BB-3ζ. Compared with murine CAR, the humanized CAR significantly reduces immunogenicity, noticeably decreases the production of anti-scFv antibodies, and avoids the HAMA reaction. Research results show that Hu3A4CAR-T cells can specifically bind to the CD45RA-highly expressing myeloid leukemia cell line KG1a. Hu3A4CAR-T cells can target and kill 3A4-positive cell lines and leukemia cells, alleviate the human anti-mouse antibody response, and significantly reduce the production of anti-scFv antibodies, thereby ensuring the CAR's killing activity.

Description

Technical Field

[0001] This invention pertains to biotechnology and relates to the preparation and use of a non-natural humanized chimeric anti-human CD45RA antigen receptor. It describes the creation of Hu3A4CAR-T cells by transfecting or infecting human T cells with the humanized chimeric anti-human CD45RA antigen receptor (Hu3A4CAR) gene via a vector, and its use in the preparation of drugs for targeted therapy of hematologic malignancies. Background Technology

[0002] According to data from the International Agency for Research on Cancer (IARC) of the World Health Organization, in 2012, there were over 14 million new cases of malignant tumors worldwide, and approximately 8 million people died from them, making it a leading cause of death globally. Malignant tumors have become a major public health problem; since 2010, they have surpassed cardiovascular and cerebrovascular diseases to become the leading cause of death. Leukemia is one of the most common hematologic malignancies, with a global incidence rate of approximately 4.62 per 100,000 and a local incidence rate of approximately 5.17 per 100,000, ranking first in both pediatric malignant tumor incidence and mortality. While the prognosis of leukemia has significantly improved with in-depth research into its diagnosis and treatment, the continuous emergence of new anti-tumor drugs, and the implementation of hematopoietic stem cell transplantation, a significant number of deaths still occur each year, primarily due to tumor recurrence and treatment-related complications. It is reported that the overall survival rate of adult acute lymphoblastic leukemia (ALL) is only 30%-40%, and 20% of children with ALL still relapse. For relapsed and drug-resistant patients, there is a lack of effective alternative drugs, which brings great difficulties to clinical treatment.

[0003] In recent years, with the advancements in molecular biology and antibody engineering technology, targeted therapy for leukemia that kills tumor cells without damaging normal cells has received widespread attention and development. CAR-T cell therapy utilizes CAR-modified T lymphocytes to target and eliminate malignant tumors in a major histocompatibility complex-independent manner. It combines the specificity of CAR antibody target markers with the potent effector mechanism of T cells. When this receptor is expressed on the surface of a patient's T cells, it can redirect T cells to target antigens on tumor cells, leading to T cell activation and tumor cell death. In patients with B-cell malignancies, anti-CD19 CAR-T cell targeted therapy has been shown to lead to sustained disease remission and prolonged survival. The successful application of CAR-T cells in B-cell tumors has brought new hope for the treatment of relapsed and refractory AML. However, compared with B-cell malignancies, the clinical trial results of this therapy in the treatment of AML are significantly worse than those in ALL. The most challenging task is to select ideal target molecules. Targeting antigens on AML cells present in normal bone marrow lineages can lead to severe neutropenia, which can have a devastating impact on the patient's health even if the AML has been eradicated. Few targets have been approved for clinical treatment. To date, only CD123CAR has been approved by the US FDA for the clinical treatment of relapsed / refractory AML, while CARs targeting other targets are still in the process of therapeutic trials. Current research has identified cell surface targets for CAR-T therapy that can target AML, including CD33, CD45, CD123, FLT3, Lewis-Y, and CLL-1. However, the effects are mostly unsatisfactory, and there is an urgent need to find new targets. In CAR-T therapy research, identifying a highly specific target is the primary issue in order to improve the selective killing of tumors by targeted drugs and reduce toxic side effects on non-target tissues.

[0004] CD45 antigen is an evolutionarily conserved receptor protein tyrosine phosphatase expressed on all nucleated leukocytes in the hematopoietic system, and is crucial for efficient T and B cell antigen receptor signal transduction. Multiple CD45 isoforms can be generated through complex alternating splicing of exons 4(A), 5(B), and 6(C) in the extracellular domain of the molecule. CD45 is widely expressed on the surface of leukocytes but not on other non-hematopoietic tissue cells, which largely avoids the side effects of off-target effects leading to damage to other organs. A, Schraven B, Bommhardt U. CD45 in human physiology and clinical medicine. Immunology Letters. 2018; 196:22-32. Currently, radiolabeled CD45 antibodies are used internationally for pretreatment before hematopoietic stem cell transplantation. Glatting G utilizes... 111Eight bone marrow transplant patients were pretreated with the In-labeled anti-CD45 monoclonal antibody YAML568. Biological distribution analysis showed that the concentration in red bone marrow was significantly higher than in vital tissues such as the spleen, liver, and kidneys. While significantly suppressing bone marrow function, no significant impairment was observed in the function of other vital organs. (Glatting G, Müller M, Koop B, Hohl K, Friesen C, Neumaier B, et al. Anti-CD45 monoclonal antibody YAML568: A promising radioimmunoconjugate for targeted therapy of acute leukemia. Journal of Nuclear Medicine: official publication, Society of Nuclear Medicine.)

[0005] 2006;47(8):1335-41.). In a phase I clinical trial, Phuong Vo found that in 10 patients with advanced AML and 5 patients with high-risk myelodysplastic syndrome, the use of I-anti-CD45 antibody combined with fludarabine and 2 Gy whole-body irradiation as part of the pre-transplantation conditioning regimen for HLA-matched peripheral blood stem cell transplantation achieved a relatively ideal bone marrow clearance effect (Vo P, Gooley TA, Rajendran JG, Fisher DR, Orozco JJ, Green DJ, et al. Yttrium-90-labeled anti-CD45 antibody followed by a reduced-intensity hematopoietic cell transplantation for patients with relapsed / refractory leukemia or myelodysplasia. Haematologica.

[0006] 2020;105(6):1731-7.). However, ordinary CD45 antibodies cannot be routinely used for targeted therapy of leukemia, as they can cause patients to develop severe cellular immunodeficiency or neutropenia.

[0007] CD45RA is an allosteric variant of the CD45 molecule, expressed on the surface of naive T cells, B cells, some granulocytes, and some monocytes, but not on activated and memory T cells, mature erythrocytes and platelets, or other non-hematopoietic tissue and organ cells (Li S, Tang Y, Zhang J, Guo X, Shen H. 3A4, a new potential target for band myeloid lineage leukemias. Journal of drug targeting. 2011; 19(9):797-804.). Drug-resistant leukemia stem cells (LSCs) are considered a cause of relapse after treatment for acute myeloid leukemia (AML). CD45RA is expressed in leukemia cells in most AML patients. CD45RA is a specific marker for a subpopulation of leukemia stem cells in acute myeloid leukemia (Kersten B, Valkering M, Wouters R, van Amerongen R, Hanekamp D, Kwidama Z, et al. CD45RA, a specific marker for leukaemia stem cell sub-populations in acute myeloid leukaemia. British journal of haematology. 2016; 173(2):219-35.; Li S, Shen D, Guo X, Liao C, Tang Y. Construction, Expression, and Characterization of a Novel Human-Mouse Chimeric Antibody, Hm3A4: A Potential Therapeutic Agent for B and Myeloid Lineage Leukemias. DNA and cellbiology. 2018; 37(9):778-85.). Developing CAR-T therapy targeting the CD45RA antigen may provide a new targeted treatment for leukemia in clinical practice. The success rate of CAR depends on both target modulation and the degree of humanization of the monoclonal antibody that recognizes the target.Most CARs are murine in origin. Murine CARs are immunogenic to humans. Their immunogenicity stimulates a human anti-mouse antibody (HAMA) response, which can produce human anti-mouse scFv antibodies. The CAR is neutralized, which greatly weakens the function of the CAR. HAMA also causes some allergic reactions, ranging from relatively mild symptoms such as rash, fever and dizziness to serious side effects such as shock and organ failure, which may even be fatal. Summary of the Invention

[0008] One objective of this invention is to provide a method for preparing a non-natural anti-human CD45RA humanized chimeric antigen receptor, which is a CAR-T cell preparation capable of recognizing CD45RA antigen-positive leukemia cells, namely, a method for constructing T cells (Hu3A4CAR-T) modified with the non-natural anti-human CD45RA humanized chimeric antigen receptor gene (Hu3A4CAR gene): T cells express CAR protein, which specifically binds to the human CD45RA antigen. The target recognition region of the chimeric antigen receptor is the human CD45RA antigen target. The HuCD45RACAR or Hu3A4CAR gene consists of the following structure: CD8a leader is the CAR leader sequence (SEQ ID NO 1), humanized CD45RA single-chain antibody Hu3A4scFv is the target recognition region (SEQ ID NO 3) and (SEQ ID NO 5), CD8a hinge is the hinge region (SEQ ID NO 7), CD8a transmembrane is the CAR transmembrane region (SEQ ID NO 9), and the intracellular signal transduction region of 4-1BB (CD137) (SEQ ID NO 11) and the intracellular signal transduction region of CD3ζ (SEQ ID NO 13) are linked together to form the complete humanized CD45RACAR gene, namely HuCD45RACAR or Hu3A4CAR gene.

[0009] The CAR protein is expressed by T cells and specifically binds to the human CD45RA antigen. The target recognition region of the chimeric antigen receptor is the human CD45RA antigen target. The Hu3A4CAR gene is composed of the following structure: CD8a leader (SEQ ID NO 1) is the CAR leader sequence, humanized CD45RA single-chain antibody 3A4scFv (SEQ ID NO 3)+ (SEQ ID NO 5) is the target recognition region, CD8a hinge (SEQ ID NO 7) is the hinge region, CD8a transmembrane (SEQ ID NO 9) is the CAR transmembrane region, and the intracellular signal transduction region of 4-1BB (CD137) (SEQ ID NO 11) and the intracellular signal transduction region of CD3ζ (SEQ ID NO 13) are linked together to form a complete humanized CD45RACAR gene, namely HuCD45RACAR or Hu3A4CAR gene.

[0010] Based on the existing humanized immunoglobulin against human CD45RA (ZCH-6-Hu3A4 monoclonal antibody, abbreviated as Hu3A4) gene sequence, this invention uses molecular biology techniques to develop a non-natural humanized chimeric antigen receptor against human CD45RA (Hu3A4CAR), which is prepared through the following steps:

[0011] (1) Consult comparative literature to determine the basic structure of the second-generation 3A4CAR, search the National Center for Biotechnology Information (NCBI) to verify the gene sequences of the 3A4CAR hinge region, transmembrane region and intracellular signal transduction region, and commission a company to synthesize pUC / Hu3A4scFv-4-1BB-3ζ;

[0012] (2) The lentiviral expression vector pLenti / Hu3A4-4-1BB-3ζ of Hu3A4CAR was constructed by genetic engineering and molecular cloning methods;

[0013] (3) Identification of Hu3A4-CAR lentiviral expression vector activity: detect whether the vector is expressed and whether the protein is correctly localized, and determine the transfection efficiency of the lentiviral expression vector.

[0014] This invention constructs a lentiviral expression vector containing the nucleotide sequence CD8a leader (SEQ ID NO 1)-Hu3A4scFv (SEQ ID NO 3)(SEQ ID NO 5)-CD8a hinge (SEQ ID NO 7)-CD8a transmembrane region (SEQ ID NO 9)-CD137 (SEQ ID NO 11)-CD3ζ sequence (SEQ ID NO 13). The vector is the lentiviral vector pLenti-Hu3A4CAR gene.

[0015] The present invention provides an effector cell, wherein the host cell is a human T cell, namely HuCD45RACAR-T cell or Hu3A4CAR-T cell, which is transfected by the above-mentioned expression vector.

[0016] Another object of the present invention is to provide the use of the chimeric antigen receptor in the preparation of medicaments for treating cell-mediated diseases caused by the expression of the CD45RA antigen. The diseases refer to oncological diseases related to the expression of the CD45RA membrane antigen, primarily hematological malignancies, specifically acute myeloid leukemia, acute lymphoblastic leukemia, malignant lymphoma, chronic myeloid leukemia, and chronic lymphocytic leukemia.

[0017] The lentiviral expression vector pLenti-Hu3A4CAR provided by this invention is used for the preparation of Hu3A4CAR-T cells and can be used to treat patients with CD45RA-positive leukemia, lymphoma, etc. The lentiviral expression vector pLenti-Hu3A4-4-1BB-3ζ, i.e., pLenti-CD45RACAR gene expression vector of this invention, can successfully infect human T cells, and the Hu3A4CAR-T cells developed can effectively kill CD45RA(3A4)-positive target cells KG1a cells and Raji cells. Hu3A4CAR-T cells recognize target antigens independently of the antigen presentation process and are not restricted by the major histocompatibility complex (MHC), thus overcoming tumor immune evasion and more effectively killing 3A4-positive tumor cells.

[0018] The advantages of this invention are as follows: This invention utilizes a carefully developed non-natural HuCD45RACAR (Hu3A4CAR) protein, whose antigen recognition is independent of the antigen presentation process and is not MHC-restricted, thus overcoming tumor immune evasion and more effectively killing 3A4-positive tumor cells. This invention has conducted a series of experiments using the developed non-natural HuCD45RACAR (Hu3A4CAR). After successfully infecting human T cells with Hu3A4CAR to produce Hu3A4CAR-T cells, in vitro antigen-binding activity assays showed that Hu3A4CAR-T cells could specifically bind to the CD45RA-highly expressing myeloid leukemia cell line KG1a. Hu3A4CAR-T cells can target and kill 3A4-positive cell lines and leukemia cells in newly diagnosed AML patients. Hu3A4CAR-T cells can alleviate human anti-mouse antibody responses and significantly reduce the production of anti-scFv antibodies, thereby ensuring the killing activity of CAR.

[0019] This invention proposes using a humanized CAR to reduce the response of human anti-mouse antibodies and significantly decrease the production of anti-scFv antibodies, thereby ensuring the CAR's killing activity. To this end, the 3A4 antibody has been humanized and named Hu3A4, achieving a humanization rate of 93%. Since the affinity and specificity of monoclonal antibodies are mainly determined by the amino acid sequence of the supervariable region (or complementarity-determining region, CDR) of the antibody's light and heavy chains, any monoclonal antibody with the same CDR sequence will have essentially the same specificity, affinity, and therapeutic effect in recognizing the target antigen. After obtaining a clinically valuable mouse antibody sequence, humanizing the amino acid sequence of its non-CDR region (framework region) can yield a humanized monoclonal antibody with the same biological activity. Hu3A4 can specifically bind to and kill myeloid and B-line LSCs. Constructing a Hu3A4-CAR using the Hu3A4 monoclonal antibody has significant scientific importance and clinical application prospects for targeting and killing leukemia stem cells and treating refractory AML. Attached Figure Description

[0020] Figure 1 Schematic diagram of the Hu3A4-CAR gene structure.

[0021] Figure 2 Electrophoresis images of pLenti / Hu3A4-4-1-BB-3z plasmid extraction (left) and XbaI / SalI digestion (right); Lane 1: electrophoresis of pLenti / Hu3A4-4-1BB-3ζ plasmid, Lanes 2 and 3: XbaI and SalI single enzyme digestion of pLenti / Hu3A4-4-1BB-3ζ plasmid, respectively, and Lane 4: XbaI and SalI double enzyme digestion of pLenti / Hu3A4-4-1BB-3ζ plasmid.

[0022] Figure 3 Plasmid map constructed from the lentiviral expression vector pLenti / Hu3A4-4-1BB-3ζ.

[0023] Figure 4 Flow cytometry was used to detect the expression of the pLenti / Hu3A4-4-1BB-3ζ lentiviral expression vector.

[0024] Figure 5 Western blotting confirmed that Hu3A4CAR and Mu3A4CAR were successfully expressed on T cells.

[0025] Figure 6 The killing effects of Hu3A4CAR-T, Mu3A4CAR-T and T cells on leukemia cell lines. In the figure, bar represents the mean ± standard error. Detailed Implementation

[0026] The present invention will be further described in conjunction with the accompanying drawings and embodiments.

[0027] Example 1

[0028] The nucleotide sequence of one gene in this invention:

[0029] Basic structure of Hu3A4-CAR Figure 1 The CAR sequence is as follows: CD8a leader is the leader chain, Hu3A4scFv is the target recognition region, CD8a hinge is the hinge region, CD8aTM is the transmembrane region, and the intracellular co-stimulatory signal transduction region of 4-1BB and the intracellular signal transduction region of CD3ζ are tandemly linked to form the intracellular segment of Hu3A4CAR. The sequences of each part were verified by comparison with the NCBI gene database. Specifically, CD8a Hinge+TM (NM_001768): 207 bp; 4-1BB (NM_001561) intracellular segment: 126 bp; CD3ζ (NM_198053) intracellular segment sequence: 336 bp. The nucleotide sequence (SEQ ID NO 3) and amino acid sequence (SEQ ID NO 4) of the humanized Hu3A4scFv heavy chain gene, and the nucleotide sequence (SEQ ID NO 5) and amino acid sequence (SEQ ID NO 6) of the Hu3A4scFv light chain gene are also provided.

[0030] Example 2

[0031] The pUC / Hu3A4-4-1BB-3ζ and pLenti / C427z plasmids were digested with two enzymes, respectively. The resulting target fragment Hu3A4-4-1BB-3ζ was ligated to the double-digested vector pLenti using a T4 ligation system. The ligation product was transformed into competent Transblue bacteria, and the obtained positive clones were confirmed by sequencing. The double-digestion electrophoresis bands showed the target band appearing at 1500 bp. Figure 2 This also confirms the successful construction of the lentiviral expression vector pLenti / Hu3A4-4-1BB-3ζ.

[0032] Example 3: Construction of the lentiviral expression vector pLenti / Hu3A4-4-1BB-3ζ

[0033] 1. Following the instructions of the Axygen plasmid DNA mini-extraction kit, plasmids pGEM-T / Hu3A4-4-1BB-3ζ and pLenti / C427z were extracted and purified.

[0034] 2. Double enzyme digestion reaction

[0035] (1) The plasmids pGEM-T / Hu3A4-4-1BB-3ζ and pLenti / C427z were digested with XbaI and SalI respectively, and reacted at 37℃ for 2 hours.

[0036] (2) The enzyme digestion products were electrophoresed on a 1% agarose gel at 100V for 30 min, and the results were observed and saved.

[0037] (3) Recover the target fragment from the above gel.

[0038] 3. Connection reaction

[0039] 4. Transformation of DH5α competent cells

[0040] 5. Extraction of pLenti / Hu3A4-4-1BB-3ζ plasmid

[0041] Plasmid extraction, electrophoresis, and bacterial sequencing were performed according to the Axygen plasmid DNA extraction kit instructions. Plasmids with correct sequences were stored at -80°C in 15% glycerol. The plasmid was labeled pLenti / Hu3A4-4-1BB-3ζ. The lentiviral expression vector pLenti / Hu3A4-4-1BB-3ζ plasmid map is shown below. Figure 3 .

[0042] Example 4

[0043] Activity identification of lentiviral expression vector pLenti / Hu3A4-4-1BB-3ζ

[0044] 1. Transfection-grade plasmid pLenti / Hu3A4-4-1BB-3ζ

[0045] Follow the instructions for the QIAGEN plasmid purification mini kit.

[0046] 2. CHO cell resuscitation and culture

[0047] CHO cells frozen in liquid nitrogen were quickly removed and thawed by rapid shaking in a 37°C water bath. The cells were then transferred to a 15ml test tube using a sterile pipette in a laminar flow hood. RPMI 1640 basal culture medium was added to a final volume of 15ml. The tube was capped and centrifuged at 1500 rpm for 10 minutes. The supernatant was discarded, and the cells at the bottom of the tube were retained. The cells were resuspended in 5ml of 10% FBS RPMI 1640 culture medium and aseptically transferred to a T25 culture flask. The flask was then incubated at 37°C, 5% CO2, and 100% humidity for 72 hours. Cells were then collected for further experiments.

[0048] 3. Transfection of eukaryotic cells with CHO

[0049] Following the instructions for X-treme GENE HP DNA Transfection Reagent, CHO cells were transfected. Two groups were established: an empty vector group and a pLenti / Hu3A4-4-1BB-3ζ group.

[0050] (1) One day before transfection, CHO cells were seeded into 6-well plates with antibiotic-free RPMI 1640 complete medium, with a cell suspension volume of 2 ml per well and a cell count of 8 × 10⁶ cells per well. 5 The cell confluence reaches 80%-90%;

[0051] (2) Before transfection, replace RPMI1640 medium with Opti-MEM I serum-free medium;

[0052] (3) Take 2 μg each of pLenti empty plasmid and pLenti / Hu3A4-4-1BB-3ζ transfection grade plasmid, dilute them to 200 μl with Opti-MEM I medium, and mix thoroughly.

[0053] (4) Add 4 μl (2:1) of X-treme GENE HP DNA Transfection Reagent to 200 μl of Opti-MEM I medium containing 2 μg of transfection grade plasmid, mix thoroughly, and incubate at room temperature for 15-30 minutes.

[0054] (5) Slowly add the above DNA-liposome mixture into a 6-well culture plate and mix gently;

[0055] (6) After culturing in an incubator containing 5% CO2 at 37°C and saturated humidity for 24 hours, the cells were taken out and passaged into new 6-well plates at a ratio of 1:3.

[0056] 4. Flow cytometry detection of pLenti / Hu3A4-4-1BB-3ζ protein expression

[0057] (1) Count the CHO cells 2-3 days after transfection and adjust the cell concentration to 1×10⁶. 6 / ml, take 100μl of cell suspension into flow cytometry tubes, set up empty vector group and pLenti / Hu3A4-4-1BB-3ζ group;

[0058] (2) Each group has 2 tubes. The first tube is the control tube, with 1 μl of PE Streptavidin added. The second tube is the test tube, with GAH IgG (Fab) added. ’ 2-3 μl, incubate at 4°C in the dark for 30 minutes;

[0059] (3) Wash with PBS, centrifuge at 1000 rpm for 5 min twice, add 1 μl of PE Streptavidin to the second branch tube, and incubate at 4°C in the dark for 30 min.

[0060] (4) Wash with PBS, centrifuge at 1000 rpm for 5 min twice, and then analyze by flow cytometer.

[0061] Seventy-two hours after infecting CHO cells with the lentiviral expression vector, we collected the infected CHO cells and used flow cytometry to detect the expression of Hu3A4CAR on their surface. Figure 4 The results showed that the lentiviral expression vector pLenti / Hu3A4CAR could successfully express the recombinant Hu3A4CAR protein on CHO cell membranes, with an expression efficiency of 37.6%.

[0062] Example 5

[0063] We extracted proteins from T cells transfected with Hu3A4CAR and Mu3A4CAR, as well as from T cells not transfected with CAR, and used Western blotting (primary antibody: mouse anti-human CD3ζ antibody, 1:1000 dilution; secondary antibody: goat anti-mouse HRP, 1:2000 dilution) to identify the differences in protein expression between CAR-T cells and ordinary T cells. Figure 5 The results showed that the proteins expressed by T cells transfected with Hu3A4CAR and Mu3A4CAR and untransfected CART cells were all expressed. The CD3ζ chain has a molecular weight of about 15 kDa; T cells transfected with Hu3A4CAR and Mu3A4CAR respectively also express Hu3A4CAR and Mu3A4CAR fusion proteins with CD3ζ, which have a molecular weight of about 55 kDa, consistent with the expected size.

[0064] Example 6

[0065] Targeted killing of leukemia cell lines by Hu3A4CAR-T and comparison with targeted killing by Mu3A4CAR-T

[0066] (1) Nalm-6 cells (3A4 negative), Raji cells (3A4 positive), and KG1a cells (3A4 positive) were used as target cells to perform cytotoxicity experiments. Three effector-to-target ratio gradients of 1:1, 10:1, and 20:1 were set up for each group. The Hu3A4CAR-T cell group and the Mu3A4CAR-T cell group were experimental wells under different gradients, and the corresponding ordinary T cell groups were negative control wells.

[0067] (2) Culture sufficient Raji, KG1a, and Nalm-6 cells in good condition and count the cells. Dilute Calcein-AM stock solution with PBS at a ratio of 400:1, and add an appropriate amount of the diluted solution to the target cells and mix well. After incubating at 37°C for half an hour, add RPMI 1640 medium and centrifuge at 1000 rpm for 5 min. Wash once.

[0068] (3) The target cells were added to RPMI 1640 complete culture medium to adjust the cell concentration to 1×10⁻⁶. 6 / ml, take 100μl of cell suspension and add it to the cell culture plate.

[0069] (4) After removing the magnetic beads, Hu3A4CAR-T cells, Mu3A4CAR-T cells, and T cells were centrifuged at 1000 rpm for 5 min and resuspended in RPMI 1640 complete medium to a cell concentration of 1×10⁻⁶. 6 / ml, add to target cells at the corresponding effector-target ratios of 1:1, 10:1, and 20:1, mix well by pipetting, place in a 48-well plate, and incubate at 37°C in a 5% CO2 incubator.

[0070] (5) After 24 hours, remove the cells from the 48-well plate into a flow cytometer, wash with PBS, centrifuge at 1000 rpm for 5 min, and repeat the washing once.

[0071] (6) Two tubes are set up for each group. The first tube is the isotype control tube and the second tube is the experimental tube. 3 μl each of mouse IgG1-PerCP antibody and mouse IgG1-PE antibody and 1 μl of mouse IgG1-APC antibody are added to the first tube. 3 μl each of mouse anti-human CD34-PerCP antibody and mouse anti-human CD45-PE antibody and 1 μl of mouse anti-human CD3-APC antibody are added to the experimental tube.

[0072] (7) After incubating in the dark for 30 min, add PBS to wash, centrifuge at 1000 rpm for 5 min, repeat washing and centrifugation, discard the supernatant, and then perform flow cytometer detection.

[0073] (8) Kill rate = Target cell death rate in experimental group (number of dead target cells / total target cells) - Target cell death rate in control group (number of dead target cells / total target cells)

[0074] Using 3A4-negative Nalm-6 leukemia cell line, 3A4-positive KG1a myeloid leukemia cell line, and 3A4-positive Raji lymphoma cell line as target cells, respectively, Hu3A4CAR-T or Mu3A4CAR-T cells were added for killing assays. Flow cytometry results showed that ( Figure 6Compared to untransfected CAR-T cells, Hu3A4CAR-T and Mu3A4CAR-T cells exhibit cytotoxicity against both Raji and KG1a cells, with the cytotoxicity increasing as the effector-target ratio increases. However, they show no cytotoxicity against 3A4-negative Nalm-6 cells, indicating that Hu3A4CAR-T and Mu3A4CAR-T cells have good targeted cytotoxicity.

Claims

1. Use of a non-natural anti-human CD45RA humanized chimeric antigen receptor in the manufacture of a medicament for the treatment of B lymphocytic lineage lymphoma, characterized in that, The targeting recognition region of the chimeric antigen receptor is a human CD45RA antigen target, and the chimeric antigen receptor is composed of the following structures: The CD8a leader region is the leader region of the chimeric antigen receptor, and the nucleotide sequence is shown in SEQ ID NO. 1; the humanized CD45RA single-chain antibody 3A4scFv is the targeting recognition region, the nucleotide sequence of the humanized 3A4scFv heavy chain gene is shown in SEQ ID NO 3, and the amino acid sequence is shown in SEQ ID NO 4; the nucleotide sequence of the 3A4scFv light chain gene is shown in SEQ ID NO 5, and the amino acid sequence is shown in SEQ ID NO 6; The CD8a hinge region is the hinge region of the chimeric antigen receptor, and the nucleotide sequence is shown in SEQ ID NO 7; the transmembrane region of CD8a is the transmembrane region of the chimeric antigen receptor, and the sequence is shown in SEQ ID NO 9; the intracellular signal transduction region of CD137 and the intracellular signal transduction region of CD3ζ are the intracellular signal transduction region of the chimeric antigen receptor; the sequence of the intracellular signal transduction region of CD137 is shown in SEQ ID NO 11, and the sequence of the intracellular signal transduction region of CD3ζ is shown in SEQ ID NO 13; the above structures are connected in series to form a complete humanized chimeric antigen receptor.

2. Use of a non-natural anti-human CD45RA humanized chimeric antigen receptor in the manufacture of a medicament for the treatment of acute myeloid leukemia, characterized in that, The targeting recognition region of the chimeric antigen receptor is a human CD45RA antigen target, and the chimeric antigen receptor is composed of the following structures: The CD8a leader region is the leader region of the chimeric antigen receptor, and the nucleotide sequence is shown in SEQ ID NO. 1; the humanized CD45RA single-chain antibody 3A4scFv is the targeting recognition region, the nucleotide sequence of the humanized 3A4scFv heavy chain gene is shown in SEQ ID NO 3, and the amino acid sequence is shown in SEQ ID NO 4; the nucleotide sequence of the 3A4scFv light chain gene is shown in SEQ ID NO 5, and the amino acid sequence is shown in SEQ ID NO 6; The CD8a hinge region is the hinge region of the chimeric antigen receptor, and the nucleotide sequence is shown in SEQ ID NO 7; the transmembrane region of CD8a is the transmembrane region of the chimeric antigen receptor, and the sequence is shown in SEQ ID NO 9; the intracellular signal transduction region of CD137 and the intracellular signal transduction region of CD3ζ are the intracellular signal transduction region of the chimeric antigen receptor; the sequence of the intracellular signal transduction region of CD137 is shown in SEQ ID NO 11, and the sequence of the intracellular signal transduction region of CD3ζ is shown in SEQ ID NO 13; the above structures are connected in series to form a complete humanized chimeric antigen receptor.

3. An expression vector, characterized by, The vector is a lentiviral vector pLenti-Hu3A4CAR, and the lentiviral vector pLenti-Hu3A4CAR comprises the gene of the chimeric antigen receptor of claim 1 or 2.

4. An effector cell, characterized in that, The effector cell is obtained by transfecting human T cells with the expression vector of claim 3. The effector cell is obtained by transfecting human T cells with the expression vector of claim 3.

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