A humanized single-chain antibody targeting human CD19 antigen and its chimeric antigen receptor, immune cell and application

Abstract

The present invention provides a humanized single-chain antibody targeting the human CD19 antigen, its chimeric antigen receptor, immune cells and applications. The humanized single-chain antibody targeting the human CD19 antigen is based on the mouse-derived FMC63 antibody targeting the human CD19 antigen, and the light chain and heavy chain framework regions of the antibody are humanized. The humanized single-chain antibody can bind to the human CD19 antigen with high specificity and high affinity. A chimeric antigen receptor is constructed based on the humanized single-chain antibody targeting the human CD19 antigen, which includes, in sequence, a humanized single-chain antibody targeting the human CD19 antigen, a hinge region domain, a transmembrane region domain and an intracellular region domain. The chimeric antigen receptor sequence containing the humanized single-chain antibody targeting the human CD19 antigen is transduced into immune cells, and the prepared chimeric antigen receptor immune cells containing the humanized single-chain antibody targeting the human CD19 antigen have higher transduction efficiency, stronger killing effect on human CD19-positive tumor cells, and show excellent pharmacological efficacy in animals.

Description

Technical Field

[0001] The present invention belongs to the field of immunotherapy technology, and relates to humanized CD19 antigen-binding single-chain antibodies and chimeric antigen receptor immune cells, and further their application in immunotherapy for patients with autoimmune diseases, leukemia, and lymphoma. Background Art

[0002] Chimeric antigen receptor (CAR)-modified immune cells are currently the most promising immunotherapy drugs and have been used to treat relapsed and refractory leukemia and lymphoma with good therapeutic effects. CAR is primarily composed of three functional domains: the extracellular domain, the transmembrane domain, and the intracellular domain. The extracellular domain is primarily a single-chain antibody (scFv) that recognizes and binds to antigens. The transmembrane domain primarily binds the chimeric antigen receptor to the cell membrane. The intracellular domain consists of a co-stimulatory domain and a signal transduction domain. A single-chain antibody is a genetically engineered antibody that is a small molecule composed of the variable region of the antibody heavy chain (VH) and the variable region of the light chain (VL) connected by a peptide chain. It is the smallest functional structural unit with antibody activity.

[0003] Currently, the indications for immune cell therapy are gradually expanding beyond oncology to include autoimmune diseases, which have a large market. In addition to CAR-T therapy, research on CAR-NK cell therapy for the treatment of systemic lupus erythematosus is also showing a growing trend. CAR-NK immune cell therapy has become a new frontier in fields such as cancer and autoimmune diseases, and has successively received approval from relevant authorities to enter clinical trials. In the field of autoimmune diseases, Nkarta's off-the-shelf CAR-NK cell therapy candidate NKX019 has received approval for its IND for the treatment of systemic lupus erythematosus nephritis. Artiva Biotherapeutics announced that the US FDA has approved its IND application for its allogeneic NK therapy AB-101 (AlloNK) in combination with rituximab for the treatment of systemic lupus erythematosus (SLE). This marks the first application of allogeneic NK cell therapy for the treatment of an autoimmune disease. CAR-T cells possess potent tumor-killing capabilities and specific targeting properties. Several CAR T drugs are already marketed and are highly effective in treating hematological malignancies, but they are expensive and require significant production time. As research progresses, CAR-NK cells may be a safer alternative to CAR-T cells. Compared with CAR-T cell therapy, CAR-NK cells have unique advantages in adoptive cell therapy: (1) CAR-NK cells do not cause long-term B cell deficiency, reducing the risk of infection; (2) CAR-NK cells have a low probability of cytokine storm; (3) CAR-NK cells have multiple killing mechanisms that can kill tumor cells with low or no expression of target antigens, reducing the recurrence of target antigen-negative tumors; (4) Allogeneic transplanted CAR-NK cells do not cause graft-versus-host disease (GvHD); (5) NK cells have multiple sources, meeting the needs of patients whose own cell "quality" and "quantity" are affected by multiple chemotherapy cycles; (6) CAR-NK cells can provide timely "off-the-shelf" treatment.

[0004] CD19 is a B lymphocyte surface antigen, a 95 kDa membrane protein and a member of the immunoglobulin (Ig) superfamily. CD19 expression is restricted to the surface of B lymphocytes, making it one of the earliest B-cell lineage-specific antigens discovered. CD19 is expressed on early precursor B cells and disappears upon terminal differentiation into plasma cells, making it one of the most reliable B-cell surface biomarkers. As a coreceptor of the B-cell antigen receptor complex (BCR) on B lymphocytes, CD19 is highly expressed in most B-cell lymphomas, including diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma, as well as acute lymphoblastic leukemia, chronic lymphoblastic leukemia, and some acute myeloid leukemias. CD19 is not expressed on hematopoietic stem cells, plasma cells, or other normal human tissues. Therefore, CD19-targeted therapies do not cross-react with other normal tissues. Therefore, CD19 has been demonstrated in clinical practice to be a safe and effective target for the treatment of B-cell hematological malignancies.

[0005] In clinical applications, mouse antibodies are highly immunogenic, triggering human anti-mouse antibody (HAMA) reactions, which shorten their half-life and weaken their therapeutic efficacy. Therefore, their clinical therapeutic applications are limited. Humanized design can minimize the immunogenicity of antibodies. Antibody humanization involves the humanization of mouse antibodies. Humanized sequences are designed for both the light and heavy chains. The candidate humanized sequences are expressed and purified to produce humanized antibodies. Humanized antibodies and their chimeric antigen receptor cells are less immunogenic and safer. However, antibody humanization generally results in decreased antibody function. Summary of the Invention

[0006] The present invention provides a humanized single-chain antibody targeting the human CD19 antigen, a chimeric antigen receptor, an immune cell and its application. The humanized single-chain antibody targeting the human CD19 antigen is based on the mouse-derived FMC63 antibody targeting the CD19 antigen, and the light chain and heavy chain framework regions of the antibody are humanized. The humanized single-chain antibody targeting the human CD19 antigen can bind to the CD19 antigen with high specificity and high affinity. A chimeric antigen receptor is constructed based on the humanized single-chain antibody targeting the human CD19 antigen, and is transduced into immune cells. The prepared chimeric antigen receptor immune cells targeting the human CD19 antigen have a higher transduction efficiency, a stronger killing effect on human CD19-positive tumor cells, and show excellent efficacy in animals.

[0007] The present invention adopts the following technical solutions:

[0008] A humanized single-chain antibody targeting the human CD19 antigen is based on the murine FMC63 antibody targeting the human CD19 antigen. The amino acid sequences of the CDR regions remain unchanged, and the light chain framework regions VL FR1, VL FR2, VL FR3, and VL FR4 and the heavy chain framework regions VH FR1, VH FR2, VH FR3, and VH FR4 are humanized. The amino acid sequence and nucleotide sequence of the humanized VL FR1 are shown in SEQ ID No: 1 and SEQ ID No: 2; the amino acid sequence and nucleotide sequence of the humanized VL FR2 are shown in SEQ ID No: 3 and SEQ ID No: 4; the amino acid sequence and nucleotide sequence of the humanized VL FR3 are shown in SEQ ID No: 5 and SEQ ID No: 6; and the amino acid sequence and nucleotide sequence of the humanized VL FR4 are shown in SEQ ID No: 7 and SEQ ID No: 8. The amino acid sequence and nucleotide sequence of humanized VH FR1 are shown in SEQ ID No: 9 and SEQ ID No: 10; the amino acid sequence and nucleotide sequence of humanized VH FR2 are shown in SEQ ID No: 11 and SEQ ID No: 12; the amino acid sequence and nucleotide sequence of humanized VH FR3 are shown in SEQ ID No: 13 and SEQ ID No: 14; and the amino acid sequence and nucleotide sequence of humanized VH FR4 are shown in SEQ ID No: 15 and SEQ ID No: 16.

[0009] Furthermore, the amino acid sequence and nucleotide sequence of the humanized single-chain antibody targeting human CD19 antigen obtained by humanizing the mouse FMC63 antibody targeting human CD19 antigen are shown in SEQ ID No: 17 and SEQ ID No: 18.

[0010] A chimeric antigen receptor targeting human CD19 antigen comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain comprises the above-mentioned humanized single-chain antibody targeting human CD19 antigen.

[0011] The present invention discloses a nucleic acid molecule encoding the humanized antibody targeting human CD19 or a nucleic acid molecule encoding the chimeric antigen receptor targeting human CD19 antigen, or a vector comprising the nucleic acid molecule. The vector is conventional technology, for example, the vector includes one or more of a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector, and an oncolytic viral vector.

[0012] The present invention discloses an immune effector cell comprising the chimeric antigen receptor targeting human CD19 antigen. Immune cells are known in the art, such as T cells, NK cells, γδT cells, etc.

[0013] The present invention discloses an immune cell therapy drug, comprising the above-mentioned immune effector cells, and may also comprise a pharmaceutically acceptable carrier, diluent or excipient. The dosage form includes liquid, solid, and paste, preferably a liquid preparation, such as an injection.

[0014] The present invention discloses the use of the above-mentioned humanized single-chain antibody targeting the human CD19 antigen, the above-mentioned chimeric antigen receptor targeting the human CD19 antigen, or the above-mentioned immune effector cell in the preparation of medicines, especially in the preparation of immune medicines. In particular, the preparation of medicines for treating human CD19-positive tumor diseases and CD19-positive immune diseases. Specifically, B-cell lymphomas, such as diffuse large B-cell lymphoma, follicular lymphoma and mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia; and autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, scleroderma, etc.

[0015] Furthermore, the drugs disclosed in the present invention can be used alone or in combination with other drugs, such as other drugs that kill tumor cells, including nucleic acid drugs, antibody drugs, targeted drugs, other immune cell drugs, chemotherapy drugs, etc.

[0016] In the present invention, the extracellular domain also includes a signal peptide domain; a hinge region domain is provided between the extracellular domain and the transmembrane domain; the intracellular signaling domain includes a costimulatory domain, an intracellular signal transduction domain, a self-cleavage peptide domain, and a secretory IL15-IL15Ra domain.

[0017] The chimeric antigen receptor targeting human CD19 antigen disclosed in the present invention includes a signal peptide domain, the above-mentioned humanized single-chain antibody targeting human CD19 antigen, a hinge region domain, a transmembrane region domain, a co-stimulatory domain, an intracellular signaling domain, a self-cleavage peptide domain, and a secretory IL15-IL15Ra domain.

[0018] The present invention mainly discloses a humanized single-chain antibody targeting the human CD19 antigen, which is based on the mouse-derived FMC63 antibody targeting the human CD19 antigen. The single-chain antibody is then humanized and constructed into a chimeric antigen receptor targeting the human CD19 antigen. The chimeric antigen receptor is further transduced into immune cells. The prepared chimeric antigen receptor immune cells targeting the human CD19 antigen have higher transduction efficiency, stronger killing effect on human CD19-positive tumor cells, and excellent pharmacodynamics in animals. Other domains of the chimeric antigen receptor targeting the human CD19 antigen can be designed based on existing technologies.

[0019] Preferably, the amino acid sequence and nucleotide sequence of the signal peptide domain are shown in SEQ ID No: 19 and SEQ ID No: 20; the amino acid sequence and nucleotide sequence of the hinge region domain are shown in SEQ ID No: 21 and SEQ ID No: 22; the amino acid sequence and nucleotide sequence of the transmembrane domain are shown in SEQ ID No: 23 and SEQ ID No: 24; the amino acid sequence and nucleotide sequence of the costimulatory domain are shown in SEQ ID No: 25 and SEQ ID No: 26; the amino acid sequence and nucleotide sequence of the intracellular signaling domain are shown in SEQ ID No: 27 and SEQ ID No: 28; the amino acid sequence and nucleotide sequence of the self-cleavage peptide domain are shown in SEQ ID No: 29 and SEQ ID No: 30; the amino acid sequence and nucleotide sequence of the secretory IL15-IL15Ra domain are shown in SEQ ID No: 31 and SEQ ID No: 32.

[0020] Currently, the antibody recognition sequences in most CAR-T technologies targeting CD19 are derived from mouse sources. Due to individual differences, they produce strong immunogenicity after reinfusion, causing adverse reactions such as human anti-mouse antibody (HAMA) reactions. This makes the reinfused CAR cells easily recognized and cleared by the body's immune system, affecting long-term efficacy and posing a risk of recurrence. The present invention has developed a new humanized single-chain antibody (scFv) containing a human CD19 antigen. The scFv-based CAR and corresponding immune cells can specifically target and recognize the human CD19 antigen, effectively killing and clearing tumor cells expressing the human CD19 antigen. At the same time, it has the advantages of lower immunogenicity, longer survival in the body, greater safety, and higher efficiency. Experimental results showed that the CAR positivity rates of uNK cells, PKN0105 anti-CD19 CAR NK cells, and PKN0191 anti-CD19CAR NK cells were 1.15%, 60.7%, and 95.5%, respectively. Compared with chimeric antigen receptor NK cells containing the mouse FMC63 single-chain antibody sequence targeting the human CD19 antigen (PKN0105 anti-CD19 CAR NK), chimeric antigen receptor NK cells containing the humanized single-chain antibody sequence targeting the human CD19 antigen (PKN0191 anti-CD19 CAR NK) had a higher CAR positivity rate and higher transduction efficiency. Furthermore, compared with uNK cells, PKN0105 anti-CD19 CAR NK and PKN0191 anti-CD19 CAR NK cells could effectively kill CD19-positive Raji-ffluc target cells at effector-target ratios of 1:1, 1:0.3, and 1:0.1. Moreover, at an effector-target ratio of 1:0.1, PKN0191 anti-CD19 CAR NK cells had stronger killing activity than PKN0105 anti-CD19 CAR NK cells, with a statistically significant difference (P<0.01). The results of animal experiments showed that compared with the PBS group and the uNK group, the chimeric antigen receptor NK cell group (PKN0105) containing the mouse-derived FMC63 single-chain antibody sequence targeting the human CD19 antigen and the chimeric antigen receptor cell group (PKN0191) containing the humanized single-chain antibody targeting the human CD19 antigen significantly inhibited the growth of Raji xenograft tumors. Compared with the PKN0105 group, the PKN0191 group eliminated Raji tumors more significantly on D24. By observing the time of death of mice and drawing a survival curve, it can be seen that as of D24, the survival rate of mice in the PKN0105 group was 80%, and the survival rate of mice in the PKN0191 group was 100%. At this time, all mice in the PBS group and the uNK group had died ( Figure 7The experimental results show that the chimeric antigen receptor cell group containing a humanized single-chain antibody targeting the human CD19 antigen has better in vivo tumor-killing efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the affinity test result of single-chain antibody targeting human CD19 antigen.

[0022] Figure 2 Schematic diagram of the chimeric antigen receptor structure.

[0023] Figure 3 This is the CAR positivity test result targeting human CD19 CAR NK cells.

[0024] Figure 4 This is the killing result of human CD19 CAR NK cells on CD19 antigen-positive target cells.

[0025] Figure 5 Images of Raji-luciferase xenograft tumors in the NSG mouse model targeting human CD19 CAR NK cells.

[0026] Figure 6 Inhibitory effect of human CD19 CAR NK cells on Raji-luciferase xenograft tumors in NSG mouse model.

[0027] Figure 7 Survival results of human CD19 CAR NK cells targeting Raji xenograft tumor mouse model. DETAILED DESCRIPTION

[0028] Based on the murine FMC63 antibody targeting the human CD19 antigen, the inventors humanized the light chain framework regions (VL FR1, VL FR2, VL FR3, VL FR4) and the heavy chain framework regions (VH FR1, VH FR2, VH FR3, VH FR4), while retaining the antibody fragments (CDR regions) that directly contact the antigen. This resulted in a single-chain antibody that maintains specificity and affinity while reducing immunogenicity and toxic side effects. While antibody humanization typically results in decreased antibody function, the humanized sites in this invention differ from existing humanization sites, resulting in humanized antibodies with biological functions that approach or even enhance those of non-humanized antibodies.

[0029] The humanized antibody targeting human CD19 of the present invention comprises a light chain, a connecting region, and a heavy chain. The light chain comprises three light chain complementary determining regions (VL CDRs) and four humanized light chain framework regions (VL FRs); the heavy chain comprises three heavy chain complementary determining regions (VH CDRs) and four humanized heavy chain framework regions (VH FRs). Furthermore, the sequence of connection of the segments in the light chain is: humanized VL FR1-VLCDR1-humanized VL FR2-VL CDR2-humanized VL FR3-VL CDR3-humanized VL FR4; the sequence of connection of the segments in the heavy chain is: humanized VH FR1-VH CDR1-humanized VH FR2-VH CDR2-humanized VH FR3-VH CDR3-humanized VH FR4.

[0030] The present invention also discloses a chimeric antigen receptor targeting human CD19 antigen, comprising a signal peptide domain, the above-mentioned humanized CD19 antigen-binding single-chain antibody, a hinge region domain, a transmembrane region domain, a costimulatory domain, an intracellular signaling domain, a self-cleavage peptide domain, and a secretory IL15-IL15Ra domain. Preferably, the chimeric antigen receptor comprises a CD8a signal peptide domain, a CD8a hinge region domain, a CD8a transmembrane domain, a 4-1BB costimulatory domain, a CD3ζ intracellular signaling domain, a P2A self-cleavage peptide domain amino acid, and a secretory IL15-IL15Ra domain.

[0031] The following experiments illustrate the technological advancements of the present invention. The specific raw materials, preparation procedures, and performance testing all utilize conventional techniques. Data collection and variance analysis are performed using conventional statistical methods. The present invention is inventive in that it discloses a novel humanized antibody targeting human CD19, which represents a significant improvement over murine antibodies targeting human CD19. The specific experimental methods do not affect the understanding of the technical benefits of the present invention by those skilled in the art. The murine FMC63 single-chain antibody targeting the CD19 antigen is a previously reported antibody, and its amino acid sequence is shown in SEQ ID No: 33.

[0032] Example 1 CD19 antibody humanized sequence design

[0033] The humanization of CD19 antibody is based on the sequence of FMC63 antibody, a mouse antibody targeting CD19 antigen. The framework (FR) region sequence is humanized based on the amino acid sequence of its scFv chain to design a humanized single-chain antibody sequence targeting human CD19 antigen.

[0034] Based on the murine FMC63 antibody targeting the human CD19 antigen, the amino acid sequence and nucleotide sequence of the humanized single-chain antibody targeting the human CD19 antigen are shown in SEQ ID No: 17 and SEQ ID No: 18. Among them, the amino acid sequence and nucleotide sequence of the humanized VL FR1 are shown in SEQ ID No: 1 and SEQ ID No: 2; the amino acid sequence and nucleotide sequence of the humanized VL FR2 are shown in SEQ ID No: 3 and SEQ ID No: 4; the amino acid sequence and nucleotide sequence of the humanized VL FR3 are shown in SEQ ID No: 5 and SEQ ID No: 6; and the amino acid sequence and nucleotide sequence of the humanized VL FR4 are shown in SEQ ID No: 7 and SEQ ID No: 8. The amino acid sequence and nucleotide sequence of humanized VH FR1 are shown in SEQ ID No: 9 and SEQ ID No: 10; the amino acid sequence and nucleotide sequence of humanized VH FR2 are shown in SEQ ID No: 11 and SEQ ID No: 12; the amino acid sequence and nucleotide sequence of humanized VH FR3 are shown in SEQ ID No: 13 and SEQ ID No: 14; and the amino acid sequence and nucleotide sequence of humanized VH FR4 are shown in SEQ ID No: 15 and SEQ ID No: 16.

[0035] Example 2: Construction and expression of a humanized single-chain antibody targeting human CD19 antigen using conventional techniques

[0036] The vector plasmid construction and expression of the humanized single-chain antibody targeting human CD19 antigen were completed by Bio-Ying Biotechnology Co., Ltd. The experimental steps are briefly described as follows:

[0037] The nucleotide sequences of the mouse FMC63 single-chain antibody targeting human CD19 antigen (SEQ ID No: 34) and the humanized single-chain antibody targeting human CD19 antigen (SEQ ID No: 18) were synthesized (Baiying Biotechnology Co., Ltd.) and seamlessly cloned into the pcDNA3.4 antibody expression vector plasmid via the HindIII / BamHI restriction sites.

[0038] The mouse FMC63 single-chain antibody sequence expression vector plasmid targeting human CD19 antigen and the humanized single-chain antibody sequence expression vector plasmid targeting human CD19 antigen were transfected into CHO cells to express antibodies.

[0039] The mixed transfection solution was placed in a 37°C constant temperature incubator (BLUE PARD) and cultured at 8% CO2 and 37°C for 4 days. The supernatant was collected by centrifugation (8000 rpm, 5 min), and the obtained supernatant antibody was purified by Protein A (Cytiva, 29127556) affinity chromatography column.

[0040] The mouse FMC63 single-chain antibody targeting human CD19 antigen and a humanized single-chain antibody targeting human CD19 antigen were obtained, named F22469501-CHO and F22469502-CHO, respectively.

[0041] Example 3 Conventional method for detecting antibody affinity

[0042] The murine FMC63 single-chain antibody (F22469501-CHO) targeting the human CD19 antigen and the humanized single-chain antibody (F22469502-CHO) targeting the human CD19 antigen were simultaneously subjected to full-concentration affinity testing using Biacore 8K (Cytiva). The experimental steps are briefly described as follows:

[0043] The murine FMC63 single-chain antibody targeting human CD19 antigen and the humanized single-chain antibody targeting human CD19 antigen were diluted to 2 μg / mL in 1× HBS-EP running buffer (0.1 M HEPES: 1.5 M NaCl, 30 mM EDTA, 0.05% Tween-20, pH 7.4).

[0044] 400 RU were captured on a Biacore 8K instrument at 10 μL / min.

[0045] Dilute the CD19 antigen two-fold in running buffer. Inject the diluted CD19 antigen sequentially into the experimental and reference channels at a flow rate of 30 μL / min, allowing binding and dissociation times to be determined. Both binding and dissociation steps are performed in running buffer.

[0046] Biacore Insight Evaluation Software was used to calculate the affinity values ​​of F22469501-CHO and F22469502-CHO antibodies, including ka, kd, and KD.

[0047] The experimental results showed that the affinities of the mouse FMC63 single-chain antibody targeting human CD19 antigen (F22469501-CHO) and the humanized single-chain antibody targeting human CD19 antigen (F22469502-CHO) were 2.74E-09 and 5.49E-09, respectively. Figure 1), the humanized single-chain antibody targeting human CD19 antigen still has a high affinity.

[0048] Example 4: Construction of Chimeric Antibody Receptor Vectors Using Conventional Methods

[0049] The brief description is as follows: pMSCV retroviral backbone vector plasmid was purchased from Wuhan Miaoling Biotechnology Co., Ltd., and the nucleotide sequence (DNA fragment) of the chimeric antigen receptor (CAR) was artificially synthesized at Jinweizhi. The CAR nucleotide sequence (SEQ ID No: 35) encoding the mouse FMC63 single-chain antibody targeting the human CD19 antigen and the CAR nucleotide sequence (SEQ ID No: 36) encoding the humanized single-chain antibody targeting the human CD19 antigen were digested with EcoRI and PacI respectively. The CAR DNA fragment and the pMSCV retroviral backbone vector plasmid fragment were homologously recombined using recombinase to construct the chimeric antigen receptor (PKN0105) containing the mouse FMC63 single-chain antibody sequence targeting the human CD19 antigen and the chimeric antigen receptor (PKN0191) containing the humanized single-chain antibody sequence targeting the human CD19 antigen. The chimeric antigen receptor structure is shown in Figure 2. Figure 2 As shown in A and B.

[0050] Example 5: Preparation of Chimeric Antigen Receptor Retrovirus by Conventional Method

[0051] The retroviral vector plasmids containing the chimeric antigen receptor (PKN0105) containing the mouse FMC63 single-chain antibody sequence targeting the human CD19 antigen and the chimeric antigen receptor (PKN0191) containing the humanized single-chain antibody sequence targeting the human CD19 antigen were extracted and co-transfected with the retroviral helper packaging plasmid into 293T cells to prepare retrovirus. The experimental steps are briefly described as follows:

[0052] To resuscitate and culture 293T cells, add 5E+06 cells to a T75 cell flask in DMEM medium containing 10% FBS and culture in a CO2 cell culture incubator;

[0053] When the cell confluence reaches about 80%, replace with fresh cell culture medium (DMEM medium containing 10% FBS);

[0054] Take 500ul opti-DMEM medium, add 10ug pMSCV plasmid (PKN0105 and PKN0191), 10ug pUMVC plasmid and 5ug pRD114 plasmid and mix well (pUMVC plasmid and pRD114 plasmid were purchased from Fenghui Biotechnology Co., Ltd.);

[0055] Take another 500ul of opti-DMEM medium, add 25ug of polyetherimide (PEI) solution and mix well;

[0056] Add the PEI solution to the plasmid solution in step (3) and mix well. After standing at room temperature for 20 minutes, add it to 293T cells. After incubation for 8 hours, remove the culture medium and add 15 ml of fresh cell culture medium.

[0057] 48 hours after transfection, the culture supernatant was collected and centrifuged at 400 g for 5 minutes. The supernatant virus solution was aliquoted and stored in a -80°C refrigerator for NK cell transduction.

[0058] Example 6 Retroviral transduction of NK cells to prepare Anti-CD19 CAR NK cells

[0059] NK cell isolation and activation are conventional techniques. This study used the Miltenyi Biotec NK Cell Isolation Kit (Miltenyi Biotec, 130-092-657) to isolate and activate NK cells from human cord blood mononuclear cells (CBMCs). The experimental steps are briefly described as follows:

[0060] (1) Sorting and activation of NK cells

[0061] Prepare fresh MACS buffer (1X DPBS containing 2mM EDTA and 0.5% FBS) and pre-chill at 4°C. Thaw 25 μL of human umbilical cord blood mononuclear cells (CBMCs) in a water bath at 37°C. In a biosafety cabinet, collect 10 μL of CBMCs and count them using a cell counter. Transfer the CBMCs to a 15ml centrifuge tube, add 6ml of pre-chilled 1X DPBS, and centrifuge at 400g for 5 minutes. Discard the supernatant and resuspend the CBMCs in MACS buffer (40 μL / 1E+07 cells). Add NK cell Biotin-Antibody Cocktail (10 μL / 1E+07 cells), mix thoroughly, and incubate at 4°C for 5 minutes. Add 30 μL of MACS buffer per 1E+07 cells, followed by 20 μL of NK Cell Microbeads Cocktail (1E+07 cells), mix thoroughly, and incubate at 4°C for 10 minutes. Rinse the LS column (Miltenyi Biotec, 130-042-401) with 3 ml of MACS buffer. Add the cell suspension to the LS column and immediately add 3 ml of MACS buffer. Once the cell suspension has completely flowed into a 15 ml centrifuge tube, add another 3 ml of MACS buffer and collect all cells into the 15 ml centrifuge tube. Centrifuge the cell suspension at 400 g for 10 minutes. Resuspend the cells in Ecosine NK medium supplemented with 10% FBS. Count 10 μl of cells and add K562 feeder cells to the NK cells for activation culture at a 1:1 ratio of NK to K562 feeder.

[0062] (2) Retroviral transduction

[0063] On day 5 of NK cell activation culture, 5E+05 activated NK cells were plated in triplicate onto 12-well cell culture plates. One aliquot of NK cells remained untransfected with retrovirus (uNK), while two aliquots were infused with PKN0105 and PKN0191 retroviral supernatants, respectively (MOI = 1). RetroNectin (TaKaRa, T100B) was then added to the transduction reagent at a concentration of 10 µg / mL. The cells were cultured in Ecosine NK Cell Medium (Ecosine, NE000-N012) supplemented with 5% FBS and 200 IU IL-2 at a cell density of 5E+05 cells / mL. The cells were centrifuged at 1000 g for 30 minutes and then cultured in a CO2 incubator. This yielded PKN0105 anti-CD19 CAR NK cells and PKN0191 anti-CD19 CAR NK cells. uNK cells served as a control.

[0064] Example 7 Anti-CD19 CAR NK cell CAR positive rate

[0065] uNK, PKN0105 anti-CD19CAR NK, and PKN0191 anti-CD19 CAR NK cells were cultured in Ecosine NK cell culture medium containing 5% FBS and 200 IU IL-2. On day 9 of culture, the CAR positive rate was detected to verify the viral transduction efficiency. The experimental steps are briefly described as follows:

[0066] 5E+05 cells of each uNK, PKN0105 anti-CD19 CAR NK, and PKN0191 anti-CD19 CAR NK cells were transferred to a 96-well V-bottom plate and centrifuged at 400 g for 5 minutes;

[0067] Discard the supernatant, add 200 μl of 1X DPBS to each well, and wash twice by centrifugation (400 g for 5 minutes). Then, add 100 μl of 1:100 diluted PE Anti-CD19 FMC63 antibody (ACROBiosystems, FM3-HPY53) to each well. Incubate at room temperature for 15 minutes in the dark.

[0068] After incubation, centrifuge at 400g for 5 minutes. Discard the supernatant, add 200µl of 1X DPBS to each well, and wash once by centrifugation (400g for 5 minutes). Discard the supernatant, and resuspend the cells in 100µl of 1X DPBS per well. Flow cytometry was used to analyze the CAR-positive rates of UNK, PKN0105 anti-CD19 CAR NK cells, and PKN0191 anti-CD19 CAR NK cells.

[0069] The experimental results showed that the CAR positive rates of uNK, PKN0105 anti-CD19 CAR NK, and PKN0191 anti-CD19 CAR NK cells were 1.15%, 60.7%, and 95.5%, respectively ( Figure 3 Compared with chimeric antigen receptor NK cells containing the mouse FMC63 single-chain antibody sequence targeting the human CD19 antigen (PKN0105 anti-CD19 CAR NK), the humanized chimeric antigen receptor NK cells containing the humanized single-chain antibody targeting the human CD19 antigen (PKN0191 anti-CD19 CAR NK) have a higher CAR positive rate and higher transduction efficiency.

[0070] Example 8 In vitro killing function of Anti-CD19 CAR NK cells

[0071] CD19-positive Raji tumor cells were transduced with lentiviral vectors to generate Raji-ffluc target cells stably expressing firefly luciferase (ffluc). uNK, PKN0105 anti-CD19 CAR NK, and PKN0191 anti-CD19 CAR NK cells were co-incubated with the Raji-ffluc target cells to assess their tumor cell cytotoxicity. The experimental steps are briefly described as follows:

[0072] Count the Raji-ffluc target cells and add 20,000 cells / well to a 96-well white plate in a volume of 100 μl. The culture medium is 1640 containing 10% FBS.

[0073] uNK, PKN0105 anti-CD19 CAR NK, and PKN0191 anti-CD19 CAR NK cells were counted and added to the cells according to the CAR positivity effector-target ratio (E:T ratio) of 1:1, 1:0.3, and 1:0.1, respectively. The culture medium was 1640 containing 10% FBS, 100 μl / well;

[0074] The cells were placed in a CO2 cell culture incubator and incubated for 4 hours. After incubation, 50 μl of firefly luciferase substrate ONE-Glo (Promega, E6110) was added to each well and mixed by vortexing. After incubation for 5 minutes, the fluorescence value was measured using a microplate reader (TECAN, SAPRK).

[0075] The experimental results showed that compared with uNK cells, PKN0105 anti-CD19 CAR NK and PKN0191 anti-CD19CAR NK cells could effectively kill CD19-positive Raji-ffluc target cells at effector-target ratios of 1:1, 1:0.3, and 1:0.1. Moreover, at an effector-target ratio of 1:0.1, PKN0191 anti-CD19 CAR NK cells had stronger killing activity than PKN0105 anti-CD19 CAR NK cells ( Figure 4 ), with statistically significant differences (P<0.01), indicating that PKN0191 Anti-CD19 CAR NK cells containing a chimeric antigen receptor targeting the humanized single-chain antibody sequence of human CD19 antigen have better killing effects.

[0076] Example 9 In vivo animal efficacy test of Anti-CD19 CAR NK cells

[0077] In a xenograft tumor model, the efficacy of chimeric antigen receptor NK cells (PKN0105 anti-CD19 CAR NK) containing the murine FMC63 single-chain antibody sequence targeting the human CD19 antigen and chimeric antigen receptor NK cells (PKN0191 anti-CD19 CAR NK) containing the humanized single-chain antibody sequence targeting the human CD19 antigen were compared. The experimental steps are briefly described as follows:

[0078] Twenty NCG immunodeficient mice were prepared and randomly divided into PBS group (PBS), uNK control group (uNK), chimeric antigen receptor cell group containing mouse FMC63 single-chain antibody sequence targeting human CD19 antigen (PKN0105), and chimeric antigen receptor cell group containing humanized single-chain antibody sequence targeting human CD19 antigen (PKN0191).

[0079] On D0, luciferase-labeled Raji leukemia cells (100 μl, 5E+06 cells) were injected into NCG immunodeficient mice;

[0080] On D3 and D10, PBS, uNK cells (5E+06 NK cells), PKN0105 CAR NK cells (5E+06 CAR-positive NK cells), and PKN0191 CAR NK cells (5E+06 CAR-positive NK cells) were injected, respectively;

[0081] On D3, D10, D17, and D24, tumor fluorescence signals were detected by IVIS in vivo imaging, and tumor growth curves were drawn;

[0082] The survival status of mice in each group from D0 to D24 was counted and a survival curve was drawn.

[0083] Animal test results showed that compared with the PBS group and uNK group, the chimeric antigen receptor NK cell group (PKN0105) containing the mouse-derived FMC63 single-chain antibody sequence targeting the human CD19 antigen and the chimeric antigen receptor cell group (PKN0191) containing the humanized single-chain antibody sequence targeting the human CD19 antigen significantly inhibited the growth of Raji xenograft tumors. Compared with the PKN0105 group, the PKN0191 group had a more obvious elimination of Raji tumors at D24 ( Figure 5 and Figure 6 By observing the time of death of mice and drawing a survival curve, we can see that by D24, the survival rate of mice in the PKN0105 group was 80%. Unexpectedly, the survival rate of mice in the PKN0191 group was 100%, at which time all the mice in the PBS group and the uNK group had died ( Figure 7The experimental results show that the chimeric antigen receptor cell group containing the humanized single-chain antibody sequence targeting the human CD19 antigen has better in vivo tumor-killing efficacy.

[0084] The above amino acid and nucleotide sequences are as follows.

[0085] Humanized VL FR1 amino acid sequence SEQ ID No: 1

[0086] DIQMTQSPSSLSASVGDRVTITC

[0087] Humanized VL FR1 nucleotide sequence SEQ ID No: 2

[0088] GACATTCAGATGACACAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGAGTGACAATTACCTGC

[0089] Humanized VL FR2 amino acid sequence SEQ ID No: 3

[0090] WYQQKPGKAPKLLIY

[0091] Humanized VL FR2 nucleotide sequence SEQ ID No: 4

[0092] TGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTAC

[0093] Humanized VL FR3 amino acid sequence SEQ ID No: 5

[0094] GVPSRFSGSGSGTDYTLTISSLQPEDIATYFC

[0095] Humanized VL FR3 nucleotide sequence SEQ ID No: 6

[0096] GGCGTGCCTAGCAGATTCAGCGGCTCCGGCAGCGGCACCGATTACACCCTGACCATTAGCAGCCTGCAGCCCGAGGACATCGCCACCTACTTCTGT

[0097] Humanized VL FR4 amino acid sequence SEQ ID No: 7

[0098] FGQGTKLEIK

[0099] Humanized VL FR4 nucleotide sequence SEQ ID No: 8

[0100] TTCGGCCAAGGCACCAAGCTCGGAAATTAAG

[0101] Humanized VH FR1 amino acid sequence SEQ ID No: 9

[0102] QVQLQESGPGLVKPSETLSLTCTVS

[0103] Humanized VH FR1 nucleotide sequence SEQ ID No: 10

[0104] CAAGTGCAGCTGCAAGAGTCCGGCCCCGGGCTCGTGAAGCCTAGCGAGACACTGAGCCTGACCTGCACCGTGAGC

[0105] Humanized VH FR2 amino acid sequence SEQ ID No: 11

[0106] VSWIRQPPGKGLEWIG

[0107] Humanized VH FR2 nucleotide sequence SEQ ID No: 12

[0108] GTGAGCTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGC

[0109] Humanized VH FR3 amino acid sequence SEQ ID No: 13

[0110] RVTISKDNSKSQVSLKLSSVTAADTAVYYCAK

[0111] Humanized VH FR3 nucleotide sequence SEQ ID No: 14

[0112] AGAGTGACCATCAGCAAGGACAACAGCAAGAGCCAAGTGAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAAG

[0113] Humanized VH FR4 amino acid sequence SEQ ID No: 15

[0114] WGQGTLVTVSS

[0115] Humanized VH FR4 nucleotide sequence SEQ ID No: 16

[0116] TGGGGGCAAGGCACCCTGGTCACAGTCAGCAGC

[0117] Humanized single-chain antibody amino acid sequence (Clone ID: F22469502-CHO) SEQ ID No: 17

[0118] DIQMTQSPSSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDIATYFCQQGNTLPYTFGQGTKLEIKGSTSGSGKPGSGEGS TKGQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSALKSRVTISKDNSKSQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS

[0119] Humanized single-chain antibody nucleotide sequence (Clone ID: F22469502-CHO) SEQ ID No: 18

[0120] GACATTCAGATGACACAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGAGTGACAATTACCTGCAGAGCTAGCCAAGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACCACACAAGCAGACTGCACAGCGGCGTGCCTAGCAGATTCAGCGGCTCCGGCAGCGGCACCGATTACACCCTGACCATTAGCAGCCTGCAGCCCGAGGACATCGCCACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCCAAGGCACCAAGCTGGAAATTAAGGGCAGCACAAGCGGCAGCGGGAAGCCCGGGTCCGGCGAGGGCTCCACAAAAGGCCAAGTGCAGCTGCAAGAGTCCGGCCCCGGGCTCGTGAAGCCTAGCGAGACACTGAGCCTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCAGAGTGACCATCAGCAAGGACAACAGCAAGAGCCAAGTGAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCAAGCACTACTATTACGGCGGCAGCTACGCCATGGACTATTGGGGGCAAGGCACCCTGGTCACAGTCAGCAGC

[0121] CD8a signal peptide amino acid sequence SEQ ID NO: 19

[0122] MALPVTALLLPLALLLHAARP<000,0264><000,0265>CD8a signal peptide nucleotide sequence SEQ ID NO: 20<000,0266><000,0267>[[ID=1,2]]ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG<000,0268><000,0269>CD8a hinge region amino acid sequence SEQ ID NO: 21

[0126] AAAAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0127] CD8a hinge region nucleotide sequence SEQ ID NO: 22

[0128] GCGAAGCCCACCACGACGCCAGCGCCGCCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

[0129] CD8a transmembrane region amino acid sequence SEQ ID NO: 23

[0130] IYIWAPLAGTCGVLLLSLVITLYC

[0131] CD8a transmembrane region nucleotide sequence SEQ ID NO: 24

[0132] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC

[0133] 4-1BB costimulatory domain amino acid sequence SEQ ID NO: 25

[0134] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0135] 4-1BB costimulatory domain nucleotide sequence SEQ ID NO: 26

[0136] AAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

[0137] CD3ζ intracellular signaling domain amino acid sequence SEQ ID NO: 27

[0138] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0139] CD3ζ intracellular signaling domain nucleotide sequence SEQ ID NO: 28

[0140] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAA GGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

[0141] P2A self-cleavage peptide domain amino acid sequence SEQ ID NO: 29

[0142] GSGATNFSLLKQAGDVEENPGP

[0143] P2A self-cleaving peptide domain nucleotide sequence SEQ ID NO: 30

[0144] GCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCT

[0145] Secretory IL15-IL15Ra domain amino acid sequence SEQ ID NO: 31

[0146] GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

[0147] Secreted IL15-IL15Ra domain nucleotide sequence SEQ ID NO: 32

[0148] GGCATCCACGTGTTCATCCTGGGATGCTTCAGTGCCGGCCTGCCTAAGACTGAAGCCAATTGGGTGAACGTGATCTCCGACCTGAAGAAGATCGAAGATCTGATCCAGTCAATGCACATCGACGCCACCCTGTATACCGAGAGCGACGTGCACCCATCTTGCAAAGTGACCGCCATGAAGTGTTTTCTGCTGGAGCTGCAGGTGATCAGCCTCGAGTCTGGCGACGCCAGCATCCATGACACCGTGGAGAACCTGATCATCCTGGCAAATAACTCCCTGTCTTCCAACGGCAATGTGACGGAATCCGGCTGTAAGGAATGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCTTTTGTGCACATCGTGCAGATGTTTATTAATACCTCCAGCGGAGGCGGCAGTGGCGGCGGCGGGTCCGGCGGGGGCGGCAGCGGAGGAGGGGGCAGCGGCGGCGGCTCCCTGCAGGCTCCTAGGCGGGCCCGGGGATGTCGGACTCTGGGCCTGCCTGCCCTGCTGCTGCTGCTGCTGCTGAGGCCCCCCGCAACCAGGGGAATCACTTGCCCACCACCTATGTCCGTGGAGCACGCCGACATCTGGGTGAAGTCCTACAGTCTGTACTCCAGAGAGAGATACATTTGCAACTCCGGATTCAAGAGGAAGGCCGGGACCTCCAGCCTGACAGAGTGCGTGCTGAATAAGGCCACCAACGTGGCCCACTGGACAACCCCCAGCCTGAAGTGCATCCGG

[0149] Amino acid sequence of murine - derived single - chain antibody FMC63 targeting human CD19 antigen, SEQ ID NO: 33

[0150] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGS TKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

[0151] The nucleotide sequence of the murine FMC63 single-chain antibody targeting human CD19 antigen is SEQ ID NO: 34

[0152] GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGCTCCACCTCTGGATCCGGCAAGCCCGGATCTGGCGAGGGATCCACCAAGGGCGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0153] CAR nucleotide sequence (SEQ ID No: 35) containing a murine-derived single-chain antibody targeting human CD19 antigen

[0154]

[0155] CAR nucleotide sequence containing a humanized single-chain antibody targeting human CD19 antigen (SEQ ID No: 36)

[0156]

Claims

1. A humanized single-chain antibody targeting human CD19 antigen, characterized in that: Based on the murine FMC63 antibody targeting human CD19 antigen, the light chain framework regions VL FR1, VL FR2, VL FR3, VL FR4 and the heavy chain framework regions VH FR1, VHFR2, VH FR3, and VH FR4 were humanized; the amino acid sequence of the murine FMC63 single-chain antibody targeting human CD19 antigen is SEQ ID NO: 33; the amino acid sequence of humanized VL FR1 is shown in SEQ ID No: 1; the amino acid sequence of humanized VL FR2 is shown in SEQ ID No: 3; the amino acid sequence of humanized VL FR3 is shown in SEQ ID No: 5; the amino acid sequence of humanized VL FR4 is shown in SEQ ID No: 7; the amino acid sequence of humanized VH FR1 is shown in SEQ ID No: 9; the amino acid sequence of humanized VH FR2 is shown in SEQ ID No: 11; the amino acid sequence of humanized VH FR3 is shown in SEQ ID No: 13; and the amino acid sequence of humanized VH The amino acid sequence of FR4 is shown in SEQ ID No: 15; the amino acid sequence of the CDR region remains unchanged.

2. The humanized single-chain antibody targeting human CD19 antigen according to claim 1, characterized in that: The amino acid sequence of the humanized antibody targeting human CD19 antigen is shown in SEQ ID No:

17.

3. A chimeric antigen receptor targeting human CD19 antigen, comprising an extracellular domain, a transmembrane domain and an intracellular domain, characterized in that: The extracellular domain includes the humanized single-chain antibody targeting human CD19 antigen according to claim 1 or 2.

4. A nucleic acid molecule encoding the humanized single-chain antibody targeting human CD19 antigen according to claim 1 or 2, or a nucleic acid molecule encoding the chimeric antigen receptor targeting human CD19 antigen according to claim 3, or a vector comprising the nucleic acid molecule.

5. An immune effector cell comprising the chimeric antigen receptor targeting human CD19 antigen according to claim 3.

6. An immune cell therapy drug comprising the immune effector cells according to claim 5.

7. Use of the humanized single-chain antibody targeting human CD19 antigen according to claim 1 or 2, the chimeric antigen receptor targeting human CD19 antigen according to claim 3, or the immune effector cell according to claim 5 in the preparation of a medicament for treating B-cell lymphoma, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis or scleroderma.

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