Humanized antibody B09 and its application in the preparation of CAR-T cells

Abstract

This application relates to the field of biomedicine, specifically to the humanized antibody B09 and its application in the preparation of CAR-T cells. This application first provides a new specific targeting CD20 single-domain antibody and optimizes it into a humanized antibody. The CD20 single-domain antibody of this application has high affinity and the entire sequence has been modified by humanization, enabling the production of antibodies and CAR-T cells with low immunogenicity. After the CD20 single-domain antibody of this application is concatenated with the CD19 antibody obtained by optimization and screening, a bispecific chimeric antigen receptor (CAR) is constructed and CAR-T cells are prepared. The CAR-T cells can specifically recognize CD19 and / or CD20 proteins and kill the cells expressing the corresponding protein targets.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and specifically to a humanized antibody B09 and its application in the preparation of CAR-T cells. Background Art

[0002] CD20 is a transmembrane protein mainly expressed on the surface of B cells and is involved in regulating the development, activation, and differentiation of B cells. In certain diseases, such as non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), the accumulation of abnormal B cells is a key factor in disease development. CD19 is a leukocyte differentiation antigen expressed by B cells and belongs to the Ig superfamily. All B cell lines except plasma cells, and malignant B cells and FDCs will express CD19. It is an important membrane antigen involved in B cell proliferation, differentiation, activation, and antibody production, and can also promote the signal transduction of BCR.

[0003] In CAR-T (chimeric antigen receptor T cell) therapy, CD19 and CD20 have become key targets for the treatment of certain types of B cell malignancies. The tandem CAR-T cell therapy targeting both CD19 and CD20 means targeting two antigens simultaneously in the same CAR structure. The advantages of this design are as follows: 1) Enhanced targeting: By targeting two surface antigens simultaneously, the recognition and killing ability of CAR-T cells against cancer cells can be increased. This is especially beneficial for tumor cells that may evade the attack of single-target CAR-T cells due to reduced expression of one target. 2) Reduced escape: Tumor cells may escape the attack of the immune system by reducing or altering the expression of a certain antigen. By targeting multiple antigens, the tandem CAR-T cell therapy can reduce the risk of tumor escape in patients after cell therapy. 3) Improved efficacy: The dual-target CAR-T therapy may provide a higher therapeutic effect than single-target therapy, especially in cases with limited response to traditional single-target CAR-T therapy. Summary of the Invention

[0004] To provide a more effective treatment method, this application first provides a new single-domain antibody specifically targeting CD20 and optimizes it into a humanized antibody. The humanized CD20 single-domain antibody provided by this application has high affinity and the entire sequence has been modified by humanization, enabling the production of antibodies and CAR-T cells with low immunogenicity. After concatenating the CD20 single-domain antibody of this application with the CD19 antibody obtained through optimization and screening, a bispecific chimeric antigen receptor (CAR) is constructed and CAR-T cells are prepared. The CAR-T cells can specifically recognize CD19 and / or CD20 proteins and kill the cells expressing the corresponding protein targets. Specifically, this application provides the following technical solutions:

[0005] Product

[0006] In a first aspect, the present application provides a CD20 antibody or an antigen-binding fragment thereof that targets the CD20 protein. The amino acid sequences of CDR1, CDR2, and CDR3 in the CD20 antibody or its antigen-binding fragment are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0007] Preferably, the antibody is humanized.

[0008] As used in the present application, the definition of "humanized" refers to a protein molecule that is recombinantly designed with only a part of the complementarity-determining regions (CDRs) of the variable region of an immunoglobulin molecule from a non-human species being retained, and the remaining major variable region part and the entire constant region part being of human origin. The immunogenicity of a humanized antibody should theoretically be less than that of a chimeric antibody and is more suitable for large-scale and repeated use.

[0009] Preferably, the CD20 antibody can be a single-domain antibody, or a monoclonal antibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, a single-chain Fv (scFv), a Fab fragment, an F(ab') fragment, a disulfide bond Fv (sdFv), an anti-idiotype antibody, an epitope-binding fragment of such an antibody, etc.

[0010] Preferably, the CD20 antibody is a single-domain antibody.

[0011] Preferably, the CD20 antibody has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with any one of the amino acid sequences shown in SEQ ID NO.4-9.

[0012] Without substantially affecting the antibody activity, those skilled in the art can replace, add, and / or delete (eliminate) any one or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequences of the present invention (such as SEQ ID NO.4-9) to obtain variants of functional fragments. They are all considered to be included within the scope of protection of the present invention.

[0013] Preferably, the CD20 antibody comprises or has an amino acid sequence shown in any one of SEQ ID NO.4-9. Among them, the sequence shown in SEQ ID NO.4 is the amino acid sequence of the original antibody, and SEQ ID NO.5-9 are the amino acid sequences of five humanized CD20 antibodies HM1-HM5 obtained by humanizing the original antibody.

[0014] Specifically, the antibody shown in SEQ ID NO.9 is named HM5 or B09-HM5 in this application, and the sequence of its encoding nucleic acid is shown in SEQ ID NO.10.

[0015] The CD20 antibody of this application may contain amino acid modifications that are not present in the receptor antibody or the donor antibody, such as deamidation, disulfide bond formation, terminal lysine cleavage, glycosylation, and the glycosylation modifications such as core fucose, sialic acid, high mannose levels, etc. These modifications can be carried out to further improve the performance of the antibody, such as binding affinity and killing ability against target cells.

[0016] In a second aspect, this application provides a humanized CD19 antibody, which comprises a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region is shown in SEQ ID NO.24, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.28; or,

[0017] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.25, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.29.

[0018] Specifically, the light chain variable region with the amino acid sequence shown in SEQ ID NO.24 is called VL-HM2 in this application, the light chain variable region with the amino acid sequence shown in SEQ ID NO.25 is called VL-HM3 in this application, the heavy chain variable region with the amino acid sequence shown in SEQ ID NO.28 is called VH-HM1 in this application, and the heavy chain variable region with the amino acid sequence shown in SEQ ID NO.29 is called VH-HM2 in this application; then the antibody formed by connecting VL-HM2 and VH-HM1 is called VL2+VH1 or abbreviated as CD19-21, 21 in this application; then the antibody formed by connecting VL-HM3 and VH-HM2 is called VL3+VH2 or abbreviated as CD19-32, 32 in this application. The encoding nucleic acid of VL2+VH1 or VL3+VH2 is shown in SEQ IDNO.33 / 34.

[0019] Preferably, the light chain variable region and the heavy chain variable region are connected by a linker (also known as a fusion protein linker, linker, or connector).

[0020] Preferably, the linker can be composed of G (Gly, glycine) and / or S (Ser, serine); the linker may also include other amino acids.

[0021] More preferably, the linker is (GGGGS)n, preferably n = 3.

[0022] Specifically, the structure of the CD19 antibody can be: light chain variable region - linker - heavy chain variable region or heavy chain variable region - linker - light chain variable region.

[0023] In a third aspect, the present application provides a fusion protein, which contains the CD20 antibody described in the first aspect of the present application and the CD19 antibody described in the second aspect of the present application.

[0024] More preferably, the CD20 antibody described in the first aspect of the present application and the CD19 antibody described in the second aspect of the present application are directly / indirectly connected by a linker to form a fusion protein. That is, in the fusion protein, there is one or more amino acids between the CD20 antibody described in the first aspect of the present application and the CD19 antibody described in the second aspect of the present application, and the one or more amino acids in between are the aforementioned linker.

[0025] More specifically, the fusion protein from the N-terminus to the C-terminus is in sequence: the aforementioned CD20, the first linker, the light chain variable region of the aforementioned CD19 antibody, the second linker, and the heavy chain variable region of the aforementioned CD19 antibody.

[0026] In a fourth aspect, the present application provides a chimeric antigen receptor, which includes the CD20 antibody described in the first aspect of the present application, the CD19 antibody described in the second aspect of the present application, or the fusion protein described in the third aspect of the present application.

[0027] Preferably, the chimeric antigen receptor further includes one or more of a hinge region, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

[0028] Preferably, the chimeric antigen receptor is composed of the CD20 antibody described in the first aspect of the present application, the CD19 antibody described in the second aspect of the present application, or the fusion protein described in the third aspect of the present application, and a hinge region, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

[0029] Preferably, the chimeric antigen receptor from the N-terminus to the C-terminus is in sequence: the CD20 antibody described in the first aspect of the present application / the CD19 antibody described in the second aspect of the present application / the fusion protein described in the third aspect of the present application - CD8 hinge region - CD8 transmembrane domain - 41BB co-stimulatory domain - CD3ζ intracellular signaling domain.

[0030] When the chimeric antigen receptor of the present invention contains a CD19 antibody and a CD20 antibody, it can be called a bispecific chimeric antigen receptor.

[0031] On the other hand, the present application provides a nucleic acid molecule, which encodes one or more of the aforementioned: CD20 antibody, CD19 antibody, fusion protein, chimeric antigen receptor.

[0032] On the other hand, the present application provides a recombinant vector, which contains the aforementioned nucleic acid molecule.

[0033] Furthermore, the recombinant vector includes a cloning vector and an expression vector.

[0034] On the other hand, the present application provides a genetically modified cell, which contains or expresses the aforementioned CD20 antibody, CD19 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector.

[0035] The "genetically modified cell" described in the present application may include the progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be exactly the same as the original parental cells in morphology or genome, but it is sufficient that they can express the aforementioned CD20 antibody, CD19 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector.

[0036] Preferably, the cell is an immune cell.

[0037] Preferably, the cell includes T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, white blood cells and / or peripheral blood mononuclear cells.

[0038] Preferably, the cell is a T cell. Specifically, when the T cell contains or expresses a chimeric antigen receptor, it can be called a CAR-T cell.

[0039] Preferably, the cell is a human cell.

[0040] On the other hand, the present application provides an antibody-drug conjugate, which contains a cytotoxic agent or a label, and the CD20 antibody and / or CD19 antibody described in the present application.

[0041] Preferably, the cytotoxic agent can be MMAF or MMAE.

[0042] Preferably, the label can be a fluorescent agent.

[0043] On the other hand, the present application provides a pharmaceutical composition, which contains one or more of the following: the aforementioned CD20 antibody, CD19 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell, antibody-drug conjugate.

[0044] In certain embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipient. The pharmaceutically acceptable carrier and / or excipient are described in detail in Remington's Pharmaceutical Sciences (19 th th ed., 1995). These substances are used as needed to assist in the stability of the drug or to enhance the activity of the active ingredient. The formulations that can be used in such pharmaceutical compositions can be in the form of the original compound itself, or optionally in the form of its pharmaceutically acceptable salts. The pharmaceutical composition formulated in this way can be administered to the drug in any suitable manner known to those skilled in the art as needed. The pharmaceutical composition may further comprise one or more of the group consisting of hormonal preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of costimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0045] Preferably, the pharmaceutical composition contains immune cells expressing the chimeric antigen receptor of the present invention, particularly CAR-T cells. The CAR-T cells can be dissolved in a suitable solution or cell culture medium for transportation, use, storage, preparation, etc.

[0046] On the other hand, the present application provides a detection kit containing a labeled antibody, which is the aforementioned CD19 antibody and / or CD20 antibody.

[0047] The term "labeled antibody" in the present application refers to an antibody containing one or more labels at the C-terminus, N-terminus, or any position in the molecule. The label can be any suitable label known to those skilled in the art, such as a radioactive, fluorescent, or chemiluminescent label.

[0048] Application

[0049] On the other hand, the present application provides the use of any one or more of the following products in the treatment of CD20-related cancers and in the preparation of drugs for CD20-related cancers:

[0050] The aforementioned CD20 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell, antibody-drug conjugate, pharmaceutical composition.

[0051] On the other hand, the present application provides the use of any one or more of the following products in the treatment of CD19-related cancers and in the preparation of drugs for CD19-related cancers:

[0052] The foregoing CD19 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell, antibody-drug conjugate, and pharmaceutical composition.

[0053] On the other hand, the present application provides the use of the foregoing CD20 antibody, fusion protein, or kit in detecting CD20 and in preparing a CD20-related cancer diagnostic product.

[0054] On the other hand, the present application provides the use of the foregoing CD19 antibody, fusion protein, or kit in detecting CD19 and in preparing a CD19-related cancer diagnostic product.

[0055] Method

[0056] On the other hand, the present application provides a method for preventing and / or treating CD20-related cancer, which comprises administering to a subject in need thereof an effective amount of the foregoing CD20 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell, antibody-drug conjugate, and pharmaceutical composition.

[0057] Preferably, the method is to administer to the subject an effective amount of CAR-T cells, and the CAR has the CD20 antibody and / or CD19 antibody provided in the present application.

[0058] On the other hand, the present application provides a method for preventing and / or treating CD19-related cancer, which comprises administering to a subject in need thereof an effective amount of the foregoing CD19 antibody, fusion protein, chimeric antigen receptor, nucleic acid molecule, recombinant vector, cell, antibody-drug conjugate, and pharmaceutical composition.

[0059] Preferably, the method is to administer to the subject an effective amount of CAR-T cells, and the CAR has the CD20 antibody and / or CD19 antibody provided in the present application.

[0060] The term "effective amount" in the present application refers to the amount of an active compound or a material, composition, or dosage form containing an active compound, which is effective for producing certain desired therapeutic effects commensurate with a reasonable benefit / risk ratio when administered according to the desired treatment regimen.

[0061] On the other hand, the present application provides a method for detecting CD20 protein or diagnosing CD20-related cancer, the method comprising contacting a sample with the foregoing CD20 antibody and fusion protein of the present application and detecting the complex, wherein detecting the complex indicates the expression of CD20 protein in the sample.

[0062] On the other hand, the present application provides a method for detecting CD19 protein or diagnosing CD19-related cancers, the method comprising contacting a sample with the aforementioned CD19 antibody and fusion protein of the present application and detecting the complex, wherein detecting the complex indicates the expression of CD19 protein in the sample.

[0063] Preferably, the sample includes any type of sample such as body fluid, tissue, cells, etc. from a subject.

[0064] Preferably, the detection is performed in vitro on an ex vivo sample.

[0065] Preferably, the detection is for non-diagnostic purposes.

[0066] Preferably, the detection can be qualitative, quantitative, or semi-quantitative.

[0067] General concept

[0068] The term "T cell" or "T lymphocyte" as used in the present application can be any T cell, such as cultured T cells, primary T cells, or T cells obtained from a mammal (preferably a primate, species including monkey, dog, or human). T cells can be any type of T cell, including NKT cells, and can be at any stage of development. If obtained from a mammal, then the T cells can be obtained from a number of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids.

[0069] The term "chimeric antigen receptor" or "CAR" as used in the present application generally includes three main parts: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain is responsible for recognizing the antigen, which can be a CD20 antibody and / or a CD10 antibody in the present application, and the extracellular domain can generally also include a hinge region. The main function of the transmembrane domain is to anchor the CAR molecule to the cell membrane and connect the extracellular domain of the CAR molecule with the intracellular domain. The intracellular domain can include a co-stimulatory domain and / or an intracellular signaling domain.

[0070] The term "CD20-related cancers" or "CD20-positive cancers" as used in the present application includes lymphoma, leukemia, or autoimmune diseases, more specifically including non-Hodgkin lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, B-cell lymphoma, multiple sclerosis, rheumatoid arthritis, marginal zone lymphoma, small lymphocytic lymphoma, lupus nephritis, mantle cell lymphoma, systemic lupus erythematosus, pemphigus, immune thrombocytopenia, membranous nephropathy, B-cell blood cancer, acute lymphocytic leukemia, microscopic polyangiitis, kidney transplant rejection, nephrotic syndrome.

[0071] As used herein, the term "CD19-related cancer" includes lymphoma and leukemia; preferably, the CD19-related cancer is leukemia; more preferably, the CD19-related cancer is chronic myeloid leukemia.

[0072] Specifically, in the specific embodiments of the present application, experiments were verified using the cell line of chronic myeloid leukemia as the target cell.

[0073] As used herein, the term "single-domain antibody" or "nanobody", "VHH" generally refers to a class of antibodies that lack the light chain of the antibody and only have the variable region of the heavy chain. A single-domain antibody can be composed of the variable region of the heavy chain (VH). The term "variable region of the heavy chain" generally refers to the amino-terminal domain of the heavy chain of the antigen-binding fragment. The variable region of the heavy chain can be further divided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). The variable region of the heavy chain contains a binding domain that interacts with the antigen, and specifically can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In the CDR region, small additions, deletions, insertions, substitutions, or modifications of amino acids are also allowed as long as they still retain the ability of the antibody to bind to a specific antigen.

[0074] The "hinge region" of the present application includes hinge regions derived from one or more proteins selected from the group consisting of: CD28, CD8, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8A, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and their variants. In the specific embodiments of the present application, the CD8 hinge region was used as an example for construction.

[0075] The "transmembrane domain" of the present application includes transmembrane domains derived from one or more proteins selected from the group consisting of: CD8, CD28, CD3ε (CD3e), 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, SLAM, and their variants. In the specific embodiments of the present application, the CD8 transmembrane domain was used as an example for construction.

[0076] The "costimulatory domain cell" described in this application includes an intracellular costimulatory signaling domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB (CD137), CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, MyD88, and variants thereof. In a specific embodiment of this application, the 41BB costimulatory domain is used as an example for construction.

[0077] The "intracellular signaling domain" described in this application includes an intracellular signal transduction domain derived from one or more proteins selected from the group consisting of: CD3ζ, ZAP70, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP-12, and a domain containing at least one ITAM. In a specific embodiment of this application, the CD3ζ intracellular signaling domain is used as an example for construction. Description of the Drawings

[0078] Figure 1 It is a graph showing the detection results of the binding affinity of the B09 original antibody and the humanized antibody HM1-5 to CD20.

[0079] Figure 2 It is a graph showing the detection results of the binding affinity of CD20 antibodies at different concentrations.

[0080] Figure 3 It is a graph showing the EC50 detection results of the B09 original antibody and the humanized antibody HM1-5.

[0081] Figure 4 It is a schematic diagram of the structure of the CAR constructed based on HM5.

[0082] Figure 5 It is composed of Figure 4 The detection result of the killing ability of the CAR-T cells prepared from the shown CAR against K562-CD20 cells.

[0083] Figure 6 It is a schematic diagram of the structure of Fmc63-Leu16 CAR.

[0084] Figure 7 It is composed ofFigure 4 The detection results of the killing ability of CAR-T cells prepared from the shown CAR and Fmc63-Leu16 CAR against K562-CD20 cells.

[0085] Figure 8 It is the flow cytometry detection result graph of CD19 humanized antibody against negative control CHO-K1 cells.

[0086] Figure 9 It is the flow cytometry detection result graph of CD19 humanized antibody against CHO-K1-CD19 cells.

[0087] Figure 10 It is the detection result graph of the binding ability of CD19 humanized antibody at different concentrations.

[0088] Figure 11 It is the EC50 detection result graph of CD19 humanized antibody.

[0089] Figure 12 It is the detection result graph of the affinity of VL2+VH1 and VL3+VH2.

[0090] Figure 13 It is the schematic structural diagram of CAR constructed based on CD19 humanized antibody.

[0091] Figure 14 It is the detection result of the killing ability of CAR-T cells constructed and prepared from VL2+VH1 and VL3+VH2 antibodies against K562-CD19 cells.

[0092] Figure 15 It is the schematic structural diagram of CAR constructed based on bispecific chimeric antigen receptor.

[0093] Figure 16 It is composed of Figure 15 The detection results of the killing ability of CAR-T cells prepared from the shown CAR against K562-CD19 and K562-CD20 cells.

[0094] Figure 17 It is the statistical result graph of the amplification multiple of cultured cells.

[0095] Figures 18 - 21 It is the apoptosis detection result graph of CAR-T cells on the 5th / 8th / 10th / 12th day.

[0096] Figure 22 It is the result graph of CAR-T cells secreting IL2 and IFN-γ on the 5th, 7th, and 12th day of culture. Detailed implementation methods

[0097] The present application will be further described below in conjunction with specific embodiments. However, the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present application, according to the technical solution and inventive concept of the present application, making equivalent substitutions or changes, shall be covered by the protection scope of the present application.

[0098] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions.

[0099] Recognition and binding of CD20 target by Example 1, B09 antibody and 5 humanized antibodies

[0100] Step 1: Screening of antibodies and their humanization

[0101] An antibody B09 specifically targeting CD20 (single-domain antibody, having CDRs shown in SEQ ID NO.1-3, and the full-length amino acid sequence as shown in SEQ ID NO.4) was screened. Further, the B09 antibody was optimized for humanized sequence. The humanization degree scoring system used T20score: http: / / abAnalyzer.lakepharma.com for scoring, and 5 antibodies HM1-HM5 with scores higher than 85 were selected.

[0102] The amino acid sequences of the above-mentioned HM1-HM5 are shown in SEQ ID NO.5-9 in sequence. Among them, the sequences of CDR (Complementarity-determining region) are the same as those of the original antibody B09, also shown in SEQ ID NO.1-3, but the framework region (Framework Residue, FR) has changed relative to the original antibody B09.

[0103] Table 1. T20 scores

[0104]

[0105] The above-designed humanized antibodies were respectively subjected to gene synthesis and subcloned into the pcDNA3.4-IgG1Fc expression vector. After the vector was verified by sequencing without error, endotoxin-free plasmid was prepared and the antibody was expressed.

[0106] The steps of vector expression are as follows:

[0107] 1) Take out the LVTransm transfection reagent and antibody expression vector from the refrigerator. After thawing at room temperature, pipette up and down to mix thoroughly. Take out the PBS and warm it to room temperature. Add 4 mL of PBS to one well of a 6-well plate, and add 20 μg of pcDNA3.4-IgG1Fc respectively. After pipetting up and down to mix well, add 60 μL of LVTransm, and immediately pipette up and down to mix. Let it stand at room temperature for 10 minutes.

[0108] 2) Add the above DNA / LVTransm complex to 20 mL of 293F cells, and gently shake to mix well. Place the cells in an incubator at 37 °C and 5% CO2 for further culture.

[0109] 3) After continuous culture for 5 days, centrifuge to collect the culture medium supernatant, filter it through a 0.45 μm filter membrane, collect the supernatant and purify the antibody using a Protein A affinity column. Measure the concentration of the purified antibody and perform SDS-PAGE denaturing electrophoresis simultaneously.

[0110] Step 2: Detection of binding ability

[0111] Flow cytometry detection method:

[0112] 1) Resuscitate CHO and CHO-CD20 cells from liquid nitrogen and adjust the cell state to the logarithmic growth phase.

[0113] 2) Divide the cells into several portions, with the number of cells in each portion being 3×10 5 cells. Add 100 μL of antibody expression supernatant or humanized antibody (30 μg / ml, diluted 3-fold at 10 points) respectively. After mixing well, incubate at room temperature for 1 hour.

[0114] 3) Centrifuge at 800 x g at room temperature for 5 minutes, discard the supernatant containing the antibody, and wash the cells 3 times with PBS.

[0115] 4) Add 100 μL of PE-Anti-human IgG (diluted 1:500), mix well, and incubate at room temperature in the dark for 45 min.

[0116] 5) Centrifuge at 800 x g at room temperature for 5 minutes, discard the supernatant containing the secondary antibody, and wash the cells 3 times with PBS.

[0117] 6) Resuspend the cells with 200 μL of PBS for flow cytometry analysis. Prepare CHO and CHO-CD20 cells, incubate with 100 μL of antibody expression supernatant respectively, and then incubate with the PE anti-human IgG fluorescent secondary antibody for flow cytometry detection.

[0118] Furthermore, flow cytometry was performed using the above method, and the results showed that both B09 and its humanized antibodies HM1-HM5 specifically bound to CD20, and the binding affinity of the humanized antibodies HM1-HM5 to CD20 was better than that of the original antibody B09( Figure 1 ).

[0119] According to the binding strength and humanization degree, humanized antibodies HM3 and HM5 were selected for purification and then reproduced at different concentrations according to the above protocol( Figure 2 ) and the FACS EC50( Figure 3 ) was detected. The results showed that the binding activity of the 8-B09 humanized antibody was consistent with the original one, and the flow cytometry binding of HM5 was stronger.

[0120] Example 2: Selection of B09 - HM5 for construction of CAR vector and verification of CD20 - targeted killing

[0121] Step 1: Construction of vector

[0122] B09-HM5 (nucleic acid sequence as shown in SEQ ID NO.10, amino acid sequence as shown in SEQ ID NO.9 in sequence) was selected to construct a pCDH-EF1a lentiviral expression plasmid containing CAR, and the structure of CAR was as Figure 4 shown. Specifically, the CD8 signal peptide - HM5 antibody - CD8 hinge region - CD8 transmembrane domain (TM, transmembrane) - 41BB co-stimulatory domain - CD3ζ intracellular signaling domain (labeled as CD3z in the figure) were connected in sequence. That is, the amino acid sequence of CAR expressed on the vector was SEQ ID NO.11, 9, 12 - 15 connected in sequence, and the sequence of its encoding nucleic acid was SEQ ID NO.16, 10, 17 - 20 connected in sequence. After the CAR structure, EGFRt was connected through T2A for subsequent fluorescence to reflect the positive rate.

[0123] Step 2: Preparation of lentivirus

[0124] The lentiviral system plasmids (the above pCDH-EF1α lentiviral expression plasmid, PsPAX2, pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) were transfected into adherent 293T cells in the logarithmic growth phase. The cell culture supernatant harvested 48 - 72 hours after transfection was concentrated and filtered to obtain CAR lentivirus, which was stored at -80°C for later use.

[0125] Step 3: Isolation of peripheral blood mononuclear cells

[0126] Peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood. T cells were isolated using human CD3 / 28 magnetic beads and virus transduction was performed within 72 hours after activation. After 24 hours of transduction, the culture medium was changed and the cells were cultured until day 8. The cells were collected by centrifugation and resuspended in physiological saline. Flow cytometry was used to identify the EGFRt molecule on the surface of CAR-T cells to ensure that the positive rate was greater than 30%. If the positive rate was lower, EGFRt-PE magnetic beads were used for enrichment sorting.

[0127] Step 4: Detection of killing ability

[0128] For killing, an effector-to-target ratio of 5:1 was used. K562 cells and K562 cells overexpressing CD20 (K562-CD20) were used as target cells, and CAR-T killing was performed for 16 h and detected using LDH.

[0129] The test results are as Figure 5 shown. The results prove that the CAR-T cells constructed with the B09-HM5 sequence have the ability to specifically kill K562 cells overexpressing CD20. In the figure, NCT is the negative control T cells, and F11HM2 is an antibody against other CD20s)

[0130] Example 3: The killing effect of B09 - HM5 CAR - T is higher than that of Fmc63 - Leu16 CAR - T

[0131] Step 1: Preparation of CD19 - CD20 tandem CAR - T cells (Fmc63 - Leu16 CAR - T)

[0132] Prepare the Bispecific anti-CD20, anti-CD19 CAR T cells (bispecific anti-CD20, anti-CD19 CAR-T cells) disclosed in the article "Bispecific anti-CD20, anti-CD19 CAR T cells for relapsed B cell malignancies: a phase 1 dose escalation and expansion trial". The connection diagram is as Figure 6 shown.

[0133] Step 2: Comparison of killing effects

[0134] Compare the CAR-T cells prepared based on B09-HM5 prepared in Comparative Example 2 with the Fmc63-Leu16 CAR-T prepared above.

[0135] Kill using an effector-to-target ratio of 1:1. Use K562 cells overexpressing CD20 (K562-CD20) as target cells and detect after 24 hours of CAR-T killing. The results prove that the CAR-T cells constructed with the B09-HM5 sequence have the ability to specifically kill K562 cells with overexpressed CD20, and are higher than Fmc63-Leu16 CAR-T( Figure 7 ).

[0136] Example 4: CD19 antibody fmc63 and its humanized antibody

[0137] Step 1: Humanization of heavy - chain and light - chain variable regions

[0138] Humanize the antibody fmc63 against CD19 (the amino acid sequence of the light chain variable region is shown in SEQ ID NO.21, and the heavy chain variable region is shown in SEQ ID NO.22, where the CDR sequences are marked in Table 2. Its full antibody form is marked as Chimeric in the subsequent result figures, and the single-chain antibody form is marked as Fmc63scfv in the subsequent result figures). Screen the framework sequences with a T20 score higher than 80 according to the T20 scoring system described in Example 1. The T20 scores are shown in Table 3.

[0139] Table 2. Amino acid sequence of fmc63 and CDR markers

[0140]

[0141] Table 3. T20 scores

[0142]

[0143] After codon optimization, gene synthesis of the heavy and light chains of the above-designed humanized antibody was carried out separately. The heavy chain was subcloned into the pcDNA3.4-IgG1 expression vector, and the light chain was subcloned into the pcDNA3.4-IgKc expression vector. After the vectors were verified by sequencing to be correct, endotoxin-free plasmids were prepared.

[0144] Step 2: Antibody screening

[0145] The humanized VH and VL antibody expression vectors were combined pairwise, resulting in a total of 15 antibodies, namely VL1+VH1, VL1+VH2, VL1+VH3, VL1+VH4, VL1+VH5, VL2+VH1, VL2+VH2, VL2+VH3, VL2+VH4, VL2+VH5, VL3+VH1, VL3+VH2, VL3+VH3, VL3+VH4, VL3+VH5, VL4+VH1, VL4+VH2, VL4+VH3, VL4+VH4, VL4+VH5, VL5+VH1, VL5+VH2, VL5+VH3, VL5+VH4, VL5+VH5. Among them, taking VL1+VH1 as an example, it represents a complete antibody composed of the VL-HM1 shown in SEQ ID NO.23 as the light chain variable region and the VH-HM1 described in SEQ ID NO.28 as the heavy chain variable region. The same applies to other names.

[0146] The above 15 antibodies were co-transfected transiently into 293F cells with Chimeric and FMC63scfV for expression. Subsequently, the binding of the prepared humanized antibodies to the target protein was detected by flow cytometry fluorescence-activated cell sorting (FACS). The cells used for detection were CHO-K1( Figure 8 ), CHO-K1-CD19( Figure 9 ). In the figure, the primary antibody was 100 μL of supernatant and a positive antibody of 10 μg / ml FMC63 scfv, and the secondary antibody was PE anti-human IgG.

[0147] According to Figures 8 - 9 the results, VL2+VH1, VL2+VH2, VL2+VH3, VL3+VH1, VL3+VH2, and VL3+VH3 were further detected by FACS EC50( Figure 10 ). According to the FACS EC50 detection, the binding activity of the candidate antibodies was consistent with that of the chimeric antibody. The average MFI value was statistically analyzed based on the results of flow cytometry detection. The higher the MFI value, the higher the affinity of the corresponding humanized antibody for antigen CD19( Figure 11 ).

[0148] Step 3: Further screening

[0149] Further select VL2+VH1 and VL3+VH2 for affinity detection and compare them with the original antibody fmc63. The specific steps are as follows: HIS1K sensor is immobilized with CD19-His (KaiKa Biotech, CD1-HM119) at a concentration of 5 μg / ml for 350 s. The buffer is PBST (PBS + 0.02% tween20). Chmeric / VL2+VH1 / VL3+VH2 is diluted to 12.5, 6.25, 3.13, 1.56, 0.7813, 0 nM. Affinity detection: equilibration for 60 s, binding for 180 s, dissociation for 180 s, and the detection temperature is 25 °C. The results are as Figure 12 shown.

[0150] Example 5: Construction of CAR based on VL2 + VH1, VL3 + VH2 and verification of targeted killing

[0151] Step 1: Construction of vector

[0152] For the VL2+VH1 and VL3+VH2 screened in Example 4, the pCDH-EF1a lentiviral expression plasmid expressing CAR is constructed according to the Figure 12 shown structure. That is, it is constructed according to the CAR structure in step 1 of Example 2, and the antibody in step 1 of Example 2 is replaced with VL2+VH1 screened in Example 4 (the coding nucleic acid sequence is shown in SEQ ID NO.33, where the 322-366th positions are the coding nucleic acid of GGGGSGGGGSGGGGS, and GGGGSx3 is used as a linker to connect the heavy chain variable region and the light chain variable region), VL3+VH2 (the coding nucleic acid sequence is shown in SEQ ID NO.34, where the 322-366th positions are the coding nucleic acid of GGGGSGGGGSGGGGS, and GGGGSx3 is used as a linker to connect the heavy chain variable region and the light chain variable region).

[0153] Step 2: Preparation of lentivirus

[0154] The lentiviral system plasmids (pCDH-EF1α lentiviral expression plasmid, PsPAX2, pMD2.G three-plasmid system, mixed at a mass ratio of 3:2:1) are transfected into adherent 293T cells in the logarithmic growth phase. The cell culture supernatant harvested 48-72 hours after transfection is concentrated and filtered to obtain CAR lentivirus, which is stored at -80 °C for later use. It is transfected into peripheral blood mononuclear cells according to the method of Example 2.

[0155] Step 3: Detection of killing ability

[0156] For killing, a 5:1 effector-to-target ratio is used. K562 cells and K562 cells overexpressing CD19 (K562-CD19) are used as target cells, and CAR-T killing is carried out for 16 h and detected by LDH. The results are asFigure 14 It is demonstrated that the CAR-T cells constructed with the VL2+VH1 and VL3+VH2 sequences have the ability to specifically kill CD19-overexpressing K562 cells. In the figure, NCT is the negative control T cells, and FMC63-LEU16 is the CD19-CD20 tandem CAR-T (i.e., the CD19-CD20 tandem CAR-T cells mentioned in Example 3).

[0157] Example 6: Fmc63 - VL3VH2 tandem B09 - HM5 CAR - T targets and kills K562 - CD19, K562 - CD20, K562 - CD19 / CD20 cells:

[0158] Step 1: Construction of vector based on Fmc63 - VL3 + VH2 and B09 - HM5 and preparation of CAR - T cells

[0159] Select B09-HM5 (the amino acid and nucleic acid sequences are shown in SEQ ID NO.9-10 respectively) screened in Examples 1-3 and VL3+VH2 (the antibody made from the amino acid sequences shown in SEQ ID NO.25 and 29 as the light and heavy chain variable regions respectively, and the nucleic acid sequence encoding it is shown in SEQ ID NO.34) screened in Examples 4-5 for tandem connection. The specific connection order of each component of the tandem structure is B09HM5-linker-VL3-GGGGSx3-VH2. Use this tandem structure to replace the antibody in step 1 of Example 2 to construct CAR, and the constructed CAR structure is as Figure 15 shown (i.e., the bispecific chimeric antigen receptor described in the present application).

[0160] Step 2: Detection of killing ability

[0161] Express the plasmid of the CAR constructed by connecting Fmc63-VL3VH2 and B09-HM5 above, prepare lentivirus, and obtain CAR-T cells after infecting cells. Detect its killing ability against K562 cells overexpressing CD19, CD20 or CD19+CD20 (24h) with an effector-to-target ratio of 5:1.

[0162] Figure 16 The results shown prove that the CAR-T cells constructed with the Fmc63-VL3VH2 tandem B09-HM5 sequence have the ability to specifically kill K562 cells expressing CD19 or CD20 and co-expressing CD19 and CD20. Among them, NCT in the legend is the control T cells, and 32-B09HM5 is the CAR-T constructed by connecting Fmc63-VL3VH2 and B09-HM5.

[0163] Example 7: Detection of tonic signaling

[0164] Two screened humanized single-domain CD20 antibodies and the humanized antibody VL3VH2 were respectively used to construct second-generation bispecific CAR lentiviral expression vectors. The second-generation CAR is specifically the CAR consisting of a CD8 signal peptide - antibody - CD8 hinge region - CD8 transmembrane domain - 41BB costimulatory domain - CD3ζ intracellular signaling domain as described in Example 2 above. The antibodies and their legends in the result figures are shown in Table 4.

[0165] Table 4. Antibody names, related SEQ ID NOs. in CAR, and their labels in the result figures

[0166]

[0167] After constructing the expression plasmids of CAR with the above antibodies, expressing them, preparing lentiviruses, and infecting cells to obtain CAR-T cells, the following detections were carried out, and the result figures were labeled according to the legends in Table 4.

[0168] 1. Detection of cell expansion multiple

[0169] The CAR-T cells prepared above were cultured and further detected. The specific steps of cell culture were as follows: an appropriate amount of X-Vivo 15 medium (containing 200 IU / mL of IL2, 10 ng / mL of IL7, and 5 ng / mL of IL15) was added, the cells were resuspended with a pipette, and the cell density was adjusted to 0.5 - 0.7×10^6 cells / mL.

[0170] From the 5th day to the 13th day, cell counting was performed every day to count the cell number. The cell number on the initial D0 day was used as the base number to calculate the CAR cell amplification multiple. The results are as Figure 17 shown.

[0171] 2. Detection of cell apoptosis

[0172] On the 5th / 8th / 10th / 12th day of culture, some of the CAR-T cells were taken, centrifuged, washed with pre-cooled PBS, resuspended with 300 μL of 1×Binding Buffer, then 5 μL of Annexin V-PE and 5 μL of 7AAD were added for staining. After mixing, they were incubated in the dark for 15 minutes. Blank T cells without any dye were used as the blank group. Another 400 μL of 1×Binding Buffer was added, and the cells were detected by flow cytometry. The results on the 5th / 8th / 10th / 12th day are successively as Figures 18 - 21 shown.

[0173] 3. Detection of cytokine secretion level

[0174] On the 5th, 7th, and 12th days respectively, 5×10^5 cells were taken. After being washed twice with PBS, they were resuspended with 1 ml of blank X-Vivo15 medium, placed in an incubator at 37°C and 5% CO2, and cultured for another 24 h. Then, the supernatant was collected by centrifugation and stored at -80°C. After all the supernatants were collected, the secretion of IL2 and IFN-γ was detected according to the operation instructions of the ELISA kits for IL2 and IFN-γ.

[0175] Results: Cells were taken on D5, D7, and D12 respectively, cultured with blank X-vivo15 for 24 h, and then the supernatant was taken to detect the secretion of IL2 and IFN-γ. According to the detection results, CAR-T cells hardly secreted IL-2 while secreting IFN-γ, and the secretion increased with the increase of the culture time ( Figure 22 ).

[0176] The results of the above three aspects show that the CAR-T cell basal signal (tonic signaling) prepared by cascading the CD20 antibody and the CD19 antibody screened based on this application is not strong, which can ensure the normal expansion of CAR-T cells and the apoptosis ratio within the normal range. At the same time, it will not release IL-2 without activation, and the release level of IFN-γ is controllable.

Claims

1. A CD20 antibody or an antigen-binding fragment thereof targeting the CD20 protein, wherein the amino acid sequences of CDR1, CDR2, and CDR3 in the CD20 antibody or its antigen-binding fragment are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively; The CD20 antibody is a single-domain antibody.

2. The CD20 antibody or antigen-binding fragment thereof according to claim 1, wherein, The CD20 antibody is humanized.

3. The CD20 antibody or antigen-binding fragment thereof according to claim 1, wherein The CD20 antibody has an amino acid sequence shown in any one of SEQ ID NO.4 - 9.

4. The CD20 antibody or antigen-binding fragment thereof according to claim 1, wherein The CD20 antibody has modifications such as deamidation, disulfide bond formation, terminal lysine cleavage, or glycosylation.

5. The CD20 antibody or antigen-binding fragment thereof according to claim 4, wherein, The glycosylation modification includes modifications of core fucose, sialic acid, or high-mannose levels.

6. A chimeric antigen receptor, which contains the CD20 antibody described in any one of claims 1 - 5, or contains the CD20 antibody described in any one of claims 1 - 5 and a humanized CD19 antibody; The CD19 antibody contains a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region of the CD19 antibody is shown in SEQ ID NO.24, and the amino acid sequence of the heavy chain variable region of the CD19 antibody is shown in SEQ ID NO.28; Alternatively, the amino acid sequence of the light chain variable region of the CD19 antibody is shown in SEQ ID NO.25, and the amino acid sequence of the heavy chain variable region of the CD19 antibody is shown in SEQ ID NO.

29.

7. The chimeric antigen receptor according to claim 6, wherein The chimeric antigen receptor further includes one or more of a hinge region, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

8. The chimeric antigen receptor according to claim 7, wherein, The hinge region contains a hinge region derived from one or more proteins selected from the group consisting of: CD28, CD8, IgG1, IgG4, IgD, 4 - 1BB, CD4, CD27, CD7, CD8A, PD - 1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, TIM1, SLAM, CD30, LIGHT, and their variants.

9. The chimeric antigen receptor according to claim 7, wherein The transmembrane domain contains a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD28, CD3ε, 4 - 1BB, CD4, CD27, CD7, PD - 1, TRAC, TRBC, CD3ζ, CTLA - 4, LAG - 3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L (CD154), TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, SLAM, and their variants.

10. The chimeric antigen receptor according to claim 7, characterized in that, The co-stimulatory domain comprises a co-stimulatory domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8A, CD8B, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD40, MyD88, and variants thereof.

11. The chimeric antigen receptor according to claim 7, wherein, The intracellular signaling domain comprises an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3ζ, ZAP70, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FceRIγ, FceRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus-LMP2A, simian immunodeficiency virus PBj14 Nef, DAP10, DAP-12, and a domain comprising at least one ITAM.

12. The chimeric antigen receptor according to claim 6, wherein The chimeric antigen receptor is composed of the CD20 antibody according to any one of claims 1-5, or the CD20 antibody according to any one of claims 1-5 and the CD19 antibody, and a hinge region, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

13. The chimeric antigen receptor according to claim 6, wherein, The chimeric antigen receptor, from the N-terminus to the C-terminus, is in sequence: the CD20 antibody according to any one of claims 1-5, or the CD20 antibody according to any one of claims 1-5 and the CD19 antibody - CD8 hinge region - CD8 transmembrane domain - 4-1BB co-stimulatory domain - CD3ζ intracellular signaling domain.

14. A nucleic acid molecule encoding one or more of the CD20 antibodies according to any one of claims 1-5 and the chimeric antigen receptors according to any one of claims 6-13.

15. A recombinant vector comprising the nucleic acid molecule according to claim 14.

16. The recombinant vector according to claim 15, wherein The recombinant vector is a cloning vector or an expression vector.

17. A genetically modified cell comprising or expressing the chimeric antigen receptor according to any one of claims 6-13, the nucleic acid molecule according to claim 14, and / or the recombinant vector according to claim 15 or 16; The cell is an immune cell.

18. The genetically modified cell according to claim 17, wherein The cell is a T cell, B cell, natural killer cell, macrophage, and / or NKT cell.

19. The genetically modified cell according to claim 18, characterized in that, The cell is a T cell.

20. The genetically modified cell according to claim 18, wherein The cell is a human cell.

21. A host cell expressing the CD20 antibody according to any one of claims 1-5.

22. An antibody-drug conjugate comprising a cytotoxic agent or a tag, and comprising the CD20 antibody according to any one of claims 1-5, or comprising the CD20 antibody according to any one of claims 1-5 and a humanized CD19 antibody; The CD19 antibody comprises a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region of the CD19 antibody is as shown in SEQ ID NO.24, and the amino acid sequence of the heavy chain variable region of the CD19 antibody is as shown in SEQ ID NO.28; Alternatively, the amino acid sequence of the light chain variable region of the CD19 antibody is as shown in SEQ ID NO.25, and the amino acid sequence of the heavy chain variable region of the CD19 antibody is as shown in SEQ ID NO.

29.

23. A pharmaceutical composition, which contains: one or more of the CD20 antibodies according to any one of claims 1-5, the chimeric antigen receptor according to any one of claims 6-13, the nucleic acid molecule according to claim 14, the recombinant vector according to claim 15 or 16, the cell according to any one of claims 17-20, and the antibody-drug conjugate according to claim 22.

24. A detection kit, which contains a labeled antibody, and the antibody is the CD20 antibody according to any one of claims 1-5, or the antibody is the CD20 antibody according to any one of claims 1-5 and a humanized CD19 antibody; The CD19 antibody comprises a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region of the CD19 antibody is as shown in SEQ ID NO.24, and the amino acid sequence of the heavy chain variable region of the CD19 antibody is as shown in SEQ ID NO.28; Alternatively, the amino acid sequence of the light chain variable region of the CD19 antibody is as shown in SEQ ID NO.25, and the amino acid sequence of the heavy chain variable region of the CD19 antibody is as shown in SEQ ID NO.

29.

25. Use of any one or more of the following products in the preparation of cancer drugs: The CD20 antibody according to any one of claims 1-5, the chimeric antigen receptor according to any one of claims 6-13, the nucleic acid molecule according to claim 14, the recombinant vector according to claim 15 or 16, the cell according to any one of claims 17-20, the antibody-drug conjugate according to claim 22, and the pharmaceutical composition according to claim 23; The cancer is non-Hodgkin lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma or mantle cell lymphoma.

26. Use of the CD20 antibody according to any one of claims 1-5 or the detection kit according to claim 24 in the preparation of CD20-related cancer diagnostic products; The cancer is non-Hodgkin lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma or mantle cell lymphoma.

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