CN108047332B

CN108047332B - Specific antibody with CD19 as target, CAR-NK cell, and preparation and application thereof

Info

Publication number: CN108047332B
Application number: CN201810036245.8A
Authority: CN
Inventors: 李华顺
Original assignee: Asclepius Suzhou Technology Company Group Co Ltd
Current assignee: Asclepius Suzhou Technology Company Group Co Ltd
Priority date: 2018-01-15
Filing date: 2018-01-15
Publication date: 2021-08-24
Anticipated expiration: 2038-01-15
Also published as: WO2019137518A1; CN108047332A

Abstract

The invention provides an anti-CD19 antibody or antigen-binding fragment thereof that targets CD19, wherein the antibody comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the heavy chain variable region comprises a heavy chain VHCDR1, VHCDR2, VHCDR 3; the light chain variable region comprises a light chain VLCDR1, VLCDR2, VLCDR 3. The invention provides an Anti-CD19 antibody or an antigen binding fragment thereof with CD19 as a target spot, and Anti CD19 CAR-NK cells constructed by the antibody are obtained by culturing and amplifying monoclonal cell strains, have stable properties and can be used for large-scale production and preparation. The Anti CD19 CAR-NK cell takes a CD19 molecule as a target antigen, can specifically kill or kill lymphoma cells, can be used as a therapeutic drug for lymphoma diseases, and is used for treating tumors with high expression of CD19 molecules.

Description

Specific antibody with CD19 as target, CAR-NK cell, and preparation and application thereof

Technical Field

The invention relates to the field of biological medicines, in particular to a specific antibody taking CD19 as a target spot, CAR-NK cells taking CD19 as a target spot, and preparation and application thereof.

Background

Chimeric antigen receptor (abbreviated CAR) modified immune cells use genetic engineering approaches to modify immune cells to express exogenous anti-tumor genes. The CAR gene mainly includes an extracellular recognition domain and an intracellular signaling domain: the former is used for identifying tumor surface specific molecules and the latter is used for starting immune cell response after identifying the tumor surface molecules to play a cytotoxic role, and the T-cells are mainly used as carriers.

CAR-T, collectively known as Chimeric antigen receptor T-Cell Immunotherapy, is a Chimeric antigen receptor T-Cell Immunotherapy. Chimeric antigen receptor T cells (CAR-T cells) are prepared by coupling an antigen-binding portion of an antibody recognizing a certain tumor antigen to an intracellular element such as CD 3-zeta chain or 4-1BB in vitro to form a chimeric gene, and transfecting T cells of a patient by gene transduction to express the Chimeric Antigen Receptor (CAR). After the patient's T cells are "recoded," a large number of tumor-specific CAR-T cells are generated. CD19 is a 95kD membrane penetrating glycoprotein belonging to the immunoglobulin superfamily and playing a central role in B cell regulatory responses. Numerous studies have demonstrated that CD19 can be used as a target for the treatment of B-cell lymphoma for the construction of CAR-T.

At present, a large number of clinical practices prove that the CAR-T medicament developed by taking the CD19 as a target achieves remarkable curative effect. Nowa pharmaceutical, Inc. announced its CAR-T product CTL019 to enter the FDA accelerated approval channel on 3/29/2017. On month 2015 12, the FDA granted CAR-T therapy KTE-C19 by Kite Pharma for breakthrough therapy certification (BTD) for indication therapy for the treatment of diffuse large B-cell lymphoma (DLBCL). On 1/4.2017, Kite Pharma also announced a rolling declaration of biological product approval (BLA) for the official submission of CAR-T therapy KTE-C19 (hereinafter "axicabtagene ciloleucel") to the FDA.

However, some problems still exist in the current CD19 CAR-T research. For example: first, CAR-T producing T cells can only be derived from the patient himself, not foreign body reinfusion; secondly, the ScFV sequence of CD19 antibody used for CAR-T preparation internationally is mostly murine antibody, and has larger immune rejection risk.

Natural Killer (NK) cells are an important component of the non-specific immune system, cells that are critical mediators of the innate immune system response. NK cells are a broad spectrum immune cells with specific functions of rapidly discovering and destroying abnormal cells (such as cancer or virus-infected cells), and exhibit potent activity of lysing abnormal cells without the need for pre-sensitization or HLA-typing. The use of immune cells, including NK cells, for the treatment of cancer is a new trend in recent years and this new therapy is expected to provide new cure hopes for tumors that are not effective in traditional surgery, chemotherapy and radiotherapy.

Disclosure of Invention

In view of the above, the invention provides an anti-CD19 antibody or an antigen-binding fragment thereof targeting CD19, a CAR-NK cell capable of expressing the domain, and preparation and application thereof, wherein the cell has stable properties, can be produced and prepared in large scale, and can be used for foreign body backtracking; on the other hand, the antibody sequence used for constructing the monoclonal antibody is a human antibody sequence, and when the monoclonal antibody is used as a medicament, the risk of immunological rejection is greatly reduced.

In one aspect, the invention provides an anti-CD19 antibody or antigen-binding fragment thereof that targets CD19, wherein the antibody comprises a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein the heavy chain variable region comprises a heavy chain VHCDR1, VHCDR2, VHCDR 3; the light chain variable region comprises light chain VLCDR1, VLCDR2, VLCDR3, wherein the amino acid sequence of VHCDR1 is as set forth in SEQ ID NO: 1 or a homologous sequence thereof; the amino acid sequence of the VHCDR2 is shown in SEQ ID NO: 2 or a homologous sequence thereof; the amino acid sequence of the VHCDR3 is shown in SEQ ID NO: 3 or a homologous sequence thereof; the amino acid sequence of the VLCDR1 is set forth in SEQ ID NO: 4 or a homologous sequence thereof; the amino acid sequence of the VLCDR2 is set forth in SEQ ID NO: 5 or a homologous sequence thereof; the amino acid sequence of the VLCDR3 is set forth in SEQ ID NO: 6 or a homologous sequence thereof.

Illustratively, the amino acid sequence of the VH chain is as set forth in SEQ ID NO: 7 or a homologous sequence thereof.

Illustratively, the amino acid sequence of the VL chain is as set forth in SEQ ID NO: 8 or a homologous sequence thereof.

Illustratively, the homology of the homologous sequence is 60% or more, e.g., about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, or less, or more than 8% or more, or less, or more than or less or more than 0.8% or more, or more than the like, Or 99.9% or more, etc.

Illustratively, the VH chain and VL chain are linked directly or via a linking peptide.

In one embodiment provided herein, an anti-CD19 antibody or antigen-binding fragment thereof that targets CD19 comprises a VH chain and a VL chain.

In one embodiment provided herein, the VH chain and VL chain are linked by a linker peptide.

In one embodiment provided herein, the anti-CD19 antibody or antigen-binding fragment thereof has a nucleotide sequence set forth in SEQ ID NO: 9 or a degenerate sequence thereof.

In one embodiment provided herein, the anti-CD19 antibody or antigen-binding fragment thereof has an affinity for CD19 of 8.473 × 10^-8M is more than M.

In another aspect, the invention provides a nucleotide sequence capable of expressing the anti-CD 33 antibody or antigen-binding fragment thereof described above.

In one embodiment provided herein, the nucleotide sequence comprises SEQ ID NO: 10 or a degenerate sequence thereof, and/or SEQ ID NO: 11 or a degenerate sequence thereof.

In one embodiment of the present invention, the nucleotide sequence is as shown in SEQ ID NO: 12 or a degenerate sequence thereof.

Illustratively, the degenerate sequence has a homology of 60% or more, e.g., about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.1 or more, 99.2 or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, or less, or more, or less, or more, or less, or more than or less, or less than or more than or less than or, Or 99.9% or more, etc.

In another aspect, the present invention provides a method for preparing the anti-CD19 antibody or the antigen-binding fragment thereof, comprising: phage antibody libraries are created and screened for antibodies or antibody fragments that bind to CD 19. The specific process comprises the following steps:

preparing M13KO7 helper phage;

establishing a phage antibody library by using M13KO7 helper phage;

thirdly, screening single-chain phage DNA from the antibody library in the second step by using CD19 antigen; and optionally also (c) a second set of one or more of,

fourthly, expressing and purifying the single-stranded phage DNA obtained in the third step.

In another aspect, the invention provides an Anti CD19 CAR-NK cell capable of expressing a chimeric antigen receptor comprising the Anti-CD19 antibody or antigen-binding fragment thereof described above.

In a specific embodiment of the invention, the chimeric antigen receptor further comprises a transmembrane domain and/or a costimulatory signaling region.

Illustratively, the transmembrane domain is selected from: a transmembrane domain of one or more of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD 154; preferably, the transmembrane domain is a CD8 transmembrane domain; and/or the presence of a gas in the gas,

the costimulatory signaling region comprises the intracellular domain of a costimulatory molecule selected from the group consisting of: one or more of CD3 ζ, CD3 γ, CD3 δ, CD3 ∈, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28, CD134, CD137, ICOS, CD154, 4-1BB, and OX 40; preferably, the costimulatory signaling region comprises the 4-1BB and CD3 zeta intracellular domains.

Preferably, the anti-CD19 antibody or antigen-binding fragment thereof is capable of specifically binding to the tumor-specific antigen CD19 and activating the NK cell via a transmembrane domain and a costimulatory signaling region.

Illustratively, the chimeric antigen receptor is a fusion protein with the structure of SCFV-CD8-4-1BB-CD3 zeta, and the fusion protein can specifically recognize CD19 molecules.

In one embodiment of the invention, the amino acid sequence of the SCFV-CD8-4-1BB-CD3 zeta fusion protein is represented by SEQ ID NO: 13 or a homologous sequence thereof, and the like.

Illustratively, the sequence of the nucleotide of the SCFV-CD8-4-1BB-CD3 zeta fusion protein is shown in SEQ ID NO: 14 or a degenerate sequence thereof, and the like.

In a specific embodiment of the invention, the Anti CD19 CAR-NK cells are capable of effectively killing or killing Raji cells and the like.

The invention also provides a preparation method of the Anti CD19 CAR-NK cell, which comprises the following steps:

(1) synthesizing and amplifying a SCFV-CD8-4-1BB-CD3 zeta fusion protein gene, and cloning the SCFV-CD8-4-1BB-CD3 zeta fusion protein gene onto a lentivirus expression vector;

(2) infecting 293T cells by using a lentivirus packaging plasmid and the lentivirus expression vector plasmid obtained in the step (1), packaging and preparing lentivirus;

(3) infecting NK-92 cells by using the lentivirus obtained in the step (2) to obtain CD19 CAR-NK cells.

The invention also provides the application of the Anti-CD19 antibody or an antigen binding fragment thereof, and/or the Anti-CD19 antibody or an antigen binding fragment thereof expressed by the nucleotide sequence, and/or the Anti CD19 CAR-NK cell in preparing medicines for treating and/or preventing tumors with high expression of CD19 molecules and related diseases.

Exemplarily, the Anti CD19 CAR-NK cell is applied to the preparation of a medicine for treating lymphoma with high expression of CD19 molecules.

The invention also provides a pharmaceutical composition, which comprises the Anti-CD19 antibody or an antigen-binding fragment thereof, and/or the Anti-CD19 antibody or an antigen-binding fragment thereof expressed by the nucleotide sequence, and/or the Anti CD19 CAR-NK cell, and optionally pharmaceutically acceptable auxiliary materials.

The invention has at least one of the following advantages:

the invention provides an Anti-CD19 antibody or an antigen binding fragment thereof with CD19 as a target spot, and Anti CD19 CAR-NK cells constructed by the antibody are obtained by culturing and amplifying monoclonal cell strains, have stable properties and can be used for large-scale production and preparation. The Anti CD19 CAR-NK cell takes a CD19 molecule as a target antigen, can specifically kill or kill lymphoma cells, can be used as a therapeutic drug for lymphoma diseases, and is used for treating the lymphoma with high expression of a CD19 molecule.

Drawings

FIG. 1 is a schematic structural diagram of pCDNA3.4-ScFV (anti-CD19) expression vector provided in the embodiment of the present invention.

FIG. 2 is a schematic diagram showing the structure of pRRSLIN-ScFV (2-27) -CD8TM-4-1BB-CD3 zeta lentivirus transfection vector provided in the embodiment of the present invention.

FIG. 3 is a graph showing the results of detecting the positive rate of CD19 CAR-NK cells by flow cytometry; FIG. 3a is a NK-92 control group, and FIG. 3b is a CD19 NK-92 experimental group.

FIG. 4 is a graph showing the results of experiments on killing Raji cells by CD19 CAR-NK cells according to the present invention.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Specifically, the nucleotide sequence encoding the SCFV-CD8-4-1BB-CD3 zeta fusion protein is any DNA sequence capable of encoding the fusion protein, preferably, the sequence is SEQ ID NO: 14 or the complement thereof. In another aspect, the nucleotide sequence encoding the SCFV-CD8-4-1BB-CD3 zeta fusion protein according to the invention may be a nucleotide sequence that hybridizes under stringent conditions with the full complement of the sequence defined by SEQ ID NO: 14, and a polynucleotide encoding the fusion protein or a complementary sequence thereof;

the "stringent conditions" as used herein may be any of low stringency conditions, medium stringency conditions or high stringency conditions, and preferably high stringency conditions. Illustratively, "low stringency conditions" can be conditions of 30 ℃, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 52% formamide; "Medium stringency conditions" can be 40 ℃, 5 XSSC, 5 XDenhardt solution, 0.5% SDS, 52% formamide; the "high stringency conditions" may be 50 ℃ in 5 XSSC, 5 XDenhardt's solution, 0.5% SDS, 52% formamide. It will be appreciated by those skilled in the art that higher temperatures will result in polynucleotides with high homology. In addition, one skilled in the art can select the result of combining multiple factors, such as temperature, probe concentration, probe length, ionic strength, time, salt concentration, etc., that affect the stringency of hybridization to achieve the corresponding stringency.

The polynucleotides that can hybridize to each other may have about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1 or more, 99.2 or more, or the polynucleotides encoding sequence ID No. 6, or more, or the same, when calculated by FASTA, BLAST-equivalent homology search software using default parameters set by a system, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more, identical polynucleotides.

The identity of nucleotide sequences can be determined using the algorithm rules BLAST of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-. The programs BLASTN, BLASTX based on the rules of the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215:403,1990). When BLASTN is used to analyze a nucleotide sequence, the parameters are set to score 100 and workength 12; when BLASTX is used to analyze an amino acid sequence, the parameters are set to score 50 and Wordlength 3; when BLAST and Gapped BLAST programs are used, default parameter values can be set for the system using each program.

In the present invention, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The Antibodies of the invention may exist In a variety of forms including, for example, polyclonal, monoclonal, Fv, Fab and F (ab)2, as well as single chain and humanized Antibodies and the like (Harlow et al, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl. Acad. Sci.USA 85: 5879-.

The term "antibody fragment" refers to a portion of an intact antibody and refers to the epitope variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab ', F (ab')2, and Fv fragments, linear antibodies formed from antibody fragments, scFv antibodies, and multispecific antibodies.

Unless otherwise specified, "coding nucleotides" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The nucleotide sequence encoding the protein may include an intron.

The term "lentivirus" refers to a genus of the family retroviridae that is capable of efficiently infecting non-periodic and post-mitotic cells; they can transmit significant amounts of genetic information into the DNA of the host cell, so that they are one of the most efficient methods of gene delivery vectors.

The term "vector" is a composition of matter that includes an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphipathic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

The term "cancer" is defined as a disease characterized by rapid and uncontrolled growth of aberrated cells. Cancer cells can spread locally or through the blood stream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. The term "comprising" in any of the expressions herein, particularly in describing the method, use or product of the invention, is to be understood as including those products, methods and uses which consist essentially of and consist of the recited components or elements or steps. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

English names appearing herein are case-insensitive; CD19 CAR-NK, CD19-CAR NK have the same meaning; CD19 CAR-NK has the same meaning as Anti CD19-CAR NK, both of which represent CAR-NK cells against CD19 molecules; NK-92 and NK92 both represent NK92 cells.

The 'NK' is a normal NK cell or NKT cell or NK cell line of a human body, and comprises an NK-92 cell, a YT cell, an NKL cell, a HANK-1 cell, an NK-YS cell, a KHYG-1 cell, an SNK-6 cell, an IMC-1 cell and the like. NK-92 cells are exemplified in the specific examples of the present invention.

For a more clear illustration of the invention, reference is now made in detail to the following examples, which are intended to be purely exemplary of the invention and are not to be interpreted as limiting the application.

Example 1 screening of human antibodies to CD19

2 × YT liquid medium: 16g of peptone, 10g of yeast extract, 5g of sodium chloride and 800mL of water, adjusting the pH to 7.0 by using sodium hydroxide, adding water to a constant volume of 1000mL, and sterilizing at 121 ℃ for 20 min.

2 XYT-G: 2% glucose was added to 2 XYT medium.

2 XYT-AK: 2 XYT medium was supplemented with 100g/mL ampicillin and 50g/mL kanamycin.

First, M13KO7 helper phage was prepared

1. TG1 bacterial cells in the logarithmic growth phase were infected with different dilutions of the helper phage M13K07 for 30 minutes at 37 ℃ before plating on agar plates.

2. TG1 plaque clones were picked into 3mL of liquid 2YT medium for culture. The culture was carried out at 37 ℃ for 2 hours.

3. The culture in step 2 was transferred to 1L of 2YT medium, kanamycin was added to 50. mu.g/m, and cultured at 37 ℃ for 16 hours.

4. The bacterial cells were removed by centrifugation (10min at 5000g) and phage was collected by adding phage precipitant to the supernatant.

5. The titer of the phage is calculated,then it was diluted to 1X 10¹³pfu/mL and stored in-20 degree refrigerator for use.

Secondly, preparing a phage antibody library

1. Inoculating the phage library glycerol strain into 500ml 2YT-G culture medium, and adjusting OD value to 0.8-0.9 at 37 deg.C and 250 rpm.

2. The M13KO7 prepared in step one was added to the above step 1 to a final concentration of 5X 10⁹pfu/mL, standing at 37 ℃ for 30min, and then shaking-culturing at 200rpm for 30 min.

3. The culture broth from step 2 was centrifuged (2200g, 15min), bacterial cells were collected, and the bacterial sludge was resuspended in 2YT-AK medium and cultured overnight at 30 ℃ at 300 rmp.

4. The culture broth in step 3 was centrifuged (7000g, 15min, 4 ℃) to remove bacterial cells, and the supernatant was collected into a 1L flask.

5. And 4, adding a phage precipitator into the supernatant obtained in the step 4, and collecting phage precipitates.

6. Centrifugation (7000g, 15min) removed the supernatant, phage pellet collected and added to 8ml PBS.

7. Bacterial debris was removed by re-centrifugation (12,000g, 10min) and phage was retained in the supernatant for use.

Third, antibody screening

1. The CD19 antigen was coated onto the well plate at a concentration of 5. mu.g/mL for 2h at 4 ℃ and then washed three times. Blocking was performed overnight at 4 ℃ using blocking solution.

2. The blocking solution was decanted and washed, and the phage (prepared in step two) and 100. mu.g/mL human IgG protein were resuspended in blocking solution (containing 2% milk) and added to the well plate for 1h incubation at room temperature, followed by washing.

3. Phage bound to the well plate were trypsinized for a subsequent second round of screening.

4. Three rounds of selection were repeated as described in the above procedures 1-3.

5. Obtaining single-chain phage DNA, namely a single-chain antibody gene sequence through precipitation, and sequencing and identifying.

Example 2: expression and characterization of CD19 antibody

The single-stranded phage DNA obtained in example 1 was cloned into pCDNA3.4 vector to construct pCDNA3.4-ScFV (anti-CD19) expression vector shown in FIG. 1, the vector was transiently transferred to CHO cell for expression, and the single-stranded antibody sequence was purified by nickel strain for subsequent analysis.

The affinity of the selected single-chain antibody to the CD19 protein was identified by the SPR method, and the single-chain antibody having the highest affinity to the CD19 protein was selected and named as single-chain antibody 2-27. The affinity of the single-chain antibodies 2-27 for the CD19 molecule is shown in Table 1.

Table 1: single chain antibody 2-27 with affinity to CD19 molecule

sequence	Ka(1/Ms)	Kd(1/s)	KD(M)
				2-27	4.984E+4	0.004223	8.473E-8

Wherein, the amino acid sequence of the VH chain of the single-chain antibody 2-27 is shown as SEQ ID NO.7, and the nucleotide sequence thereof is shown as SEQ ID NO. 10; the amino acid sequence of the VL chain of the single-chain antibody 2-27 is shown in SEQ ID NO.8, and the nucleotide sequence thereof is shown in SEQ ID NO. 11; the amino acid sequence of the single-chain antibody 3-3 is shown in SEQ ID NO.9, and the nucleotide sequence of the single-chain antibody 2-27 is shown in SEQ ID NO. 12.

EXAMPLE 3 preparation of lentiviral expression vectors

The gene synthesizes the fusion gene sequence of SCFV (2-27) -CD8-4-1BB-CD3 zeta (the amino acid sequence is shown as SEQ ID NO: 13, and the gene sequence is shown as SEQ ID NO: 14). The DNA fragment is converted and connected into a PRRSV IN vector through enzyme digestion, and the upstream of the gene is an EP-1 alpha promoter. The vector is transformed into Stbl3 escherichia coli strain, ampicillin is screened to obtain positive clone, plasmid is extracted, and restriction enzyme digestion identification clone is carried out to obtain PRRLSIN-SCFV (2-27) -CD8-4-1BB-CD3 zeta lentivirus transfection vector (shown in figure 2).

Example 4 preparation of lentiviruses

(1) 24 hours before transfection, at about 8X 10 per dish⁶293T cells were seeded into 15cm dishes. Ensure that the cells are confluent at around 80% and evenly distributed in the culture dish during transfection.

(2) Preparing solution A and solution B

Solution A: 6.25ml of 2 XHEPES buffer (5 large dishes packaged together, best).

Solution B: the following mixtures of plasmids were added separately: 112.5. mu.g PRRLSIN-SCFV (2-27) -CD8-4-1BB-CD3 ζ (target plasmid); 39.5 μ G pMD2.G (VSV-G envelop); 73. mu.g pCMVR8.74(gag, pol, tat, rev); 625. mu.l of 2M calcium ion solution. Total volume of solution B: 6.25 ml.

And (3) fully mixing the solution B, adding the solution B dropwise while slightly swirling the solution A, and standing for 5-15 minutes. The mixed solution of A and B was vortexed gently, added dropwise to a culture dish containing 293T cells, and the dish was shaken gently back and forth to uniformly distribute the mixture of DNA and calcium ions. (without rotating the dish) was placed in an incubator for 16-18 hours. The culture was continued by replacing the fresh medium, and the virus-containing supernatants were collected after 48 hours and 72 hours, respectively. 500g, centrifuged at 25 ℃ for 10 minutes. PES membrane (0.45 μm) filtration. Beckmann Coulter Ultra-clear SW28 centrifuge tubes were sterilized with 70% ethanol and placed under an ultraviolet lamp for 30 minutes. The filtered lentivirus-containing supernatant was transferred to a centrifuge tube. A20% layer of sucrose (1 ml of sucrose per 8ml of supernatant) was carefully applied to the bottom of the tube. Centrifuge tubes were equilibrated with PBS, 25000rpm (82, 700g), and centrifuged at 4 ℃ for 2 hours. Carefully remove the tube, pour off the supernatant, invert the tube to remove the residual liquid. Add 100. mu.l PBS, seal the tube, stand at 4 ℃ for 2 hours, gently vortex every 20 minutes, centrifuge at 500g for 1 minute (25 ℃), and collect the viral supernatant. Cooling on ice, and storing at-80 deg.C.

Example 5 preparation of CD19 CAR-NK-92 cells

Adjusting NK-92 cell density to 2-3X 10⁵Per ml, in volume ratio (V/V) viral vector: viral vectors (prepared in example 4) were added to the cell culture medium at a ratio of 1:5-10, and 8. mu.g/ml polybrene was added. After 4h, the cell density was adjusted to 1X 10 by feeding an equal amount of fresh complete medium⁵The culture was continued at a concentration of/ml. The next day, all cells were centrifuged, fresh medium was added and culture continued. Supplementing every 1-2 days to maintain cell density at 2-3 × 10⁵And/ml. CAR antibody staining was performed after 72h, while CD19 CAR NK-92 positive cells were flow sorted and expanded in culture. The color change, cell density, cell morphology of the culture medium were observed daily and recorded accordingly.

The flow detection CAR NK-92 cell positive rate is utilized, and the flow detection result is shown in figure 3a and figure 3 b. In fig. 3a and 3b, the antibody used was fluorescent labeled with APC and is shown on the abscissa, and the signal value of NK92 cells will be significantly increased if they successfully express the CAR molecule. As can be seen from FIGS. 3a and 3b, the signal value of APC fluorescence labeling is significantly increased, indicating that NK-92 cells successfully express the CAR molecule, and the CAR-NK92 positive rate is 99.91%.

Example 6 evaluation of CD19 CAR-NK cells for killing of tumors in vitro

The killing effect of CD19 CAR-NK cells on Raji cells is detected by using a CCK-8 method. The experimental operating method is as follows:

(1) 1ml of Raji cell suspension (2X10^ 4/well) was prepared in 24-well plates. The plates were pre-incubated in an incubator for 12 h.

(2) The culture supernatant of the 24-well plate was discarded and 1ml of effector cells were added per well, the ratio of the number of effector cells to target cells being 1:1 or 0.5: 1. Medium control wells were filled with only 1ml of medium, and triplicate wells were placed for each experiment. Effector cells were incubated with target cells for 4 hours or 2 hours, and experimental groups are shown in table 2 below.

TABLE 2 experimental grouping of the killing effect of CD19 CAR-NK cells on Raji cells

(3) 100ul of CCK-8 solution was added to each well and the plates were incubated in an incubator for 2 h.

(4) Absorbance at 450nm was measured with a microplate reader.

(5) The killing rate is (As-Ab)/(Ac-Ab) X100%;

as: test wells (tumor cell-containing medium, CCK-8, CAR-NK);

ac: control wells (tumor cell-containing medium, CCK-8);

ab: blank control (medium without cells and CAR-NK, CCK-8);

the experimental results of the evaluation of the in vitro tumor killing effect of CD19 CAR-NK cells are shown in fig. 4.

As shown in fig. 4, the experimental results show that compared with NK92 control group, CD19 CAR NK-92 cells prepared by the present invention can significantly kill Raji target cell strain.

The CD19 CAR NK-92 is obtained by infecting CD19 CAR molecules into an NK92 cell strain, obtaining single clone cells through flow screening, culturing and amplifying the CAR NK92 single clone cell strain with high stable killing activity. The cells can be prepared for large-scale production, can be used for different patients and do not generate GVHR rejection. Compared with the CAR-T cells, the CD19 CAR NK-92 cells do not need to separate Peripheral Blood Mononuclear Cells (PBMC) of patients, do not need to specifically activate T cells and prepare CAR-T cells (the process needs the patients to wait for more than 10 days), do not need to be customized, can be used for a plurality of patients, and shorten the time, and the CD19 CAR-NK92 cells can be prepared and cultured in a large scale, so that the patients can use the cells immediately; on the other hand, the conventionally prepared CAR-T cells are prepared by separating T cells of patients and infecting the T cells with viruses, the T cells are not from the same monoclonal source, and the separated CAR-NK92 cells are derived from the same monoclonal, have uniform and stable properties and activities, and are convenient for large-scale production and quality control; compared with NK92 cells, the CAR vector is introduced, so that the killing activity is specific and the tumor treatment effect is obvious.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.

Sequence listing

<110> Zhejiang Acisco Biotech Ltd

<120> specific antibody taking CD19 as target, CAR-NK cell and preparation and application thereof

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VHCDR1

<400> 1

Ser Tyr Ala Met Ser

1 5

<210> 2

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VHCDR2

<400> 2

Ser Ile Asp Ala Gln Gly Leu Pro Thr Arg Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly Arg Phe Thr

20

<210> 3

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VHCDR3

<400> 3

Ser Gly Thr Pro Phe Asp Tyr

1 5

<210> 4

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VLCDR1

<400> 4

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VLCDR2

<400> 5

Ser Ala Ser His Leu Gln Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VLCDR3

<400> 6

Gln Gln Ala Asn Thr Ser Pro Thr Thr

1 5

<210> 7

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH

<400> 7

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Asp Ala Gln Gly Leu Pro Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Gly Thr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 8

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL

<400> 8

Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val

1 5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser

20 25 30

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Ala Ser His Leu Gln Ser Gly Val Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Thr Ser Pro

85 90 95

Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 9

<211> 240

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Anti CD19

<400> 9

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Asp Ala Gln Gly Leu Pro Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Gly Thr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

130 135 140

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

145 150 155 160

Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

165 170 175

Lys Leu Leu Ile Tyr Ser Ala Ser His Leu Gln Ser Gly Val Pro Ser

180 185 190

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

195 200 205

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn

210 215 220

Thr Ser Pro Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

225 230 235 240

<210> 10

<211> 348

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VH

<400> 10

gaggtgcagc tgctggaatc aggaggagga ctggtgcagc caggaggatc tctgagactg 60

tcttgcgccg cttccggctt caccttctct tcctacgcca tgtcttgggt ccgacaggct 120

ccaggaaagg gactggagtg ggtgtcctct atcgacgcac agggcctgcc taccagatac 180

gccgattccg tgaagggcag gttcaccatc tcccgggaca actccaagaa caccctgtac 240

ctgcagatga actccctgag ggccgaggat accgcagtgt actattgcgc caagagcggc 300

acccctttcg actattgggg ccagggaacc ctcgtgacag tgtctagc 348

<210> 11

<211> 327

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> VL

<400> 11

accgacatcc agatgaccca gtccccctct tctctgagcg cttccgtggg cgatagggtg 60

accatcactt gcagagcctc ccagtccatc tcctcctacc tgaattggta ccagcagaag 120

ccaggcaagg cccctaagct gctgatctac tccgcttctc atctgcagag cggcgtgcct 180

tctagatttt ccggctccgg atccggcacc gatttcaccc tgaccatctc ctccctgcag 240

ccagaggact tcgccaccta ctattgccag caggccaaca cctcccctac aaccttcgga 300

cagggcacca aggtggagat caagagg 327

<210> 12

<211> 795

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Anti CD19

<400> 12

atggagaccg acacactgct cctgtgggtc ctgctcctct gggtgccagg aagtacagga 60

ggaggaggag gatctgaggt gcagctgctg gaatcaggag gaggactggt gcagccagga 120

ggatctctga gactgtcttg cgccgcttcc ggcttcacct tctcttccta cgccatgtct 180

tgggtccgac aggctccagg aaagggactg gagtgggtgt cctctatcga cgcacagggc 240

ctgcctacca gatacgccga ttccgtgaag ggcaggttca ccatctcccg ggacaactcc 300

aagaacaccc tgtacctgca gatgaactcc ctgagggccg aggataccgc agtgtactat 360

tgcgccaaga gcggcacccc tttcgactat tggggccagg gaaccctcgt gacagtgtct 420

agcggaggag gaggatctgg aggaggagga tccggaggag gaggatctac cgacatccag 480

atgacccagt ccccctcttc tctgagcgct tccgtgggcg atagggtgac catcacttgc 540

agagcctccc agtccatctc ctcctacctg aattggtacc agcagaagcc aggcaaggcc 600

cctaagctgc tgatctactc cgcttctcat ctgcagagcg gcgtgccttc tagattttcc 660

ggctccggat ccggcaccga tttcaccctg accatctcct ccctgcagcc agaggacttc 720

gccacctact attgccagca ggccaacacc tcccctacaa ccttcggaca gggcaccaag 780

gtggagatca agagg 795

<210> 13

<211> 464

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ScFV(2-27)- CD8TM-4-1BB-CD3ζ

<400> 13

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Asp Ala Gln Gly Leu Pro Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Gly Thr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

130 135 140

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

145 150 155 160

Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

165 170 175

Lys Leu Leu Ile Tyr Ser Ala Ser His Leu Gln Ser Gly Val Pro Ser

180 185 190

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

195 200 205

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn

210 215 220

Thr Ser Pro Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

225 230 235 240

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

245 250 255

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

260 265 270

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

275 280 285

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

290 295 300

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

305 310 315 320

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

325 330 335

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

340 345 350

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

355 360 365

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

370 375 380

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

385 390 395 400

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

405 410 415

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

420 425 430

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

435 440 445

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Trp

450 455 460

<210> 14

<211> 1467

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ScFV(2-27)- CD8TM-4-1BB-CD3ζ

<400> 14

atggagaccg acacactgct cctgtgggtc ctgctcctct gggtgccagg aagtacagga 60

ggaggaggag gatctgaggt gcagctgctg gaatcaggag gaggactggt gcagccagga 120

ggatctctga gactgtcttg cgccgcttcc ggcttcacct tctcttccta cgccatgtct 180

tgggtccgac aggctccagg aaagggactg gagtgggtgt cctctatcga cgcacagggc 240

ctgcctacca gatacgccga ttccgtgaag ggcaggttca ccatctcccg ggacaactcc 300

aagaacaccc tgtacctgca gatgaactcc ctgagggccg aggataccgc agtgtactat 360

tgcgccaaga gcggcacccc tttcgactat tggggccagg gaaccctcgt gacagtgtct 420

agcggaggag gaggatctgg aggaggagga tccggaggag gaggatctac cgacatccag 480

atgacccagt ccccctcttc tctgagcgct tccgtgggcg atagggtgac catcacttgc 540

agagcctccc agtccatctc ctcctacctg aattggtacc agcagaagcc aggcaaggcc 600

cctaagctgc tgatctactc cgcttctcat ctgcagagcg gcgtgccttc tagattttcc 660

ggctccggat ccggcaccga tttcaccctg accatctcct ccctgcagcc agaggacttc 720

gccacctact attgccagca ggccaacacc tcccctacaa ccttcggaca gggcaccaag 780

gtggagatca agaggaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 840

gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 900

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 960

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 1020

ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1080

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1140

agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1200

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1260

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1320

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1380

gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1440

cacatgcagg ccctgccccc tcgctaa 1467

Claims

1. An anti-CD19 antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises heavy chain VHCDR1, VHCDR2, VHCDR 3; the light chain variable region comprises light chain VLCDR1, VLCDR2, VLCDR3, wherein the amino acid sequence of VHCDR1 is as set forth in SEQ ID NO: 1; the amino acid sequence of the VHCDR2 is shown in SEQ ID NO: 2; the amino acid sequence of the VHCDR3 is shown in SEQ ID NO: 3; the amino acid sequence of the VLCDR1 is set forth in SEQ ID NO: 4; the amino acid sequence of the VLCDR2 is set forth in SEQ ID NO: 5; the amino acid sequence of the VLCDR3 is set forth in SEQ ID NO: 6.

2. The anti-CD19 antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the VH chain is as set forth in SEQ ID NO: 7; the amino acid sequence of the VL chain is shown in SEQ ID NO: 8, or a sequence shown in figure 8.

3. The anti-CD19 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the VH chain and VL chain are linked directly or through a linking peptide.

4. The anti-CD19 antibody or antigen-binding fragment thereof of claim 1 or 2, the amino acid sequence of the anti-CD19 antibody or antigen-binding fragment thereof is set forth in SEQ ID NO: 9, and (c) 9.

5. The anti-CD19 antibody or the antigen-binding fragment thereof according to claim 1 or 2, wherein the anti-CD19 antibody or the antigen-binding fragment thereof has an affinity of 8.473 x10 to CD19^-8M is more than M.

6. A polynucleotide encoding the anti-CD19 antibody or antigen-binding fragment thereof of any one of claims 1-5.

7. The polynucleotide of claim 6, wherein the sequence of the polynucleotide comprises the sequence of SEQ ID NO: 10 or a degenerate sequence thereof, and SEQ ID NO: 11 or a degenerate sequence thereof.

8. The polynucleotide of claim 6, having the sequence set forth in SEQ ID NO: 12 or a degenerate sequence thereof.

9. A polynucleotide according to claim 7 wherein the degenerate sequence shares 60% or more identity with the original sequence.

10. A polynucleotide according to claim 9 wherein the degenerate sequence shares 90% or more identity with the original sequence.

11. A method of making the anti-CD19 antibody or antigen-binding fragment thereof of any one of claims 1-5 or the anti-CD19 antibody or antigen-binding fragment thereof encoded by the polynucleotide of any one of claims 6-10, comprising: expressing the polynucleotide of any one of claims 6-10 in a host cell.

An Anti CD19 CAR-NK cell capable of expressing a chimeric antigen receptor comprising the Anti-CD19 antibody or antigen-binding fragment thereof of any one of claims 1-5, and/or the Anti-CD19 antibody or antigen-binding fragment thereof encoded by the polynucleotide of any one of claims 6-10, and/or the Anti-CD19 antibody or antigen-binding fragment thereof prepared of claim 11.

13. The Anti CD19 CAR-NK cell of claim 12, further comprising a transmembrane domain and a costimulatory signaling region; the transmembrane domain is selected from: a transmembrane domain of one or more of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD 154;

the costimulatory signaling region comprises the intracellular domain of a costimulatory molecule selected from the group consisting of: one or more of CD3 ζ, CD3 γ, CD3 δ, CD3 ∈, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD28, CD134, CD137, ICOS, CD154, 4-1BB, and OX 40.

14. The Anti CD19 CAR-NK cell of claim 13, wherein said transmembrane domain is a CD8 transmembrane domain;

the costimulatory signaling region comprises the 4-1BB and CD3 zeta intracellular domains.

15. The Anti CD19 CAR-NK cell of claim 13, wherein the Anti CD19 antibody or antigen-binding fragment thereof is capable of specifically binding to the tumor-specific antigen CD19 and activating the NK cell via a transmembrane domain and a costimulatory signaling region.

16. The Anti CD19 CAR-NK cell of claim 13, wherein the chimeric antigen receptor is a fusion protein of the structure scFV-CD8-4-1BB-CD3 ζ, wherein the scFV is capable of specifically binding to a CD19 molecule.

17. The Anti CD19 CAR-NK cell of claim 16, wherein the amino acid sequence of the SCFV-CD8-4-1BB-CD3 ζ fusion protein is as set forth in SEQ ID NO: 13.

18. the Anti CD19 CAR-NK cell of claim 16, wherein the sequence of the polynucleotide encoding the SCFV-CD8-4-1BB-CD3 ζ fusion protein is as set forth in SEQ ID NO: 14 or a degenerate sequence thereof.

19. The Anti CD19 CAR-NK cell of any one of claims 12-18, which is capable of effectively killing and/or killing Raji cells.

20. A method of producing Anti CD19 CAR-NK cells as defined in any one of claims 12 to 19, comprising the steps of:

(1) synthesizing and amplifying a polynucleotide encoding a scFV-CD8-4-1BB-CD3 ζ fusion protein of any one of claims 16-19 and cloning the polynucleotide into a lentiviral expression vector;

(2) infecting cells by using a lentivirus packaging plasmid and the lentivirus expression vector plasmid obtained in the step (1), packaging and preparing lentivirus;

(3) and (3) infecting NK-92 cells by using the lentivirus obtained in the step (2) to obtain CAR-NK cells.

21. A pharmaceutical composition comprising an Anti-CD19 antibody or antigen-binding fragment thereof according to any one of claims 1-5, and/or an Anti-CD19 antibody or antigen-binding fragment thereof encoded by the polynucleotide of any one of claims 6-10, and/or an Anti-CD19 antibody or antigen-binding fragment thereof prepared according to claim 11, and/or an Anti CD19 CAR-NK cell of any one of claims 12-19, and/or an Anti CD19 CAR-NK cell prepared according to claim 20.

22. The pharmaceutical composition of claim 21, further comprising a pharmaceutically acceptable excipient.

23. Use of the Anti-CD19 antibody or antigen-binding fragment thereof according to any one of claims 1-5, and/or the Anti-CD19 antibody or antigen-binding fragment thereof encoded by the polynucleotide of any one of claims 6-10, and/or the Anti-CD19 antibody or antigen-binding fragment thereof prepared according to claim 11, and/or the Anti CD19 CAR-NK cells according to any one of claims 12-19, and/or the Anti CD19 CAR-NK cells prepared according to claim 20, for the preparation of a medicament for the treatment and/or prevention of CD 19-highly expressing tumors and associated diseases.

24. The use according to claim 23, wherein the tumor highly expressing CD19 is a lymphoma.