CN108047332A - Using CD19 as the specific antibody of target spot, CAR-NK cells and its preparation and application - Google Patents

Abstract

The present invention provides a kind of using CD19 as the anti-CD 19 antibodies of target spot or its antigen-binding fragment, wherein described antibody includes heavy chain variable region (VH) and/or light chain variable region (VL), wherein the heavy chain variable region includes heavy chain VHCDR1, VHCDR2, VHCDR3；The light chain variable region includes light chain VLCDR1, VLCDR2, VLCDR3.The present invention is provided using CD19 as the anti-CD 19 antibodies of target spot or its antigen-binding fragment, and as monoclonal cell strain culture and expands gained using the Anti CD19 CAR NK cells constructed by this, and character is stablized, and is prepared available for large-scale production.Anti CD19 CAR NK cells specifically can kill or kill lymphoma cell using CD19 molecules as target antigen, can be as the medicine of lymthoma class disease, for the treatment of the tumour of the high expression of CD19 molecules.

Description

Specific antibody targeting CD19, CAR-NK cells and their preparation and application

technical field

The invention relates to the field of biomedicine, in particular to a specific antibody targeting CD19, a CAR-NK cell targeting CD19, and its preparation and application.

Background technique

Chimeric antigen receptor (chimeric antigen receptor, CAR) modified immune cells use genetic engineering to modify immune cells to express exogenous anti-tumor genes. The CAR gene mainly includes an extracellular recognition domain and an intracellular signal transduction domain: the former is used to recognize specific molecules on the tumor surface and the latter is used to initiate immune cell responses after recognizing tumor surface molecules and exert cytotoxicity. T-cells are the vectors.

CAR-T, the full name is Chimeric AntigenReceptor T-Cell Immunotherapy, chimeric antigen receptor T cell immunotherapy. Chimeric antigen receptor T cells (CAR-T cells) combine the antigen-binding part of an antibody that can recognize a certain tumor antigen with intracellular elements such as CD3-ζ chain or 4-1BB to form a chimeric gene in vitro. The patient's T cells are transfected by gene transduction to express 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 95 kD transmembrane glycoprotein that belongs to the immunoglobulin superfamily and plays a central role in B cell regulatory responses. A large number of studies have proved 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 amount of clinical practice has proved that the CAR-T drug developed with this CD19 as the target has achieved remarkable curative effect. Novartis Pharmaceuticals announced on March 29, 2017 that its CAR-T product CTL019 entered the FDA accelerated approval channel. In December 2015, the FDA granted Breakthrough Therapy Designation (BTD) to Kite Pharma's CAR-T therapy KTE-C19 for the indication therapy of diffuse large B-cell lymphoma (DLBCL). On April 1, 2017, Kite Pharma also announced that it had officially completed the rolling submission of the Biologics License Application (BLA) for the CAR-T therapy KTE-C19 (later renamed "axicabtagene ciloleucel") to the FDA.

However, there are still some problems in CD19 CAR-T research. For example: first, the T cells used to prepare CAR-T can only come from the patient himself, and cannot be reinfused; Risk of immune rejection.

Natural killer (NK) cells are an important part of the non-specific immune system and the key mediator cells of the innate immune system response. NK cells are a kind of broad-spectrum immune cells that have the specific function of quickly finding and destroying abnormal cells (such as cancer or virus-infected cells), and can display a powerful ability to dissolve abnormal cells without prior sensitization or HLA matching active. Using immune cells (including NK cells) to treat cancer is a new trend in recent years. This new therapy is expected to provide new hope for the cure of tumors that are ineffective to traditional surgery, chemotherapy and radiotherapy.

Contents of the invention

In view of this, the present invention provides an anti-CD19 antibody or its antigen-binding fragment targeting CD19, as well as CAR-NK cells capable of expressing the domain and its preparation and application. On the one hand, the cells have stable properties and can Large-scale production and preparation can be used for allogeneic backtracking; on the other hand, the antibody sequence used to construct it is a human antibody sequence, which greatly reduces the risk of immune rejection when it is used as a drug.

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

Exemplarily, the amino acid sequence of the VH chain is shown in SEQ ID NO: 7 or its homologous sequence.

Exemplarily, the amino acid sequence of the VL chain is shown in SEQ ID NO: 8 or its homologous sequence.

Exemplarily, the homology of the homologous sequence and the original sequence is more than 60%, for example, 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 above, 85% or above, 86% or above, 87% or above, 88% or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% or above, 96% or above, 97% or above, 98% or above, 99% or above, 99.1 or above, 99.2 or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or above, 99.7% or above, 99.8% or above, or 99.9% or above etc.

Exemplarily, the VH chain and VL chain are linked directly or via a linker peptide.

In a specific embodiment provided by the present invention, the anti-CD19 antibody or antigen-binding fragment thereof targeting CD19 includes a VH chain and a VL chain.

In a specific embodiment provided by the present invention, the VH chain and VL chain are linked by a connecting peptide.

In a specific embodiment provided by the present invention, the nucleotide sequence of the anti-CD19 antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 9 or a degenerate sequence thereof.

In a specific embodiment provided by the present invention, the affinity of the anti-CD19 antibody or antigen-binding fragment thereof to CD19 is above 8.473×10 ^-8 M.

Another aspect of the present invention provides a nucleotide sequence capable of expressing the above-mentioned anti-CD33 antibody or antigen-binding fragment thereof.

In a specific embodiment provided by the present invention, the nucleotide sequence comprises the sequence shown in SEQ ID NO: 10 or its degenerate sequence, and/or the sequence shown in SEQ ID NO: 11 or its degenerate sequence sequence.

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

Exemplarily, the homology between the degenerate sequence and the original sequence is more than 60%, for example, 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 above, 85% or above, 86% or above, 87% or above, 88% or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% or above, 96% or above, 97% or above, 98% or above, 99% or above, 99.1 or above, 99.2 or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or above, 99.7% or above, 99.8% or above, or 99.9% or above etc.

Another aspect of the present invention provides a method for preparing the above-mentioned anti-CD19 antibody or antigen-binding fragment thereof, which includes: establishing a phage antibody library and screening the antibody or antibody fragment that can bind to CD19. Its specific process includes:

① Preparation of M13KO7 helper phage;

②Using M13KO7 helper phage to establish a phage antibody library;

③ using CD19 antigen to screen single-stranded phage DNA from the antibody library in step ②; and optionally,

④ Express and purify the single-stranded phage DNA obtained in step ③.

Another aspect of the present invention provides an Anti CD19 CAR-NK cell, which can express a chimeric antigen receptor, and the chimeric antigen receptor includes the above-mentioned anti-CD19 antibody or an antigen-binding fragment thereof.

In a specific embodiment of the present invention, the chimeric antigen receptor further comprises a transmembrane domain and/or a co-stimulatory signal transduction region.

Exemplarily, the transmembrane domain is selected from one or more of CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154 The transmembrane domain; preferably, the transmembrane domain is CD8 transmembrane domain; and/or,

The co-stimulatory signal transduction region comprises intracellular domains of co-stimulatory molecules, and the co-stimulatory molecules are selected from the group consisting of: CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5 , one or more of CD28, CD134, CD137, ICOS, CD154, 4-1BB and OX40; preferably, the co-stimulatory signaling region includes 4-1BB and CD3ζ intracellular domains.

Preferably, the anti-CD19 antibody or antigen-binding fragment thereof can specifically bind to the tumor-specific antigen CD19, and activate the NK cells through the transmembrane domain and costimulatory signal transduction region.

Exemplarily, the chimeric antigen receptor is a fusion protein with the structure of SCFV-CD8-4-1BB-CD3ζ, and the fusion protein can specifically recognize CD19 molecule.

In a specific embodiment of the present invention, the amino acid sequence of the SCFV-CD8-4-1BB-CD3ζ fusion protein is shown in SEQ ID NO: 13 or its homologous sequence, etc.

Exemplarily, the nucleotide sequence of the SCFV-CD8-4-1BB-CD3ζ fusion protein is shown in SEQ ID NO: 14 or its degenerate sequence, etc.

In a specific embodiment of the present invention, the Anti CD19 CAR-NK cells can effectively kill or kill Raji cells and the like.

Another aspect of the present invention provides a method for preparing the above-mentioned Anti CD19 CAR-NK cells, which includes the following steps:

(1) Synthesize and amplify the SCFV-CD8-4-1BB-CD3ζ fusion protein gene, and clone the SCFV-CD8-4-1BB-CD3ζ fusion protein gene onto the lentiviral expression vector;

(2) using the lentiviral packaging plasmid and the lentiviral expression vector plasmid obtained in step (1) to infect 293T cells, packaging and preparing the lentivirus;

(3) Infect NK-92 cells with the lentivirus obtained in step (2) to obtain CD19 CAR-NK cells.

The present invention also provides the above-mentioned anti-CD19 antibody or its antigen-binding fragment, and/or the anti-CD19 antibody or its antigen-binding fragment expressed by the above-mentioned nucleotide sequence, and/or the above-mentioned Anti CD19 CAR-NK cell in the preparation of treatment and/or prevention Application in drugs for tumors and related diseases with high expression of CD19 molecules.

Exemplarily, the application of Anti CD19 CAR-NK cells in the preparation of medicaments for treating lymphomas highly expressing CD19 molecules.

The present invention also provides a pharmaceutical composition, which includes the above-mentioned anti-CD19 antibody or its antigen-binding fragment, and/or the anti-CD19 antibody or its antigen-binding fragment expressed by the above-mentioned nucleotide sequence, and/or the above-mentioned Anti CD19 CAR- NK cells, and optionally, pharmaceutically acceptable excipients.

The present invention has at least one of the following advantages:

The present invention provides an anti-CD19 antibody or an antigen-binding fragment thereof targeting CD19, and the AntiCD19 CAR-NK cells constructed therefrom are cultured and amplified as a monoclonal cell line with stable properties and can be used for large-scale production and preparation . Anti CD19 CAR-NK cells use CD19 molecule as the target antigen, can specifically kill or kill lymphoma cells, and can be used as a therapeutic drug for lymphoma diseases, and can be used for the treatment of lymphoma with high expression of CD19 molecules.

Description of drawings

Fig. 1 is a schematic diagram showing the structure of the expression vector of pCDNA3.4-ScFV (anti-CD19) provided by the embodiment of the present invention.

Fig. 2 is a schematic diagram showing the structure of the pRRSLIN-ScFV(2-27)-CD8TM-4-1BB-CD3ζ lentiviral transfection vector provided in the embodiment of the present invention.

Figure 3 shows the results of CD19 CAR-NK cell positive rate detected by flow cytometry provided by the embodiment of the present invention; Figure 3a shows the NK-92 control group, and Figure 3b shows the CD19 NK-92 experimental group.

Figure 4 is a diagram showing the experimental results of CD19 CAR-NK cells killing Raji cells provided in the embodiment of the present invention.

Detailed ways

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-CD3ζ fusion protein described in the present invention is any DNA sequence capable of encoding the fusion protein, preferably, the sequence is SEQ ID NO: 14 or its complementary sequence. On the other hand, the nucleotide sequence encoding the SCFV-CD8-4-1BB-CD3ζ fusion protein described in the present invention can hybridize with the nucleotide sequence of SEQ ID NO: 14 under stringent conditions, and encode The polynucleotide of the fusion protein or its complementary sequence;

The "stringent conditions" mentioned herein can be any one of low stringency conditions, medium stringency conditions and high stringency conditions, preferably high stringency conditions. Exemplarily, "low stringent conditions" can be 30°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, 52% formamide; "medium stringent conditions" can be 40°C, 5×SSC, 5× Denhardt solution, 0.5% SDS, 52% formamide conditions; "high stringency conditions" can be 50°C, 5×SSC, 5×Denhardt solution, 0.5% SDS, 52% formamide conditions. Those skilled in the art should understand that the higher the temperature, the more highly homologous polynucleotides can be obtained. In addition, those skilled in the art can choose the comprehensive result formed by multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration that affect the stringency of hybridization to achieve the corresponding stringency.

In addition, the hybridizable polynucleotide can also be, when calculated by FASTA, BLAST and other homology search software with the default parameters set by the system, it has about 60% or more with the polynucleotide of coding sequence number 6 , 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 Above, 80% or above, 81% or above, 82% or above, 83% or above, 84% or above, 85% or above, 86% or above, 87% or above, 88% or above, 89% or Above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% or above, 96% or above, 97% or above, 98% or above, 99% or above Polynucleosides of more than, 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, or 99.9% or more identity acid.

The identity of the nucleotide sequence can use the algorithm rule BLAST of Karlin and Altschul (Proc.Natl.Acad.Sci.USA 87:2264-2268,1990; Proc.Natl.Acad.Sci.USA 90:5873,1993) to make sure. Programs BLASTN, BLASTX based on the rules of the BLAST algorithm have been developed (Altschul SF, et al: JMol Biol 215:403, 1990). When using BLASTN to analyze the base sequence, if the parameters are score=100, wordlength=12; in addition, when using BLASTX to analyze the amino acid sequence, such as setting the parameters to be score=50, wordlength=3; when using BLAST and Gapped BLAST programs, use each The program's system can set default parameter values.

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. Antibodies in the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies, etc. (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 85:5879-5883; Bird et al., 1988, Science 242:423-426).

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

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

The term "lentivirus" refers to a genus of the Retroviridae family that is capable of efficiently infecting aperiodic and post-mitotic cells; they can transfer significant amounts of genetic information into the host cell's DNA, so that they are the most suitable vectors for gene delivery. one of the effective methods.

The term "vector" is a composition of matter that includes an isolated nucleic acid and that 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 amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. 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 the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. 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 "comprising", "comprising" or "characterized by", and is inclusive or open-ended and does not exclude additional unstated elements or method steps. The term "comprising" in any expression herein, especially when describing the method, use or product of the present invention, should be understood as including consisting essentially of said components or elements or steps and consisting of said components or elements or Those products, methods and uses that consist of steps. The invention exemplarily described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The terms and expressions which have been employed herein are used as terms of description rather than limitation, and in the use of such terms and expressions it is not intended to exclude any equivalents of the features shown and described, or parts thereof, and various modifications are to be recognized. It is possible within the scope of the claimed invention. Accordingly, it should be understood that although the invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be employed by those skilled in the art and that such modifications and variations are considered to be defined within the scope of the invention.

The English names appearing in this article are not case-sensitive; CD19 CAR-NK and CD19-CAR NK have the same meaning; CD19 CAR-NK and Anti CD19-CAR NK have the same meaning, and both represent CAR-NK cells that resist CD19 molecules ; NK-92 and NK92 both represent NK92 cells.

"NK" in the present invention refers to human normal NK cells or NKT cells or NK cell lines, which include NK-92 cells, YT cells, NKL cells, HANK-1 cells, NK-YS cells, KHYG-1 cells, SNK -6 cells and IMC-1 cells, etc. In the specific embodiments of the present invention, NK-92 cells are taken as an example for illustration.

In order to illustrate the present invention more clearly, the following examples are now described in detail, but these examples are only exemplary descriptions of the present invention and should not be construed as limitations on the present application.

Example 1 Screening of CD19 Human Antibody

2×YT liquid medium: 16g peptone, 10g yeast extract, 5g sodium chloride, 800mL water, adjust pH to 7.0 with sodium hydroxide, add water to 1000mL, sterilize at 121°C for 20min.

2×YT-G: Add 2% glucose to 2×YT medium.

2×YT-AK: Add 100 g/mL ampicillin and 50 g/mL kanamycin to 2×YT medium.

1. Preparation of M13KO7 helper phage

1. Under the condition of 37° C., the TG1 bacterial cells in logarithmic growth phase were infected with different dilution concentrations of helper phage M13K07 for 30 minutes, and then spread on the agar plate.

2. Pick the TG1 plaque clone into 3mL liquid 2YT medium for culture. Incubate at 37°C for 2 hours.

3. Transfer the culture in step 2 to 1L 2YT medium, add kanamycin to 50 μg/m, and culture at 37°C for 16 hours.

4. Centrifuge (10min at 5000g) to remove bacterial cells, add phage precipitation agent to the supernatant to collect phages.

5. Calculate the phage titer, then dilute it to 1×10 ¹³ pfu/mL and store it in a -20 degree refrigerator for later use.

2. Prepare phage antibody library

1. Inoculate 500ml of 2YT-G medium with phage library Glycerol, and inoculate at 37°C and 250rpm until the OD value is 0.8-0.9.

2. Add the M13KO7 prepared in step 1 to the above step 1 with a final concentration of 5×10 ⁹ pfu/mL, let stand at 37°C for 30 minutes, and then incubate on a constant temperature shaker at 200 rpm for 30 minutes.

3. Centrifuge (2200g, 15min) the culture solution in step 2 to collect the bacterial cells, resuspend the sludge with 2YT-AK medium, and culture overnight at 30°C and 300rmp.

4. Centrifuge (7000g, 15min, 4°C) the culture solution in step 3 to remove bacterial cells, and collect the supernatant into a 1L bottle.

5. Add the phage precipitation agent to the supernatant in step 4, and collect the phage precipitation.

6. Centrifuge (7000g, 15min), remove the supernatant, collect the phage pellet, add the phage pellet to 8ml PBS.

7. Re-centrifuge (12,000g, 10min) to remove bacterial debris, and keep the phage in the supernatant for later use.

3. Antibody screening

1. Coat the well plate with CD19 antigen at a concentration of 5 μg/mL at 4°C for 2 hours, and then wash three times. Block overnight at 4°C with blocking solution.

2. Pour off the blocking solution and wash, resuspend the phage (prepared in step 2) and 100 μg/mL human IgG protein in the blocking solution (containing 2% milk) and add to the well plate to incubate at room temperature for 1 h, then wash.

3. Digest the phage bound on the well plate by trypsin for the subsequent second round of screening.

4. Repeat the three rounds of screening according to the process in 1-3 above.

5. Obtain single-stranded phage DNA, that is, single-chain antibody gene sequence by precipitation, and sequence and identify it.

Example 2: Expression and Identification of CD19 Antibody

The single-stranded phage DNA obtained in Example 1 was cloned into the pCDNA3.4 vector to construct the pCDNA3.4-ScFV (anti-CD19) expression vector shown in Figure 1, and the vector was transiently transferred to CHO cells for expression. Nickel strain purified the single-chain antibody sequence for subsequent analysis.

The affinity of the screened single-chain antibody to CD19 protein was identified by SPR method, and the single-chain antibody with the highest affinity to CD19 protein was selected and named as single-chain antibody 2-27. The molecular affinity between single chain antibody 2-27 and CD19 is shown in Table 1.

Table 1: Molecular affinity of scFv 2-27 to CD19

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 in SEQ ID NO.7, and its nucleotide sequence is shown in SEQ ID NO.10; the amino acid sequence of the VL chain of the single-chain antibody 2-27 is shown in Shown in SEQ ID NO.8, its nucleotide sequence 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 The sequence is shown in SEQ ID NO.12.

Example 3 Preparation of lentiviral expression vector

Gene synthesis SCFV(2-27)-CD8-4-1BB-CD3ζ fusion gene sequence (the amino acid sequence is shown in SEQ ID NO: 13, and the gene sequence is shown in SEQ ID NO: 14). Through enzyme digestion, it was transformed and connected to the PRRSL IN vector, and the upstream of the gene was the EP-1α promoter. The vector was transformed into Stbl3 Escherichia coli strain, screened with ampicillin, and positive clones were obtained, plasmids were extracted, and clones were identified by enzyme digestion to obtain the PRRLSIN-SCFV(2-27)-CD8-4-1BB-CD3ζ lentiviral transfection vector (as shown in Figure 2 Show).

The preparation of embodiment 4 lentiviruses

(1) 24 hours before transfection, inoculate 293T cells into a 15cm culture dish at about 8×10 ⁶ per dish. Make sure that the cells are at about 80% confluence and evenly distributed in the culture dish during transfection.

(2) Prepare solution A and solution B

Solution A: 6.25ml 2×HEPES buffer (the amount packed in 5 large dishes, the effect is the best).

Solution B: Add the mixture of the following plasmids respectively: 112.5 μg PRRLSIN-SCFV(2-27)-CD8-4-1BB-CD3ζ (target plasmid); 39.5 μg pMD2.G (VSV-G envelope); 73 μg pCMVR8.74 ( gag, pol, tat, rev); 625 μl of 2M calcium ion solution. Total volume of solution B: 6.25ml.

Mix solution B thoroughly, and while vortexing solution A gently, add solution B drop by drop and let stand for 5-15 minutes. Gently vortex the above mixed solution of A and B, add dropwise to the culture dish containing 293T cells, gently shake the culture dish back and forth to make the mixture of DNA and calcium ions evenly distributed. (Do not rotate the Petri dish) Place in the incubator for 16-18 hours. Replace with fresh medium, continue culturing, and collect virus-containing supernatant after 48 hours and 72 hours, respectively. Centrifuge at 500g for 10 minutes at 25°C. PES membrane (0.45 μm) filtration. Sterilize Beckman Coulter Ultra-clear SW28 centrifuge tubes with 70% ethanol and place them under UV light for 30 minutes. Transfer the filtered lentivirus-containing supernatant to a centrifuge tube. Carefully spread a layer of 20% sucrose on the bottom of the centrifuge tube (1ml sucrose per 8ml supernatant). Equilibrate the centrifuge tube with PBS, centrifuge at 25000rpm (82, 700g) at 4°C for 2 hours. Carefully remove the centrifuge tube, discard the supernatant, and invert the centrifuge tube to remove residual liquid. Add 100 μl PBS, seal the centrifuge tube, place at 4°C for 2 hours, vortex gently every 20 minutes, centrifuge at 500g for 1 minute (25°C), and collect the virus supernatant. After cooling on ice, store at -80°C.

Example 5 Preparation of CD19 CAR-NK-92 cells

Adjust the NK-92 cell density to 2-3×10 ⁵ /ml, and add the virus vector (prepared in Example 4) according to the ratio of volume ratio (V/V) virus vector: cell culture medium = 1:5-10 , while adding polybrene 8μg/ml. After 4 hours, an equal amount of fresh complete medium was added to adjust the cell density to 1×10 ⁵ /ml to continue culturing. The next day, all the cells were centrifuged, fresh medium was added, and the culture was continued. Replenish fluid every 1-2 days to maintain the cell density at 2-3×10 ⁵ /ml. After 72 hours, CAR antibody staining was performed, and CD19 CAR NK-92 positive cells were sorted by flow cytometry and expanded for culture. Observe the color change of the medium, cell density, and cell shape every day and make corresponding records.

The positive rate of CAR NK-92 cells was detected by flow cytometry, and the results of flow cytometry are shown in Figure 3a and Figure 3b. In Figure 3a and Figure 3b, the antibody used is fluorescently labeled with APC, which is represented on the abscissa. If NK92 cells successfully express the CAR molecule, the signal value will increase significantly. It can be seen from Figure 3a and Figure 3b that the signal value of APC fluorescent labeling increased significantly, indicating that NK-92 cells successfully expressed CAR molecules, and the positive rate of CAR-NK92 was 99.91%.

Example 6 Evaluation of CD19 CAR-NK cells on tumor killing effect in vitro

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

(1) Prepare 1ml of Raji cell suspension (2X10^4 cells/well) in a 24-well plate. The culture plate was pre-incubated for 12 h in the incubator.

(2) Discard the culture supernatant of the 24-well plate, add 1ml of effector cells to each well, and the ratio of the number of effector cells to target cells is 1:1 or 0.5:1. Only 1ml of medium was added to medium control wells, and three replicate wells were set for each experiment. The effector cells were co-incubated with the target cells for 4 hours or 2 hours, and the experimental groups were as shown in Table 2.

Table 2 Grouping of CD19 CAR-NK cells killing effect on Raji cells

(3) Add 100ul of CCK-8 solution to each well, and incubate the culture plate in the incubator for 2h.

(4) Measure the absorbance at 450 nm with a microplate reader.

(5) Killing rate=(As-Ab)/(Ac-Ab)×100%;

As: test well (medium containing tumor cells, CCK-8, CAR-NK);

Ac: control well (medium containing tumor cells, CCK-8);

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

The experimental results of evaluating the tumor killing effect of CD19 CAR-NK cells in vitro are shown in Figure 4.

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

The CD19 CAR NK-92 of the present invention is obtained by infecting NK92 cell lines with CD19 CAR molecules and obtaining single clone cells through flow cytometric screening, and then culturing and expanding CAR NK92 monoclonal cell lines with stable properties and high killing activity. The cells can be used for large-scale production and can be used for different patients without GVHR rejection. Compared with CAR-T cells, CD19 CAR NK-92 cells do not need to separate peripheral blood mononuclear cells (PBMC) from patients, specifically activate T cells and prepare CAR-T cells (this process requires patients to wait for more than 10 days), and do not It needs to be customized and can be used for multiple patients, which shortens the time. CD19 CAR-NK92 cells can be prepared and cultured in large quantities, and the patients can use them immediately; The cells are prepared by virus infection. The T cells are not from the same monoclonal source, but the sorted CAR-NK92 cells are from the same monoclonal, with uniform and stable traits and activities, which is convenient for large-scale production and quality control; Compared with NK92 cells, due to the introduction of CAR vector, its killing activity is specific, and the tumor treatment effect is obvious.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention within.

sequence listing

<110> Zhejiang Asikeli Biotechnology Co., Ltd.

<120> Specific antibodies targeting CD19, CAR-NK cells and their preparation and application

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> VHCDR1

<400> 1

Ser Tyr Ala Met Ser

1 5

<210> 2

<211> 20

<212> PRT

<213> 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

<220>

<223> VHCDR3

<400> 3

Ser Gly Thr Pro Phe Asp Tyr

1 5

<210> 4

<211> 11

<212> PRT

<213> 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

<220>

<223> VLCDR2

<400> 5

Ser Ala Ser His Leu Gln Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> 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

<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

<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

<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

<220>

<223> VH

<400> 10

gaggtgcagc tgctggaatc aggagggagga 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

<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

<220>

<223> Anti-CD19

<400> 12

atggagaccg aacacactgct 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

<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 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

<220>

<223> ScFV(2-27)-CD8TM-4-1BB-CD3ζ

<400> 14

atggagaccg aacacactgct 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 agaggacac gacgccagcg ccgcgaccac caacaccggc gccccaccatc 840

gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 900

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccggggact 960

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 1020

ctcctgtata tattcaaaca accattatg 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 aggacccta cgacgccctt 1440

cacatgcagg ccctgccccc tcgctaa 1467

Claims (18)

1. anti-CD 19 antibodies or its antigen-binding fragment, wherein the antibody includes heavy chain variable region (VH) and/or light chain variable Area (VL), wherein the heavy chain variable region includes heavy chain VHCDR1, VHCDR2, VHCDR3；The light chain variable region includes light chain VLCDR1, VLCDR2, VLCDR3, wherein the amino acid sequence of the VHCDR1 such as SEQ ID NO：Sequence shown in 1 or it is same Source sequence；The amino acid sequence of the VHCDR2 such as SEQ ID NO：Sequence or its homologous sequence shown in 2；The VHCDR3's Amino acid sequence such as SEQ ID NO：Sequence or its homologous sequence shown in 3；The amino acid sequence of the VLCDR1 such as SEQ ID NO：Sequence or its homologous sequence shown in 4；The amino acid sequence of the VLCDR2 such as SEQ ID NO：Sequence shown in 5 or its Homologous sequence；The amino acid sequence of the VLCDR3 such as SEQ ID NO：Sequence or its homologous sequence shown in 6.

2. anti-CD 19 antibodies as described in claim 1 or its antigen-binding fragment, the amino acid sequence such as SEQ of the VH chains ID NO：Sequence or its homologous sequence shown in 7；And optionally, the amino acid sequence of the VL chains such as SEQ ID NO：8 institutes The sequence shown or its homologous sequence.

3. anti-CD 33 antibody as claimed in claim 1 or 2 or its antigen-binding fragment, the homologous sequence is same with former sequence Source property is more than 60%, it is preferable that the homology is more than 90%.

4. anti-CD 33 antibody as claimed in claim 1 or 2 or its antigen-binding fragment, the VH chains and VL chains be directly connected to or It is connected by connecting peptide, it is preferable that the amino acid sequence of the anti-CD 33 antibody or its antigen-binding fragment such as SEQ ID NO：9 Shown sequence or its homologous sequence.

5. anti-CD 19 antibodies as claimed in claim 1 or 2 or its antigen-binding fragment, the anti-CD 19 antibodies or its antigen knot The affinity for closing segment and CD19 reaches 8.473 × 10^-8More than M.

6. express the nucleotide sequence of the anti-CD 19 antibodies or its antigen-binding fragment any one of claim 1-5.

7. nucleotide sequence as claimed in claim 6, wherein the nucleotide sequence includes SEQ ID NO：Shown in 10 Sequence or its degenerate sequence and/or SEQ ID NO：Sequence or its degenerate sequence shown in 11, it is preferable that the nucleotides sequence Row such as SEQ ID NO：Sequence or its degenerate sequence shown in 12.

8. the homology of nucleotide sequence as claimed in claim 7, the degenerate sequence and former sequence is more than 60%, preferably Ground, the homology are more than 90%.

It is 9. any in the anti-CD 19 antibodies or its antigen-binding fragment or claim 6-8 any one of claim 1-5 Described in nucleotide expressed by anti-CD 19 antibodies or its antigen-binding fragment preparation method, including：In host cell Nucleotide any one of middle expression claim 6-8.

10.Anti CD19 CAR-NK cells, can express Chimeric antigen receptor, and the Chimeric antigen receptor will including right It asks any one of anti-CD 19 antibodies or its antigen-binding fragment and/or the claim 6-8 any one of 1-5 Anti-CD 19 antibodies prepared by anti-CD 19 antibodies or its antigen-binding fragment and/or claim 9 expressed by nucleotide or its Antigen-binding fragment.

11. Anti CD19 CAR-NK cells as claimed in claim 10, the Chimeric antigen receptor also includes transmembrane structure Domain and/or costimulatory signal conducting region；Preferably, the transmembrane domain is selected from：CD28、CD3ε、CD45、CD4、CD5、 One or more transmembrane structures in CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS and CD154 Domain；Preferably, the transmembrane domain is CD8 transmembrane domains；And/or

The costimulatory signal conducting region includes the intracellular domain of costimulatory molecules, and the costimulatory molecules is selected from： CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、 One or more in ICOS, CD154,4-1BB and OX40；Preferably, the costimulatory signal conducting region include 4-1BB and CD3 ζ intracellular domain；Preferably, the anti-CD 19 antibodies or its antigen-binding fragment can specifically bind tumour-specific Antigens c D19, and pass through transmembrane domain and costimulatory signal conducting region activates the NK cells.

12. Anti CD19 CAR-NK cells as claimed in claim 11, the Chimeric antigen receptor is that structure is scFV- The fusion protein of CD8-4-1BB-CD3 ζ, wherein the scFV can specifically bind CD19 molecules, it is preferable that described The sequence of the amino acid of SCFV-CD8-4-1BB-CD3 ζ fusion proteins such as SEQ ID NO：13 or its homologous sequence.

13. Anti CD19 CAR-NK cells as claimed in claim 12, the SCFV-CD8-4-1BB-CD3 ζ fusion proteins Coding nucleotide sequence such as SEQ ID NO：14 or its degenerate sequence.

14. the Anti CD19 CAR-NK cells as any one of claim 10-13, can effectively kill and/ Or kill Raji cells.

15. the preparation method of Anti CD19 CAR-NK cells any one of claim 10-14, including following step Suddenly：

(1) synthesis and the nucleotide of the scFV-CD8-4-1BB-CD3 ζ fusion proteins described in amplification coding claim 12 or 13, And the nucleotide is cloned on Lentiviral；

(2) the Lentiviral plasmid infection cell obtained using slow virus packaging plasmid and step (1), packaging and preparation Slow virus；

(3) the slow-virus infection NK-92 cells obtained using step (2), obtain CAR-NK cells.

16. a kind of pharmaceutical composition, it includes the anti-CD 19 antibodies according to any one of claim 1-5 or its antigen knots Close segment and/or the encoded anti-CD 33 antibody of nucleotide sequence any one of claim 6-8 or its antigen binding Appoint in anti-CD 19 antibodies or its antigen-binding fragment and/or claim 10-14 prepared by segment and/or claim 9 The Anti CD19 CAR-NK cells prepared by Anti CD19 CAR-NK cells and/or claim 15 described in one.

17. pharmaceutical composition as claimed in claim 16 further includes pharmaceutically acceptable auxiliary material.

18. in the anti-CD 19 antibodies or its antigen-binding fragment and/or claim 6-8 any one of claim 1-5 Prepared by the encoded anti-CD 33 antibody of any one of them nucleotide sequence or its antigen-binding fragment and/or claim 9 Anti-CD 19 antibodies or its antigen-binding fragment and/or the Anti CD19 CAR-NK any one of claim 10-14 Anti CD19 CAR-NK cells prepared by cell and/or claim 15 are preparing treatment and/or the high expression CD19 of prevention Tumour and relevant disease drug in purposes；Preferably, the tumour of the high expression CD19 is lymthoma.