WO2019137518A1 - 以cd19为靶点的特异性抗体、car-nk细胞及其制备方法和应用 - Google Patents

以cd19为靶点的特异性抗体、car-nk细胞及其制备方法和应用 Download PDF

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WO2019137518A1
WO2019137518A1 PCT/CN2019/071559 CN2019071559W WO2019137518A1 WO 2019137518 A1 WO2019137518 A1 WO 2019137518A1 CN 2019071559 W CN2019071559 W CN 2019071559W WO 2019137518 A1 WO2019137518 A1 WO 2019137518A1
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sequence
car
cells
antibody
antigen
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李华顺
任宝永
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李华顺
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the invention relates to the field of biomedicine, in particular to a specific antibody targeting CD19, CAR-NK cells, a preparation method and application thereof.
  • Immune cells modified by chimeric antigen receptor 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 identify a tumor surface specific molecule and the latter is used to initiate an immune cell response after recognizing a tumor surface molecule, and exerts a cytotoxic effect, which is mainly T-cells are vectors.
  • CAR-T Chimeric Antigen Receptor T-Cell Immunotherapy, chimeric antigen receptor T cell immunotherapy.
  • a chimeric antigen receptor T cell (CAR-T cell) is an antigen binding portion of an antibody capable of recognizing a certain tumor antigen and is coupled to a CD3- ⁇ chain or an intracellular element such as 4-1BB in vitro as a chimeric gene.
  • the patient's T cells are transfected by gene transduction to express a chimeric antigen receptor (CAR). After the patient's T cells are "recoded", a large number of tumor-specific CAR-T cells are produced.
  • CAR chimeric antigen receptor
  • 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.
  • the T cells for the preparation of CAR-T can only be derived from the patient itself, and cannot be returned to the body.
  • the CD19 antibody for CAR-T preparation is internationally used. ScFv sequences are mostly murine antibodies and there is a greater risk of immunological rejection.
  • T cells are modified for specific patients, some patients may not be able to collect T cells, or there is not enough time to wait for the T cell preparation process, although CAR-T is currently developing toward the general-purpose CAR-T, but in fact It also increases clinical risk and operational difficulty.
  • these limitations may lead to some patients who are expected to benefit from CAR-T immunotherapy.
  • NK cells Natural killer cells are an important component of the non-specific immune system, the key mediator of the innate immune system response.
  • NK cells are a broad-spectrum immune cell that has the unique function of rapidly detecting and destroying abnormal cells (such as cancer or virus-infected cells) and exhibits strong lysis of abnormal cells without pre-sensitization or HLA matching. active.
  • the use of immune cells (including NK cells) to treat cancer is a new trend in recent years, and this new treatment is expected to provide new hopes for tumors that are ineffective in traditional surgery, chemotherapy and radiotherapy.
  • the present invention provides a specific antibody, CAR-NK cell targeting CD19, and a preparation method and application thereof, specifically an anti-CD19 antibody or antigen-binding fragment thereof targeting CD19, and capable of The CAR-NK cell expressing the domain and the preparation method and application thereof, the cell is stable on the one hand, can be mass-produced and prepared, can be subjected to allogeneic reinfusion; on the other hand, for constructing the anti-CD19 antibody or antigen thereof
  • the antibody sequence of the binding fragment is a human antibody sequence which, when used as a drug, greatly reduces the risk of immune rejection.
  • the invention provides an anti-CD19 antibody or antigen-binding fragment thereof targeting CD19, comprising a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein said heavy chain is variable
  • the region comprises a heavy chain VHCDR1, VHCDR2, VHCDR3;
  • the light chain variable region comprises a light chain VLCDR1, VLCDR2, VLCDR3, wherein the amino acid sequence of the VH CDR1 is the sequence set forth in SEQ ID NO: 1 or a homologous sequence thereof;
  • the amino acid sequence of the VHCDR2 is the sequence shown in SEQ ID NO: 2 or a homologous sequence thereof;
  • the amino acid sequence of the VHCDR3 is the sequence shown in SEQ ID NO: 3 or a homologous sequence thereof;
  • the amino acid sequence of the VLCDR1 is as The sequence represented by SEQ ID NO: 4 or a homologous sequence thereof;
  • the amino acid sequence of the VLCDR2 is the sequence shown in SEQ ID NO: 5 or
  • amino acid sequence of the VH chain is the sequence set forth in SEQ ID NO: 7 or a homologous sequence thereof.
  • amino acid sequence of the VL chain is the sequence set forth in SEQ ID NO: 8 or a homologous sequence thereof.
  • the homologous sequence has 95% or more homology with the original sequence, 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, or 99.9% or more, and the like.
  • VH chain and the VL chain are directly linked or linked by a linker peptide.
  • the CD19-targeting anti-CD19 antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain.
  • the VH chain and the VL chain are linked by a linker peptide.
  • the nucleotide sequence of the anti-CD19 antibody or antigen-binding fragment thereof is the sequence shown in SEQ ID NO: 9 or a degenerate sequence thereof.
  • the anti-CD19 antibody or antigen-binding fragment thereof has an affinity for CD19 of 8.473 ⁇ 10 -8 M or more.
  • Another aspect of the invention provides a nucleotide sequence which is capable of encoding the above anti-CD19 antibody or antigen-binding fragment thereof.
  • the nucleotide sequence comprises the sequence shown in SEQ ID NO: 10 or a degenerate sequence thereof, and/or the sequence shown in SEQ ID NO: 11 or its degeneracy sequence.
  • the nucleotide sequence is the sequence shown in SEQ ID NO: 12 or a degenerate sequence thereof.
  • the degenerate sequence has 95% or more homology with the original sequence, 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, or 99.9% or more, and the like.
  • Another aspect of the present invention provides a method for producing the above anti-CD19 antibody or antigen-binding fragment thereof, which comprises: establishing a phage antibody library and screening an antibody or antibody fragment capable of binding to CD19; the specific process comprises:
  • the single-stranded phage DNA obtained in the step 3 was expressed and purified.
  • Another aspect of the present invention provides an Anti CD19 CAR-NK cell capable of expressing a chimeric antigen receptor comprising the above anti-CD19 antibody or antigen-binding fragment thereof.
  • the chimeric antigen receptor further comprises a transmembrane domain and/or a costimulatory signaling region, and the anti-CD19 antibody or antigen-binding fragment thereof is capable of specifically binding to tumor specificity
  • the antigen CD19, and the Anti CD19 CAR-NK cells are activated by a transmembrane domain and a costimulatory signaling domain.
  • the transmembrane domain is selected from one or more of the group consisting of: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154.
  • Transmembrane domain preferably, the transmembrane domain is a CD8 transmembrane domain; and/or,
  • the costimulatory signaling region comprises an intracellular domain of a costimulatory molecule selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5 And one or more of CD28, CD134, CD137, ICOS, CD154, 4-1BB and OX40; preferably, said costimulatory signaling region comprises a 4-1BB and a CD3 ⁇ intracellular domain.
  • a costimulatory molecule selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5 And one or more of CD28, CD134, CD137, ICOS, CD154, 4-1BB and OX40; preferably, said costimulatory signaling region comprises a 4-1BB and a CD3 ⁇ intracellular domain.
  • the chimeric antigen receptor is a fusion protein of the structure ScFv-CD8-4-1BB-CD3 ⁇ , which is capable of specifically recognizing a CD19 molecule.
  • amino acid sequence of the ScFv-CD8-4-1BB-CD3 ⁇ fusion protein is represented by SEQ ID NO: 13 or a homologous sequence thereof.
  • sequence of the nucleotide of the ScFv-CD8-4-1BB-CD3 ⁇ fusion protein is as shown in SEQ ID NO: 14 or its degenerate sequence and the like.
  • the Anti CD19 CAR-NK cells are capable of effectively killing or killing lymphoma cells.
  • the lymphoma cells are Jurkat, K562, Raji or THP1 cells.
  • the Anti CD19 CAR-NK cells are capable of effectively killing or killing Raji cells and the like.
  • Another aspect of the present invention provides a method for producing the above Anti CD19 CAR-NK cell, comprising the steps of:
  • NK-92 cells were infected with the lentivirus obtained in the step (2) to obtain CD19 CAR-NK cells.
  • the nucleotide sequence is a nucleotide sequence encoding a ScFv-CD8-4-1BB-CD3 ⁇ fusion protein.
  • the present invention also provides the use of the above Anti CD19 CAR-NK cells for the preparation of a medicament for treating and/or preventing tumors and related diseases which highly express CD19 molecules.
  • the tumor that highly expresses CD19 is a lymphoma; preferably, the lymphoma is Jurkat, K562, Raji or THP1 cells; more preferably, the lymphoma is Raji cells.
  • the invention also provides a pharmaceutical composition comprising a therapeutically effective amount of AntiCD19 CAR-NK cells, and optionally a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is in the form of a liquid.
  • the ratio of the effective target of the Anti CD19 CAR-NK cell to the target cell is (0.5-20):1.
  • the tumor that highly expresses CD19 is a lymphoma.
  • the lymphoma is a Jurkat, K562, Raji or THP1 cell.
  • the lymphoma is a Raji cell.
  • the present invention also provides a method for treating and/or preventing a tumor and a related disease having high expression of CD19 by the above Anti CD19 CAR-NK cell, the method comprising ingesting a drug containing an effective amount of an anti-CD19 CAR-NK cell into a patient in vivo.
  • the method for ingesting the CD19 antibody or antigen-binding fragment thereof or AntiCD19 CAR-NK cell is intratumoral injection, intravenous injection, intrathoracic injection or local intervention.
  • the tumor that highly expresses CD19 is a lymphoma.
  • the lymphoma is a Jurkat, K562, Raji or THP1 cell.
  • the lymphoma is a Raji cell.
  • the present invention provides an anti-CD19 antibody or an antigen-binding fragment thereof targeting CD19, and the AntiCD19 CAR-NK cell constructed thereby is cultured and amplified by a monoclonal cell strain, and the trait is stable, and can be used for mass production. preparation.
  • the Anti-CD19 CAR-NK cell uses CD19 molecule as a target antigen to specifically kill or kill lymphoma cells, and can be used as a therapeutic drug for lymphoma diseases for the treatment of lymphoma with high CD19 expression;
  • the experiment showed that compared with NK92 cells, the targeting of the CAR vector was increased, the killing activity was specific, and the tumor treatment effect was obvious; that is, the CD19 CAR-NK cells constructed by the present invention were subjected to NK92 cells. Modification, after loading CAR, not only increased its targeting, but also because of the use of NK cells, compared with CAR-T, the side effects are small, the safety is high, the clinical risk and operability are low, and the cost is low
  • FIG. 1 is a schematic view showing the structure of a pCDNA3.4-ScFV (anti-CD19) expression vector provided by an embodiment of the present invention.
  • FIG. 2 is a schematic view showing the structure of a pRRSLIN-ScFV(2-27)-CD8TM-4-1BB-CD3 ⁇ lentiviral transfection vector according to an 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 according to an embodiment of the present invention; wherein, FIG. 3a is a NK-92 control group, and FIG. 3b is a CD19NK-92 experimental group.
  • FIG. 4 is a graph showing experimental results of killing Raji cells by CD19 CAR-NK cells according to an embodiment of the present invention.
  • FIG. 5 is a graph showing experimental results of killing different lymphoma cells by CD19 CAR-NK cells according to an embodiment of the present invention.
  • sequence of the nucleotide encoding the ScFv-CD8-4-1BB-CD3 ⁇ fusion protein of 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.
  • sequence of the nucleotide encoding the ScFv-CD8-4-1BB-CD3 ⁇ fusion protein of the present invention can be hybridized under the stringent conditions with the nucleotide sequence of SEQ ID NO: 14 and encoded. a polynucleotide of the fusion protein or a complement thereof;
  • the "stringent conditions” described herein may be any of low stringent conditions, medium stringent conditions, and high stringency conditions, preferably high stringency conditions.
  • the "low stringency conditions” may be 30 ° C, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 52% formamide
  • “medium stringent conditions” may be 40 ° C, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 52% formamide
  • “high stringency conditions” may be 50 ° C, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 52% formamide.
  • the higher the temperature the more highly homologous polynucleotides can be obtained.
  • one skilled in the art can select a comprehensive result of a plurality of factors affecting the stringency of hybridization, probe concentration, probe length, ionic strength, time, salt concentration, etc. to achieve a corresponding stringency.
  • hybridizable polynucleotide may be about 60% or more with the polynucleotide of the coding sequence number 6 when calculated by a system-determined default parameter by a homology search software such as FASTA or BLAST.
  • the term "antibody” refers to an immunoglobulin molecule that specifically binds to an antigen.
  • the antibody may be a complete immunoglobulin derived from a natural source or derived from a recombinant source, and may be an immunoreactive portion of a complete immunoglobulin.
  • the antibody is typically a tetramer of immunoglobulin molecules.
  • the antibodies of the present invention may 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 (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. 85: 5879-5883; Bird et al, 1988, Science 242: 423-426).
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable region of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies formed from antibody fragments, ScFv antibodies, and multispecific antibodies.
  • a "coding nucleotide” includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.
  • the nucleotide sequence encoding the protein can include an intron.
  • lentivirus refers to the genus of the family Retroviridae, which is capable of efficiently infecting non-cyclical and post-mitotic cells; they can deliver significant amounts of genetic information into the host cell's DNA so that they are the most gene delivery vectors. One of the effective methods.
  • vector is a composition of matter that includes an isolated nucleic acid and which can be used to deliver an isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • the term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.
  • cancer is defined as a disease characterized by rapid and uncontrolled growth of distorted cells. Cancer cells can spread locally or through the bloodstream 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.
  • "Inclusion&quot is used synonymously with "includes", "includes" or "characterize" and is intended to be inclusive or open and does not exclude additional unreported elements or method steps.
  • Any expression of the term "comprising”, particularly when describing a method, use, or product of the invention, is understood to include consisting essentially of and consisting of the component or element or component or The products, methods, and uses of the steps.
  • the invention exemplarily described herein may suitably be practiced without any one or more of the elements, one or more limitations not specifically disclosed herein.
  • 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 of anti-CD19 molecule.
  • NK-92 and NK92 all represent NK92 cells; SCFV has the same meaning as ScFv.
  • the "NK” according to the present invention is a human normal NK cell or NKT cell or NK cell line, and includes NK-92 cells, YT cells, NKL cells, HANK-1 cells, NK-YS cells, KHYG-1 cells, SNK. -6 cells and IMC-1 cells, etc.
  • NK-92 cells are taken as an example for description.
  • NK-92 cells CRL-2407 TM
  • Jurkat cells K562 cells
  • Raji cells THP1 cells
  • helper phage M13K07 were purchased from the Cell Resource Center of Shanghai Academy of Sciences, Chinese Academy of Sciences.
  • 2 ⁇ YT liquid medium 16 g peptone, 10 g yeast extract, 5 g sodium chloride, 800 mL water, adjusted to pH 7.0 with sodium hydroxide, made up to 1000 mL with water, and sterilized at 121 ° C for 20 min.
  • 2 ⁇ YT-AK 2 ⁇ YT medium was added with 100 g/mL of ampicillin and 50 g/mL of kanamycin.
  • TG1 bacterial cells in logarithmic growth phase were infected with different dilutions of helper phage M13K07 for 30 minutes at 37 ° C, then plated on agar plates and incubated at 37 ° C with constant temperature.
  • TG1 plaque clone from step 1 was picked into 3 mL of liquid 2YT medium and incubated at 37 ° C for 2 hours.
  • step 2 Transfer the culture in step 2 to 1 L 2YT medium, continue to culture for 1 h, add kanamycin to a final concentration of 50 ⁇ g/ml, and incubate at 37 ° C for 16 h.
  • the bacterial culture medium cultured for 16 hours was centrifuged (10 min at 5000 g) to remove bacterial cells, and the phage was collected by adding a phage precipitant to the supernatant to obtain an amplified M13KO7 helper phage.
  • the constructed anti-CD19 ScFv phage glycerol bacteria were inoculated into 500 ml of 2YT-G medium at 37 ° C, 250 rpm to an OD value of 0.8-0.9.
  • the M13KO7 helper phage amplified in the first step was added to the above step 1, the final concentration was 5 ⁇ 10 9 pfu/mL, and the mixture was allowed to stand at 37 ° C for 30 min, and then cultured at 200 rpm for 30 min under a constant temperature shaker.
  • step 6 Add the phage precipitant to the supernatant in step 4, centrifuge (7000 g, 15 min), remove the supernatant, collect the phage pellet, and resuspend the phage pellet in 8 ml PBS.
  • the CD19 antigen was coated at a concentration of 5 ⁇ g/mL at 4 ° C for 2 h, followed by three washes. Block overnight at 4 °C with blocking solution.
  • step 2 4. Pour off the blocking solution and wash, resuspend the phage antibody library (prepared in step 2) with 100 ⁇ g / mL of human IgG protein in blocking solution (containing 2% milk) and add to the well plate for 1 h at room temperature, then wash .
  • Single-stranded phage DNA a single-chain antibody gene sequence, was obtained by precipitation and identified by sequencing.
  • the single-stranded phage DNA obtained in Example 1 was cloned into the pCDNA3.4 vector, and the pCDNA3.4-ScFV (anti-CD19) expression vector shown in Fig. 1 was constructed, and the vector was transiently transferred to CHO cells for expression.
  • the nickel strain was purified for the subsequent analysis.
  • the affinity of the selected single-chain antibody to the CD19 protein was identified by the SPR method (see “USP39 General Information/(1105) Immunological Test Methods-Surface Plasmon Resonance"), and the single-chain antibody having the highest affinity to the CD19 protein was selected. It was named as single-chain antibody 2-27.
  • the affinity of the single-chain antibody 2-27 to the CD19 molecule is shown in Table 1.
  • amino acid sequence of the VH chain of the single-chain antibody 2-27 is as shown in SEQ ID NO. 7, and the nucleotide sequence thereof is shown in SEQ ID NO. 10; the amino acid sequence of the VL chain of the single-chain antibody 2-27 is as SEQ ID NO. 8, the nucleotide sequence of which is shown in SEQ ID NO. 11; the amino acid sequence of single-chain antibody 2-27 is shown in SEQ ID NO. 9, and the nucleotide of single-chain antibody 2-27 The sequence is shown in SEQ ID NO.
  • the gene synthesizes the SCFV(2-27)-CD8-4-1BB-CD3 ⁇ fusion gene sequence (the amino acid sequence of which is shown in SEQ ID NO: 13, and the gene sequence is shown in SEQ ID NO: 14).
  • the gene was transformed into a PRRSLIN vector by restriction enzyme digestion, and the upstream of the gene was the EP-1 ⁇ promoter.
  • the vector was transformed into Stbl3 Escherichia coli strain, ampicillin was screened, positive clone was obtained, plasmid was extracted, and the clone was identified by restriction enzyme digestion to obtain PRRLSIN-SCFV(2-27)-CD8-4-1BB-CD3 ⁇ lentiviral transfection vector (Fig. 2 Show).
  • Solution A 6.25 ml 2 x HEPES buffer buffer (5 large dishes packed together, the best effect).
  • Solution B a mixture obtained by separately adding the following plasmid: 112.5 ⁇ g of PRRLSIN-SCFV(2-27)-CD8-4-1BB-CD3 ⁇ (target plasmid); 39.5 ⁇ g of pMD2.G (VSV-G envelop); 73 ⁇ g of pCMVR8. 74 (gag, pol, tat, rev); 625 ⁇ l 2M calcium ion solution. Total volume of solution B: 6.25 ml.
  • the solution B was thoroughly mixed, and while the solution A was gently vortexed, the solution B was added dropwise to obtain a mixed solution of A and B, which was allowed to stand for 5-15 minutes. Then, the above mixed solution of A and B was gently vortexed, and added dropwise to a Petri dish containing 293T cells, and the culture dish was gently shaken back and forth to uniformly distribute the mixture of DNA and calcium ions. Then, (do not rotate the culture dish) and place it in an incubator for 16-18 hours. The fresh medium was replaced, the culture was continued, and the virus-containing supernatant was collected after 48 hours and 72 hours, respectively.
  • the collected supernatant was further centrifuged at 500 g for 10 minutes at 25 ° C, and filtered through a PES membrane (0.45 ⁇ m) to obtain a filtered supernatant.
  • the Beckman Coulter Ultra-clear SW28centrifuge tubes were then sterilized with 70% ethanol and sterilized under UV light for 30 minutes to obtain a sterile centrifuge tube.
  • the filtered lentivirus-containing supernatant is then transferred to a sterile centrifuge tube. Also carefully spread a layer of 20% sucrose at the bottom of the tube (every 8 ml of supernatant plus 1 ml of sucrose).
  • the centrifuge tube was equilibrated with PBS, 25,000 rpm (82, 700 g), and centrifuged at 4 ° C for 2 hours. Carefully remove the tube, pour off the supernatant, and invert the tube to remove any residual liquid. Then, 100 ⁇ l of PBS was added to the centrifuge tube with the residual liquid, and the centrifuge tube was sealed, placed at 4 ° C for 2 hours, gently vortexed every 20 minutes, centrifuged at 500 g for 1 minute (25 ° C), and the virus supernatant was collected to obtain a slow virus. After cooling on ice, store at -80 °C.
  • V/V lentivirus by volume ratio
  • medium containing NK-92 cells 1:5-10 (Example 4 prepared)
  • polybrene 8 ⁇ g / ml an equal amount of fresh complete medium was added to adjust the density of NK-92 cells to 1 ⁇ 10 5 /ml to continue the culture.
  • CAR antibody staining was performed, and CD19 CAR NK-92 positive cells were sorted by flow and expanded. The color change, cell density, and cell morphology
  • the positive rate of CAR NK-92 cells was detected by flow cytometry, and the flow detection results are shown in Fig. 3a and Fig. 3b.
  • the antibody used is an APC fluorescent marker, indicated on the abscissa, and the signal value will be significantly increased if the NK92 cell successfully expresses the CAR molecule.
  • the signal value of APC fluorescent label was significantly increased, indicating that NK-92 cells successfully expressed CAR molecules, and the positive rate of CAR-NK92 was 99.91%.
  • CD19 CAR- using the CCK-8 method see: Human Leukocyte Antigen-G Inhibits the Anti-Tumor Effect of Natural Killer Cells via Immunoglobulin-Like Transcript 2 in Gastric Cancer, Rui Wan Zi-Wei Wang Hui Li, et al.
  • the killing effect of NK cells on lymphoma cells-Raji cells is as follows:
  • a 1 ml Raji cell suspension (2 ⁇ 10 4 /well) was prepared in a 24-well plate. The plates were preincubated for 12 h in an incubator.
  • test well (medium containing tumor cells, CCK-8, CAR-NK);
  • control well (medium containing tumor cells, CCK-8);
  • the CD19 CAR NK-92 cells prepared by the present invention were able to significantly kill the Raji target cell strain as compared with the NK92 control group.
  • CD19 CAR-NK cells On different lymphoma cells Jurkat, K562, Raji and THP1 was also detected according to the same procedure as above. The results are shown in Fig. 5. As can be seen from the figure, CD19 CAR-NK The cells have a killing effect on different lymphoma cells, and the killing effect on Raji is most obvious.
  • the CD19 CAR NK-92 of the present invention is obtained by infecting a NK92 cell line with a CD19 CAR molecule and obtaining a single cloned cell by flow cytometry, and culturing and amplifying the CAR NK92 monoclonal cell strain with high trait stability and killing activity.
  • the cells can be used for large scale production and can be used in different patients without GVHR rejection.
  • CD19 CAR NK-92 cells do not need to isolate peripheral blood mononuclear cells (PBMC), and do not need to specifically activate T cells and prepare CAR-T cells (this process requires patients to wait for more than 10 days), It needs to be customized and can be used in multiple patients, shortening the time.
  • PBMC peripheral blood mononuclear cells
  • CD19 CAR-NK92 cells can be prepared in large quantities and ready for use.
  • conventionally prepared CAR-T cells are isolated from patients. The cells are prepared by virus infection, and the T cells are not the same monoclonal source, and the sorted CAR-NK92 cells are derived from the same single clone, and the traits and activities are uniform and stable, which is convenient for large-scale production and quality control; Compared with NK92 cells, the killing activity was specific due to the introduction of the CAR vector, and the tumor treatment effect was obvious.

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Abstract

提供一种以CD19为靶点的特异性抗体、CAR-NK细胞及其制备方法和应用，其中所述抗体包含重链可变区(VH)和/或轻链可变区(VL)，其中所述重链可变区包含重链VHCDR1、VHCDR2、VHCDR3；所述轻链可变区包含轻链VLCDR1、VLCDR2、VLCDR3。提供以CD19为靶点的抗CD19抗体或其抗原结合片段，并以此所构建的Anti CD19 CAR-NK细胞为单克隆细胞株培养并扩增所得，其性状稳定，可用于大规模生产制备。该细胞以CD19分子为靶抗原，能够特异性地杀死或杀伤淋巴瘤细胞，可作为淋巴瘤类疾病的治疗药物，用于CD19分子高表达的肿瘤的治疗。

Description

以CD19为靶点的特异性抗体、CAR-NK细胞及其制备方法和应用 技术领域
本发明涉及生物医药领域，具体涉及一种以CD19为靶点的特异性抗体、CAR-NK细胞及其制备方法和应用。
背景技术
嵌合抗原受体(chimeric antigen receptor，缩写CAR)修饰的免疫细胞使用遗传工程手段修饰免疫细胞使其表达外源性抗肿瘤基因。CAR基因主要包括细胞外识别域和细胞内信号转导结构域：前者用于识别肿瘤表面特异性分子和后者用于启动识别肿瘤表面分子后的免疫细胞应答，发挥细胞毒作用，其主要以T-细胞为载体。
CAR-T，全称是Chimeric AntigenReceptor T-Cell Immunotherapy，嵌合抗原受体T细胞免疫疗法。嵌合抗原受体T细胞(CAR-T细胞)是将能识别某种肿瘤抗原的抗体的抗原结合部与CD3-ζ链或4-1BB等胞内元件在体外偶联为一个嵌合基因，通过基因转导的方法转染患者的T细胞，使其表达嵌合抗原受体(CAR)。患者的T细胞被“重编码”后，生成大量肿瘤特异性的CAR-T细胞。CD19是95kD的穿膜糖蛋白，它属于免疫球蛋白超家族并且在B细胞调节应答中起核心作用。大量的研究证明CD19可作为治疗B细胞淋巴瘤的靶点用于CAR-T的构建。
目前经过大量的临床实践证明，以该CD19为靶点开发的CAR-T药物取得了显著的疗效。诺华制药公司2017年3月29日宣布其CAR-T产品CTL019进入FDA加速审批通道。2015年12月，FDA授予了Kite Pharma的CAR-T疗法KTE-C19的突破性疗法认证(BTD)，用于治疗弥漫性大B细胞淋巴瘤(DLBCL)的适应症疗法。2017年4月1日，Kite Pharma也宣布正式向FDA完成提交CAR-T疗法KTE-C19(后改名为“axicabtagene ciloleucel”)的生物制品许可申请(BLA)滚动申报。
但目前CD19 CAR-T研究仍然存在一些问题，例如：第一，制备CAR-T的T细胞只能来自患者自身，不能异体回输；第二，国际上用于CAR-T制备的CD19抗体的ScFv序列大多为鼠源抗体，存在较大的免疫排斥反应风险。而且，由于T细胞是针对特定患者进行修改，但是一些患者可能无法采集T细胞，或者没有足够的时间等待T细胞的准备过程，虽然目前CAR-T在向通用型的CAR-T发展，但其实又增加了临床风险以及操作难度。此外，面对CAR-T的高额费用，这些局限性可能会导致一些有望受益的患者无法接受CAR-T免疫疗法。
自然杀伤(natural killer，缩写NK)细胞是非特异性免疫系统的重要组成部分，先天免疫系统反应的关键性介质细胞。NK细胞是一种广谱免疫细胞，具有快速发现和摧毁异常细胞(如癌症或病毒感染的细胞)的特异功能，而且不需要提前致敏或HLA配型，即可展示强大的溶解异常细胞的活性。使用免疫细胞(包括NK细胞)来治疗癌症是近年来的新趋势，这种新疗法有望为对传统手术、化疗和放疗无效的肿瘤提供了新的治愈希望。
发明内容
有鉴于此，本发明提供了一种以CD19为靶点的特异性抗体、CAR-NK细胞及其制备方法和应用，具体为以CD19为靶点的抗CD19抗体或其抗原结合片段，以及能够表达该结构域的CAR-NK细胞及其制备方法和应用，该细胞一方面性状稳定，可大批量生产和制备，可进行异体回输；另一方面，用于构建该抗CD19抗体或其抗原结合片段的抗体序列为人源抗体序列，其作为药物应用时，极大地降低了免疫排斥反应的风险。
本发明一方面提供一种以CD19为靶点的抗CD19抗体或其抗原结合片段，包含重链可变区(VH)和/或轻链可变区(VL)，其中所述重链可变区包含重链VHCDR1、VHCDR2、VHCDR3；所述轻链可变区包含轻链VLCDR1、VLCDR2、VLCDR3，其中所述VHCDR1的氨基酸序列如SEQ ID NO：1所示的序列或其同源序列；所述VHCDR2的氨基酸序列如SEQ ID NO：2所示的序列或其同源序列；所述VHCDR3的氨基酸序列如SEQ ID NO：3所示的序列或其同源序列；所述VLCDR1的氨基酸序列如SEQ ID NO：4所示的序列或其同源序列；所述VLCDR2的氨基酸序列如SEQ ID NO：5所示的序列或其同源序列；所述VLCDR3的氨基酸序列如SEQ ID NO：6所示的序列或其同源序列。
示例性地，所述VH链的氨基酸序列如SEQ ID NO：7所示的序列或其同源序列。
示例性地，所述VL链的氨基酸序列如SEQ ID NO：8所示的序列或其同源序列。
示例性地，所述同源序列与原序列的同源性为95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1或以上、99.2或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上等。
示例性地，所述VH链和VL链直接连接或通过连接肽连接。
在本发明提供的一个具体实施方式中，以CD19为靶点的抗CD19抗体或其抗原结合片段包括VH链和VL链。
在本发明提供的一个具体实施方式中，所述VH链和VL链通过连接肽连接。
在本发明提供的一个具体实施方式中，所述抗CD19抗体或其抗原结合片段的核苷酸序列如SEQ ID NO：9所示的序列或其简并序列。
在本发明提供的一个具体实施方式中，所述抗CD19抗体或其抗原结合片段与CD19的亲和力达到8.473×10 -8M以上。
本发明另一方面提供一种核苷酸序列，其能够编码上述抗CD19抗体或其抗原结合片 段。
在本发明提供的一个具体实施方式中，所述的核苷酸序列包含SEQ ID NO：10所示的序列或其简并序列，和/或SEQ ID NO：11所示的序列或其简并序列。
在本发明提供的一个具体实施方式中，所述的核苷酸序列如SEQ ID NO：12所示的序列或其简并序列。
示例性地，所述简并序列与原序列的同源性为95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1或以上、99.2或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上等。
本发明另一方面提供上述抗CD19抗体或其抗原结合片段的制备方法，其包括：建立噬菌体抗体库并从中筛选能与CD19结合的抗体或抗体片段；其具体过程包括：
①扩增M13KO7辅助噬菌体；
②利用扩增后的M13KO7辅助噬菌体和抗CD19的ScFv噬菌体库建立噬菌体抗体库；
③利用CD19抗原从步骤②中的噬菌体抗体库中筛选单链噬菌体DNA；以及任选地，
④将步骤③中获得的单链噬菌体DNA进行表达，并纯化。
本发明另一方面提供一种Anti CD19 CAR-NK细胞，该细胞能够表达嵌合抗原受体，所述嵌合抗原受体包括上述抗CD19抗体或其抗原结合片段。
在本发明的一个具体实施方式中，所述嵌合抗原受体还包含跨膜结构域和/或共刺激信号传导区，所述的抗CD19抗体或其抗原结合片段能够特异性结合肿瘤特异性抗原CD19，并通过跨膜结构域和共刺激信号传导区激活所述Anti CD19 CAR-NK细胞。
示例性地，所述的跨膜结构域选自：CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154中的一种或多种的跨膜结构域；优选地，所述的跨膜结构域为CD8跨膜结构域；和/或，
所述的共刺激信号传导区包含共刺激分子的细胞内结构域，所述的共刺激分子选自：CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB和OX40中的一种或多种；优选地，所述的共刺激信号传导区包含4-1BB和CD3ζ胞内结构域。
示例性地，所述嵌合抗原受体是结构为ScFv-CD8-4-1BB-CD3ζ的融合蛋白，该融合蛋白能够特异性识别CD19分子。
在本发明的一个具体实施方式中，所述ScFv-CD8-4-1BB-CD3ζ融合蛋白的氨基酸的序列如SEQ ID NO：13所示或其同源序列等。
示例性地，所述ScFv-CD8-4-1BB-CD3ζ融合蛋白的核苷酸的序列如SEQ ID NO：14所示或其简并序列等。
示例性地，所述Anti CD19 CAR-NK细胞能够有效地杀伤或杀死淋巴瘤细胞。
在本发明的一个优选实施方式中，所述淋巴瘤细胞为Jurkat、K562、Raji或THP1细 胞。
在本发明的一个具体实施方式中，所述Anti CD19 CAR-NK细胞能够有效地杀伤或杀死Raji细胞等。
本发明另一方面提供上述Anti CD19 CAR-NK细胞的制备方法，其包括如下步骤：
(1)合成和扩增编码上述的抗CD19抗体或其抗原结合片段的核苷酸序列，将上述的嵌合抗原受体的核苷酸序列克隆到慢病毒表达载体上；
(2)利用慢病毒包装质粒和步骤(1)得到的慢病毒表达载体质粒感染293T细胞，包装和制备慢病毒；
(3)利用步骤(2)得到的慢病毒感染NK-92细胞，得到CD19 CAR-NK细胞。
在本发明的一个具体实施方式中，所述核苷酸序列为编码ScFv-CD8-4-1BB-CD3ζ融合蛋白的核苷酸序列。
本发明还提供上述Anti CD19 CAR-NK细胞在制备治疗和/或预防高表达CD19分子的肿瘤及相关疾病的药物中的应用。
示例性地，所述高表达CD19的肿瘤为淋巴瘤；优选地，所述淋巴瘤为Jurkat、K562、Raji或THP1细胞；更优选地，所述淋巴瘤为Raji细胞。
本发明还提供了一种药物组合物，其包括有效治疗量的Anti CD19 CAR-NK细胞，以及任选地，药学上可接受的辅料。
示例性地，所述药物组合物的剂型为水剂。
示例性地，所述药物组合物用于治疗或预防肿瘤时，所述Anti CD19 CAR-NK细胞与所述靶细胞的效靶比为(0.5～20)：1。
示例性地，所述高表达CD19的肿瘤为淋巴瘤。
在一个优选实施方式中，所述淋巴瘤为Jurkat、K562、Raji或THP1细胞。
在一个具体实施方式中，所述淋巴瘤为Raji细胞。
本发明还提供一种上述Anti CD19 CAR-NK细胞用于治疗和/或预防高表达CD19的肿瘤及相关疾病方法，所述方法包括将含有有效量的Anti CD19 CAR-NK细胞的药物摄入患者体内。
在一个优选实施方式中，所述CD19抗体或其抗原结合片段或Anti CD19 CAR-NK细胞的摄入方法为瘤内注射、静脉注射、胸腔内注射或局部介入。
示例性地，所述高表达CD19的肿瘤为淋巴瘤。
示例性地，所述淋巴瘤为Jurkat、K562、Raji或THP1细胞。
在本发明的一个具体实施方式中，所述淋巴瘤为Raji细胞。
本发明至少具有如下优势之一：
本发明提供以CD19为靶点的抗CD19抗体或其抗原结合片段，并以此所构建的Anti CD19 CAR-NK细胞为单克隆细胞株培养并扩增所得，性状也稳定，可用于大规模生产制 备。该Anti CD19 CAR-NK细胞以CD19分子为靶抗原，能够特异性地杀死或杀伤淋巴瘤细胞，可作为淋巴瘤类疾病的治疗药物，用于CD19分子高表达的淋巴瘤的治疗；同时通过实验表明，与NK92细胞相比，由于进行了CAR载体导入，增加了靶向性，其杀伤活性特异，肿瘤治疗效果明显；也就是说本发明构建的CD19 CAR-NK细胞，通过对NK92细胞进行改造，装上CAR后不但增加了其靶向性，而且由于使用的是NK细胞，与CAR-T相比副作用小，安全性高，临床风险和操作性低，成本低。
附图说明
图1所示为本发明实施例提供的pCDNA3.4-ScFV(anti-CD19)表达载体结构示意图。
图2所示为本发明实施例提供的pRRSLIN-ScFV(2-27)-CD8TM-4-1BB-CD3ζ慢病毒转染载体的结构示意图。
图3所示为本发明实施例提供的流式细胞仪检测CD19 CAR-NK细胞阳性率的结果图；其中，图3a为NK-92对照组，图3b为CD19NK-92实验组。
图4所示为本发明实施例提供的CD19 CAR-NK细胞杀伤Raji细胞的实验结果图。
图5所示为本发明实施例提供的CD19 CAR-NK细胞杀伤不同淋巴瘤细胞的实验结果图。
具体实施方式
除非另有定义，本发明中使用的所有技术和科学术语具有与本发明所述技术领域的普通技术人员通常理解的相同含义。
具体而言，本发明所述的编码ScFv-CD8-4-1BB-CD3ζ融合蛋白的核苷酸的序列是任何能够编码该融合蛋白的任何DNA序列，优选地，该序列为SEQ ID NO：14或其互补序列。另一方面，本发明所述的编码ScFv-CD8-4-1BB-CD3ζ融合蛋白的核苷酸的序列可为在严谨条件下与由SEQ ID NO：14的核苷酸序列进行杂交、且编码该融合蛋白的多核苷酸或其互补序列；
本文所述的“严谨条件”，可以为低严谨条件、中严谨条件、高严谨条件中的任一种，优选为高严谨条件。示例性地，“低严谨条件”可为30℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件；“中严谨条件”可为40℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件；“高严谨条件”可为50℃、5×SSC、5×Denhardt液、0.5％SDS、52％甲酰胺的条件。本领域技术人员应当理解温度越高越能得到高同源性的多核苷酸。另外，本领域技术人员可以选择影响杂交的严谨度的温度、探针浓度、探针长度、离子强度、时间、盐浓度等多个因素形成的综合结果来实现相应的严谨度。
除此之外可杂交的多核苷酸还可以为，通过FASTA、BLAST等同源性检索软件用系统设定的默认参数进行计算时，与编码序列号6的多核苷酸具有约60％或以上、约70％或以上、71％或以上、72％或以上、73％或以上、74％或以上、75％或以上、76％或以上、77％或以上、 78％或以上、79％或以上、80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1或以上、99.2或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上同一性的多核苷酸。
核苷酸序列的同一性，可以使用Karlin及Altschul的算法规则BLAST(Proc.Natl.Acad.Sci.USA 87：2264-2268,1990；Proc.Natl.Acad.Sci.USA 90:5873,1993)来确定。基于BLAST算法规则的程序BLASTN、BLASTX已被开发(Altschul SF,et al:J Mol Biol215:403,1990)。使用BLASTN分析碱基序列时，如使参数为score＝100、wordlength＝12；此外使用BLASTX分析氨基酸序列时，如使参数为score＝50、wordlength＝3；使用BLAST和Gapped BLAST程序时，采用各程序的系统可设定默认参数值。
本发明中，术语“抗体”指的是与抗原特异性结合的免疫球蛋白分子。抗体可为源于自然源或源于重组源的完整的免疫球蛋白，并可为完整免疫球蛋白的免疫反应部分。抗体通常为免疫球蛋白分子的四聚物。本发明中的抗体可以以多种形式存在，包括例如，多克隆抗体、单克隆抗体、Fv、Fab和F(ab)2，以及单链抗体和人源化抗体等(Harlow等，1999,In:Using Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,NY；Harlow等，1989,In:Antibodies:A Laboratory Manual,Cold Spring Harbor,New York；Houston等，1988,Proc.Natl.Acad.Sci.USA 85:5879-5883；Bird等，1988,Science 242:423-426)。
术语“抗体片段”指的是完整抗体的一部分，并指的是完整抗体的抗原决定可变区。抗体片段的例子包括但不限于Fab、Fab'、F(ab')2和Fv片段，由抗体片段形成的线性抗体、ScFv抗体和多特异性抗体。
除非另有规定，“编码核苷酸”包括为彼此简并版本并编码相同的氨基酸序列的所有核苷酸序列。编码蛋白质的核苷酸序列可包括内含子。
术语“慢病毒”指的是逆转录病毒科的属，其能够有效感染非周期性和有丝分裂后的细胞；它们可传递显著量的遗传信息进入宿主细胞的DNA，以便它们是基因传递载体的最有效的方法之一。
术语“载体”为物质组合物，其包括分离的核酸，并且其可用于传递分离的核酸至细胞内部。很多载体在本领域中是已知的，包括但不限于线性多核苷酸、与离子或两性分子化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。该术语也应被解释为包括便于将核酸转移入细胞的非质粒和非病毒化合物，诸如例如聚赖氨酸化合物、脂质体等等。病毒载体的例子包括但不限于，腺病毒载体、腺伴随病毒载体、逆转录病毒载体等等。
术语“癌症”被定义为以畸变细胞的快速和失控生长为特征的疾病。癌症细胞可局部蔓延或通过血流和淋巴系统蔓延至身体的其他部分。各种癌症的例子包括但不限于乳腺癌、 前列腺癌、卵巢癌、子宫颈癌、皮肤癌、胰腺癌、结肠直肠癌、肾癌、肝癌、脑癌、淋巴瘤、白血病、肺癌等等。
如本文所使用的，“包含”与“包括”、“含有”或“特征在于”同义，并且是包括在内的或开放性的，并且不排除另外的未陈述的元件或方法步骤。术语“包含”在本文中的任何表述，特别是在描述本发明的方法、用途或产品时，应理解为包括基本上由所述组分或元件或步骤组成和由所述组分或元件或步骤组成的那些产品、方法和用途。本文示例性描述的本发明适当地可以在不存在本文未具体公开的任何一种或多种元件、一种或多种限制的情况下进行实践。
本文已采用的术语和表述用作描述性而不是限制性术语，并且在此种术语和表述的使用中不预期排除所示和所述特征或其部分的任何等价物，但应认识到各种修饰在请求保护的本发明的范围内是可能的。因此，应当理解尽管本发明已通过优选实施方案和任选特征具体公开，但本领域技术人员可以采用本文公开的概念的修饰和变化，并且此类修饰和变化被视为在如由附加权利要求定义的本发明的范围内。
本文中出现的英文名称不区分大小写；CD19 CAR-NK、CD19-CAR NK表示的含义相同；CD19 CAR-NK与Anti CD19-CAR NK表示相同的含义，均表示抗CD19分子的CAR-NK细胞；NK-92、NK92均表示NK92细胞；SCFV与ScFv表示的含义相同。
本发明所述的“NK”为人体正常NK细胞或NKT细胞或NK细胞系，其包括NK-92细胞，YT细胞，NKL细胞，HANK-1细胞，NK-YS细胞，KHYG-1细胞，SNK-6细胞和IMC-1细胞等。本发明的具体实施例中以NK-92细胞为例进行说明。
为更清楚地说明本发明，现结合如下实施例进行详细说明，但这些实施例仅仅是对本发明的示例性描述，并不能解释为对本申请的限制。
下述实施例中的材料来源：
NK-92细胞(
Figure PCTCN2019071559-appb-000001
CRL-2407 TM)，Jurkat细胞、K562细胞、Raji细胞、THP1细胞、辅助噬菌体M13K07均购自中国科学院上海生科院细胞资源中心。
实施例1 CD19人源抗体的筛选
2×YT液体培养基：16g蛋白胨，10g酵母提取物，5g氯化钠，800mL水，用氢氧化钠调pH至7.0，加水定容至1000mL，121℃灭菌20min。
2×YT-G：2×YT培养基中加入2％的葡萄糖。
2×YT-AK：2×YT培养基中加入100g/mL的氨苄青霉素和50g/mL的卡那霉素。
一、扩增M13KO7辅助噬菌体
1.在37℃条件下，用不同的稀释浓度的辅助噬菌体M13K07感染对数生长期的TG1细菌细胞30分钟，之后将其涂布在琼脂平板上，置于37℃恒温培养。
2.将步骤1中的TG1噬菌斑克隆挑到3mL液体2YT培养基中，于37℃下培养2小时。
3.将步骤2中的培养物转移到1L 2YT培养基中，继续培养1h，加入卡那霉素至终浓度为50μg/ml，37℃培养16h。
4.将培养16h的细菌培养液离心(10min at 5000g)去除细菌细胞，在上清中加入噬菌体沉淀剂收集噬菌体，得到扩增好的M13KO7辅助噬菌体。
5.计算扩增好的M13KO7辅助噬菌体的滴度，之后将其稀释到1×10 13pfu/mL并保存在-20℃冰箱中，备用。
二、制备噬菌体抗体库
1.分离健康人血PBMC，扩增抗体重链和轻链基因，构建抗CD19的ScFv噬菌体库,然后加入甘油，于-80℃保存。
2.取构建好的抗CD19的ScFv噬菌体库甘油菌接种到500ml 2YT-G培养基中，在37℃、250rpm条件下至OD值为0.8-0.9。
3.将步骤一中扩增好的M13KO7辅助噬菌体加入上述步骤1中，其终浓度为5×10 9pfu/mL，37℃静置30min，之后在200rpm转速下，恒温摇床培养30min。
4.离心(2200g，15min)步骤2中的培养液，收集细菌细胞，用2YT-AK培养基重悬菌泥，在30℃条件下300rmp转速下过夜培养。
5.离心(7000g，15min，4℃)步骤3中的培养液，去除细菌细胞，将上清收集到1L瓶中。
6.向步骤4中的上清中加入噬菌体沉淀剂，离心(7000g，15min)，去除上清，收集噬菌体沉淀，再将噬菌体沉淀8ml PBS重悬。
7.重新离心(12,000g，10min)去除细菌碎片，取上清，得到制备好的噬菌体抗体库，备用。
三、抗体筛选
1.将CD19抗原以5μg/mL的浓度，在4℃条件下包被孔板2h，之后洗涤三次。利用封闭液在4℃条件下封闭过夜。
2.倒掉封闭液并洗涤，将噬菌体抗体库(步骤二中制备)与100μg/mL的人IgG蛋白重悬在封闭液中(含2％牛奶)并加入孔板中室温孵育1h，之后洗涤。
3.通过胰蛋白酶消化结合在孔板上噬菌体，用于后续第二轮筛选。
4.按上述1-3中的流程重复三轮筛选。
5.通过沉淀获得单链噬菌体DNA，即单链抗体基因序列，并测序鉴定。
实施例2：CD19抗体的表达与鉴定
将实施例1获得的单链噬菌体DNA克隆到pCDNA3.4载体上，构建如图1所示的pCDNA3.4-ScFV(anti-CD19)表达载体，将该载体瞬转至CHO细胞进行表达，通过镍株纯化该单链抗体序列用于后续分析。
用SPR方法(参见「美国药典USP39General Information/(1105)Immunological Test Methods-Surface Plasmon Resonance章节」)鉴定筛选到的单链抗体与CD19蛋白的亲和力，从中选择与CD19蛋白的亲和力最高的单链抗体，并将其命名为单链抗体2-27。单链 抗体2-27与CD19分子亲和力如表1所示。
表1：单链抗体2-27与CD19分子亲和力
sequence Ka(1/Ms) Kd(1/s) KD(M)
2-27 4.984E+4 0.004223 8.473E-8
其中，单链抗体2-27的VH链的氨基酸序列如SEQ ID NO.7所示，其核苷酸序列如SEQ ID NO.10所示；单链抗体2-27的VL链的氨基酸序列如SEQ ID NO.8所示，其核苷酸序列如SEQ ID NO.11所示；单链抗体2-27的氨基酸序列如SEQ ID NO.9所示，单链抗体2-27的核苷酸序列如SEQ ID NO.12所示。
实施例3：慢病毒表达载体的制备
基因合成SCFV(2-27)-CD8-4-1BB-CD3ζ融合基因序列(其氨基酸序列如SEQ ID NO：13所示，基因序列如SEQ ID NO：14所示)。通过酶切，将其转化连接到PRRSLIN载体中，基因上游为EP-1α启动子。载体转化Stbl3大肠杆菌菌株，氨苄青霉素筛选，获得阳性克隆，提取质粒，酶切鉴定克隆，获得PRRLSIN-SCFV(2-27)-CD8-4-1BB-CD3ζ慢病毒转染载体(如图2所示)。
实施例4：慢病毒的制备
(1)转染前24小时，以每皿约8×10 6个将293T细胞接种至15cm培养皿中。确保转染时细胞在80％左右的汇合度，且转染的细胞均匀分布于培养皿中。
(2)准备溶液A和溶液B
溶液A：6.25ml 2×HEPES buffer缓冲液(5个大皿一起包装的量，效果最好)。
溶液B：为分别加入以下质粒得到的混合物：112.5μg PRRLSIN-SCFV(2-27)-CD8-4-1BB-CD3ζ(target plasmid)；39.5μg pMD2.G(VSV-G envelop)；73μg pCMVR8.74(gag，pol，tat，rev)；625μl 2M钙离子溶液。溶液B总体积：6.25ml。
充分混匀溶液B，在轻轻涡旋溶液A的同时，逐滴加入溶液B，得到A和B的混合溶液，静置5-15分钟。再轻轻涡旋上述A和B的混合溶液，并将其逐滴加入含293T细胞的培养皿中，轻轻前后晃动培养皿使DNA与钙离子的混合物均匀分布。然后，(不要旋转培养皿)放置于培养箱中培养16-18小时。更换新鲜培养基，继续培养，分别在48小时和72小时后收集含病毒的上清液。再将收集的上清液，于500g，25℃离心10分钟，PES膜(0.45μm)过滤，得到过滤后的上清液。然后以70％乙醇消毒贝克曼库尔特Ultra-clear SW28centrifuge tubes，并置于紫外灯下消毒30分钟，得到消毒后的离心管。再将已过滤的含慢病毒的上清液转移至消毒后的离心管中。并且在离心管底部小心铺上一层20％蔗糖(每8ml上清液加1ml蔗糖)。以PBS平衡离心管，25000rpm(82，700g)，4℃离心2小时。小心取出离心管，倒掉上清液，倒置离心管去掉残余液体。再在倒掉残余液体的离心管中加入100μl PBS，密封离心管，在4℃放置2小时，每20分钟轻轻涡旋一次，500g 离心1分钟(25℃)，收集病毒上清，得到慢病毒。冰上冷却后，置于-80℃保存。
实施例5：CD19 CAR-NK-92细胞的制备
将NK-92细胞密度调整至2-3×10 5/ml，按体积比(V/V)慢病毒：含有NK-92细胞的培养基＝1:5-10的比例添加慢病毒(实施例4所制备的)，同时添加聚凝胺8μg/ml。4h之后，补加等量的新鲜的完全培养基将NK-92细胞密度调整至1×10 5/ml继续培养。次日，将所有的细胞离心，加入新鲜的培养基，继续培养。每隔1-2天进行补液，使维持细胞密度在2-3×10 5/ml。72h后进行CAR抗体染色，同时流式分选CD19 CAR NK-92阳性细胞并扩大培养。每天观察培养基的颜色变化、细胞密度、细胞形态并作相应记录。
利用流式检测CAR NK-92细胞阳性率，流式检测结果如图3a和图3b所示。图3a和图3b中，所采用的抗体为APC荧光标记，在横坐标上进行表示，NK92细胞若成功表达CAR分子，该信号值将显著升高。从图3a和图3b中可以看出，APC荧光标记的信号值显著升高，表明NK-92细胞成功表达出CAR分子，CAR-NK92阳性率为99.91％。
实施例6：CD19 CAR-NK细胞对体外肿瘤杀伤效果的评估
利用CCK-8方法(参见：Human Leukocyte Antigen-G Inhibits the Anti-Tumor Effect of Natural Killer Cells via Immunoglobulin-Like Transcript 2 in Gastric Cancer,Rui Wan Zi-Wei Wang Hui Li,et al.)检测CD19 CAR-NK细胞对淋巴瘤细胞-Raji细胞的杀伤效果。实验操作方法如下：
(1)在24孔板中配制1ml Raji细胞悬液(2×10 4个/孔)。将培养板在培养箱预培养12h。
(2)弃去24孔板的培养上清，每孔加入1ml效应细胞(CD19 CAR-NK-92细胞)，效应细胞与靶细胞数目的比例为1:1或0.5:1。培养基对照孔只加1ml培养基，每个实验置三个复孔。效应细胞与靶细胞共孵育4小时或2h，实验分组如下述表2所示。
表2 CD19 CAR-NK细胞对Raji细胞的杀伤效果试验分组
组别 效应细胞 效靶比(E：T) 杀伤时间(小时)
1 NK92 0.5:1 2
2 CD19 CAR NK-92 0.5:1 2
3 NK92 0.5:1 2
4 CD19 CAR NK-92 0.5:1 2
5 NK92 1:1 4
6 CD19 CAR NK-92 1:1 4
7 NK92 1:1 4
8 CD19 CAR NK-92 1:1 4
(3)每孔加入100ul CCK-8溶液，将培养板在培养箱内孵育2h。
(4)用酶标仪测定在450nm处的吸光度。
(5)杀伤率＝(As-Ab)/(Ac-Ab)×100％；
As：试验孔(含有肿瘤细胞的培养基、CCK-8、CAR-NK)；
Ac：对照孔(含有肿瘤细胞的培养基、CCK-8)；
Ab：空白对照(不含细胞和CAR-NK的培养基、CCK-8)；
CD19 CAR-NK细胞体外肿瘤杀伤效果评估的实验结果如图4所示。
如图4所示实验结果显示，与NK92对照组比较，本发明所制备的CD19 CAR NK-92细胞能够显著杀伤Raji靶细胞株。
并且，还按照上述同样的操作方法同时检测CD19 CAR-NK细胞对不同淋巴瘤细胞Jurkat、K562、Raji和THP1的杀伤效果，结果如图5所示，从图中可以看出，CD19 CAR-NK细胞对不同的淋巴瘤细胞均能起到杀伤效果，对Raji的杀伤效果最明显。
本发明的CD19 CAR NK-92是将CD19 CAR分子侵染NK92细胞株并经过流式筛选获得单一克隆细胞，将性状稳定杀伤活性高的CAR NK92单克隆细胞株培养并扩增获得。该细胞可以用于大规模生产制备，可以用于不同病人且不会产生GVHR排斥。CD19 CAR NK-92细胞与CAR-T细胞相比，无需分离病人外周血单核细胞(PBMC)，无需特异活化T细胞和制备CAR-T细胞(该过程需要病人等待10天以上时间)，不需要个性定制且能用于多个病人，缩短了时间，CD19 CAR-NK92细胞可以大批量制备培养，病人即来即用；另一方面，常规制备的CAR-T细胞，由于是分离病人的T细胞经病毒感染而制备的，T细胞不是同一个单克隆来源，而分选出的CAR-NK92细胞来源于同一个单克隆，性状和活性均一且稳定，便于大规模生产和质控；同时与NK92细胞相比，由于进行了CAR载体导入，其杀伤活性特异，肿瘤治疗效果明显。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
Figure PCTCN2019071559-appb-000002
Figure PCTCN2019071559-appb-000003
Figure PCTCN2019071559-appb-000004
Figure PCTCN2019071559-appb-000005
Figure PCTCN2019071559-appb-000006
Figure PCTCN2019071559-appb-000007
Figure PCTCN2019071559-appb-000008

Claims (28)

  1. 一种抗CD19抗体或其抗原结合片段，包含重链可变区(VH)和/或轻链可变区(VL)，其中所述重链可变区包含重链VHCDR1、VHCDR2、VHCDR3；所述轻链可变区包含轻链VLCDR1、VLCDR2、VLCDR3，其中所述VHCDR1的氨基酸序列如SEQ ID NO：1所示的序列或其同源序列；所述VHCDR2的氨基酸序列如SEQ ID NO：2所示的序列或其同源序列；所述VHCDR3的氨基酸序列如SEQ ID NO：3所示的序列或其同源序列；所述VLCDR1的氨基酸序列如SEQ ID NO：4所示的序列或其同源序列；所述VLCDR2的氨基酸序列如SEQ ID NO：5所示的序列或其同源序列；所述VLCDR3的氨基酸序列如SEQ ID NO：6所示的序列或其同源序列。
  2. 根据权利要求1所述的抗CD19抗体或其抗原结合片段，其特征在于，所述VH链的氨基酸序列如SEQ ID NO：7所示的序列或其同源序列；以及任选地，所述VL链的氨基酸序列如SEQ ID NO：8所示的序列或其同源序列。
  3. 根据权利要求1或2所述的抗CD19抗体或其抗原结合片段，其特征在于，所述同源序列与原序列的同源性为95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1或以上、99.2或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上等。
  4. 根据权利要求1至3中任一项所述的抗CD19抗体或其抗原结合片段，其特征在于，所述VH链和VL链直接连接或通过连接肽连接；优选地，所述抗CD19抗体或其抗原结合片段的氨基酸序列如SEQ ID NO：9所示的序列或其同源序列。
  5. 根据权利要求1至4中任一项所述的抗CD19抗体或其抗原结合片段，其特征在于，所述抗CD19抗体或其抗原结合片段与CD19的亲和力达到8.473×10 -8M以上。
  6. 一种编码权利要求1至5中任一项所述的抗CD19抗体或其抗原结合片段的核苷酸序列。
  7. 根据权利要求6所述的核苷酸序列，其特征在于，所述的核苷酸序列包含SEQ ID NO：10所示的序列或其简并序列，和/或SEQ ID NO：11所示的序列或其简并序列；优选地，所述核苷酸序列如SEQ ID NO：12所示的序列或其简并序列。
  8. 根据权利要求7所述的核苷酸序列，其特征在于，所述简并序列与原序列的同源性为95％或以上、96％或以上、97％或以上、98％或以上、99％或以上、99.1或以上、99.2或以上、99.3％或以上、99.4％或以上、99.5％或以上、99.6％或以上、99.7％或以上、99.8％或以上、或99.9％或以上等。
  9. 根据权利要求1至5中任一项所述的抗CD19抗体或其抗原结合片段或权利要求6至8中任一项所述的核苷酸所编码的抗CD19抗体或其抗原结合片段的制备方法，其包括： 建立噬菌体抗体库并从中筛选能与CD19结合的抗体或抗体片段。
  10. 根据权利要求9所述的制备方法，其特征在于，所述制备方法包括以下步骤：
    1)扩增M13KO7辅助噬菌体；
    2)利用扩增后的M13KO7辅助噬菌体和抗CD19的ScFv噬菌体库建立噬菌体抗体库；
    3)利用CD19抗原从步骤2)中的噬菌体抗体库中筛选单链噬菌体DNA；
    以及任选地，4)将步骤3)中获得的单链噬菌体DNA进行表达，并纯化。
  11. 一种Anti CD19 CAR-NK细胞，其能够表达嵌合抗原受体，所述嵌合抗原受体包括权利要求1至5中任一项所述的抗CD19抗体或其抗原结合片段，和/或权利要求6至8中任一项所述的核苷酸所编码的抗CD19抗体或其抗原结合片段，和/或权利要求9或10所制备的抗CD19抗体或其抗原结合片段。
  12. 根据权利要求11所述的Anti CD19 CAR-NK细胞，其特征在于，所述嵌合抗原受体还包含跨膜结构域和/或共刺激信号传导区；所述抗CD19抗体或其抗原结合片段能够特异性结合肿瘤特异性抗原CD19，并通过跨膜结构域和共刺激信号传导区激活所述Anti CD19 CAR-NK细胞。
  13. 根据权利要求12所述的Anti CD19 CAR-NK细胞，其特征在于，所述的跨膜结构域选自：CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD134、CD137、ICOS和CD154中的一种或多种的跨膜结构域；优选地，所述的跨膜结构域为CD8跨膜结构域；和/或，
    所述的共刺激信号传导区包含共刺激分子的细胞内结构域，所述的共刺激分子选自：CD3ζ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD66d、CD2、CD4、CD5、CD28、CD134、CD137、ICOS、CD154、4-1BB和OX40中的一种或多种；优选地，所述的共刺激信号传导区包含4-1BB和CD3ζ胞内结构域。
  14. 根据权利要求13所述的Anti CD19 CAR-NK细胞，其特征在于，所述嵌合抗原受体是结构为ScFv-CD8-4-1BB-CD3ζ的融合蛋白，其中所述的ScFv能够特异性结合CD19分子；优选地，所述ScFv-CD8-4-1BB-CD3ζ融合蛋白的氨基酸的序列如SEQ ID NO：13或其同源序列。
  15. 根据权利要求14所述的Anti CD19 CAR-NK细胞，其特征在于，所述ScFv-CD8-4-1BB-CD3ζ融合蛋白的编码核苷酸的序列如SEQ ID NO：14或其简并序列。
  16. 根据权利要求11至15中任一项所述的Anti CD19 CAR-NK细胞，其特征在于，所述Anti CD19 CAR-NK细胞能够有效地杀伤和/或杀死淋巴瘤细胞；优选地，所述淋巴瘤细胞为Jurkat、K562、Raji或THP1细胞，优选为Raji细胞。
  17. 根据权利要求11至16中任一项所述Anti CD19 CAR-NK细胞的制备方法，其包括如下步骤：
    (1)合成和扩增编码权利要求1至5任一项中所述的抗CD19抗体或其抗原结合片段的核苷酸序列或权利要求6至8中任一项所述的核苷酸序列，并将所述核苷酸序列克隆到慢病毒表达载体上；
    (2)利用慢病毒包装质粒和步骤(1)得到的慢病毒表达载体质粒感染细胞，包装和制备慢病毒；
    (3)利用步骤(2)得到的慢病毒感染NK-92细胞，得到Anti CD19 CAR-NK细胞。
  18. 根据权利要求17所述的制备方法，其特征在于，所述核苷酸序列为编码ScFv-CD8-4-1BB-CD3ζ融合蛋白的核苷酸序列。
  19. 一种药物组合物，其包含有效治疗量的权利要求11至16中任一项所述的Anti CD19 CAR-NK细胞，和/或权利要求17或18所制备的Anti CD19 CAR-NK细胞。
  20. 根据权利要求19所述的药物组合物，其特征在于，所述药物组合物用于治疗或预防肿瘤时，所述的Anti CD19 CAR-NK细胞与所述靶细胞的效靶比为(0.5～20)：1。
  21. 根据权利要求19所述的药物组合物，其特征在于，其还包括药学可接受的辅料。
  22. 根据权利要求19所述的药物组合物，其特征在于，所述药物组合物的剂型为水剂。
  23. 根据权利要求19至22中任一项所述的药物组合物，其特征在于，所述肿瘤为高表达CD19的肿瘤；优选地，所述高表达CD19的肿瘤为淋巴瘤；进一步优选地，所述淋巴瘤为Jurkat、K562、Raji或THP1细胞；更优选地，所述淋巴瘤为Raji细胞。
  24. 根据权利要求11至16中任一项所述的Anti CD19 CAR-NK细胞，和/或权利要求17或18所制备的Anti CD19 CAR-NK细胞在制备治疗和/或预防高表达CD19的肿瘤及相关疾病的药物中的应用。
  25. 根据权利要求24所述的应用，其特征在于，所述高表达CD19的肿瘤为淋巴瘤；优选地，所述淋巴瘤为Jurkat、K562、Raji或THP1细胞；更优选地，所述淋巴瘤为Raji细胞。
  26. 根据权利要求11至16中任一项所述的Anti CD19 CAR-NK细胞，和/或权利要求17或18所制备的Anti CD19 CAR-NK细胞用于治疗和/或预防高表达CD19的肿瘤及相关疾病的方法，所述方法包括将含有有效量的Anti CD19 CAR-NK细胞的药物摄入患者体内。
  27. 根据权利要求26所述的方法，其特征在于，所述CD19抗体或其抗原结合片段或Anti CD19 CAR-NK细胞的摄入方法为瘤内注射、静脉注射、胸腔内注射或局部介入。
  28. 根据权利要求26所述的方法，其特征在于，所述高表达CD19的肿瘤为淋巴瘤；优选地，所述淋巴瘤为Jurkat、K562、Raji或THP1细胞，更优选地，所述淋巴瘤为Raji细胞。
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