WO2019154391A1 - 嵌合抗原受体、表达该嵌合抗原受体的nkg2d car-nk细胞及其制备方法和应用 - Google Patents

嵌合抗原受体、表达该嵌合抗原受体的nkg2d car-nk细胞及其制备方法和应用 Download PDF

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WO2019154391A1
WO2019154391A1 PCT/CN2019/074683 CN2019074683W WO2019154391A1 WO 2019154391 A1 WO2019154391 A1 WO 2019154391A1 CN 2019074683 W CN2019074683 W CN 2019074683W WO 2019154391 A1 WO2019154391 A1 WO 2019154391A1
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nkg2d
cells
car
cell
chimeric antigen
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French (fr)
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李华顺
韩昆昆
韩俊峰
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李华顺
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Definitions

  • the invention relates to the field of biomedicine, in particular to a chimeric antigen receptor, NKG2D CAR-NK cells expressing the chimeric antigen receptor, a preparation method and application thereof.
  • Immune cells modified by chimeric antigen receptor (CAR) 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 tumor surface specific molecules and the latter is used to initiate an immune cell response after recognizing a tumor surface molecule, thereby exerting a cytotoxic effect. It mainly uses T-cells as a carrier, but when CAR-T cells treat tumors, there will be a sharp increase in cytokines such as IL-6, resulting in cytokine storm phenomenon, and there are problems such as targeted/off-target toxicity and neurotoxicity. Endanger the lives of patients.
  • T cells must be isolated from the body (this process is time consuming and costly). Moreover, since 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. In addition, in the face of the high cost of CAR-T, 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.
  • NKG2D is an activated receptor for NK cells that recognizes MHC class I molecules and plays an important role in innate immunity. NKG2D is involved in the recognition of virus-infected cells and the killing of tumor cells by NK. NKG2D belongs to the C-type lectin-like receptor family, and the receptor encoded by this gene is present in the NKG2 (naturalkiller group 2) complex. The NKG2 gene complex is located on human chromosome 12. NKG2D is a type II transmembrane protein, and NKG2D requires signal transduction via a charged residue in the transmembrane region in combination with some adaptor proteins. Human NKG2D can bind to 10 kDa of DNAX activating protein (DAP10).
  • DAP10 DNAX activating protein
  • DAP10 contains a YXXM motif (Tyr-X-X-Meth) in the cytoplasm to recruit phosphatidylinositol trihydroxykinase (PI3K) and growth factor receptor binding protein-2. All NK cells, most NKT cells, and macrophages express NKG2D. In addition, NKG2D is also present on the surface of CD8+ T cells. Under normal conditions, human and mouse CD4+ T cells do not express NKG2D. However, in patients, CD4+ T cells expressing NKG2D are mainly concentrated in tumor tissues. NKG2D binds to a number of different ligands belonging to the major histocompatibility complex class I (MHC-I)-related proteins.
  • MHC-I major histocompatibility complex class I
  • MIC-A and MIC-B Another family of human NKG2D ligands is MIC-A and MIC-B. Both MIC-A and MIC-B are polymorphic. Currently, MIC-A has 61 alleles and MIC-B has 30 alleles. The ligand of the NKG2D molecule is not expressed or expressed in normal cells, but when the cells are infected or become cancerous, the expression levels of these ligands are greatly increased.
  • CAR-NK cells for the treatment of NKG2D tumors and related diseases are not present. They are only used to treat CAR-T cells expressing NKG2D tumors and related diseases, but they have large side effects and high cost. NKG2D CAR-NK cells are bound to advance the field of cancer treatment.
  • the present invention provides a chimeric antigen receptor, NKG2D CAR-NK cells expressing the chimeric antigen receptor, and a preparation method and application thereof, which are capable of specifically recognizing and killing tumors, and are more efficient Tumor killing activity.
  • One aspect of the invention provides a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and a costimulatory signaling domain, the antigen binding domain capable of specifically binding to a tumor
  • the specific antigen NKG2D ligand activates NK cells via a transmembrane domain and a costimulatory signaling domain.
  • the antigen binding domain comprises NKG2D that specifically binds to the NKG2D ligand.
  • the NKG2D ligand is selected from one or more of a major histocompatibility complex class I (MHC-I) molecule, MIC-A, and MIC-B.
  • MHC-I major histocompatibility complex class I
  • the transmembrane domain is selected from one or more of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;
  • the transmembrane domain is a CD8 transmembrane domain;
  • the costimulatory signaling region comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, One or more of CD28, CD134, CD137, ICOS, CD154, 4-1BB, and OX40; preferably, the costimulatory signaling region comprises a 4-1BB and a CD3 sputum intracellular domain.
  • a costimulatory molecule selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, One or more of CD28, CD134, CD137, ICOS, CD154, 4-1BB, and OX40; preferably, the costimulatory signaling region comprises a 4-1BB and a CD3 sputum intracellular domain.
  • the chimeric antigen receptor structure NKG2D-CD8 ⁇ hinge-CD8 TM -4-1BB- CD3 ⁇ fusion protein is capable of specifically recognizing NKG2D ligand.
  • the homologous sequence has about 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 Above, 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.
  • Another aspect of the invention provides a nucleotide sequence which expresses a chimeric antigen receptor as described above.
  • nucleotide sequence is as set forth in SEQ ID NO: 2 or its degenerate sequence and the like.
  • the degenerate sequence has about 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 Above, 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.
  • the invention also provides a NKG2D CAR-NK cell capable of expressing a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain and a costimulatory signaling region
  • the antigen binding domain is capable of specifically binding to a tumor-specific antigen NKG2D ligand and activating the NK cell through a transmembrane domain and a costimulatory signaling region.
  • the antigen binding domain comprises NKG2D that specifically binds to the NKG2D ligand.
  • the NKG2D ligand is selected from the group consisting of major histocompatibility complex class I (MHC-I) molecules, MIC-A and MIC-B.
  • MHC-I major histocompatibility complex class I
  • the transmembrane domain is selected from one or more of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD134, CD137, ICOS, and CD154;
  • the transmembrane domain is a CD8 transmembrane domain;
  • the costimulatory signaling region comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, One or more of CD28, CD134, CD137, ICOS, CD154, 4-1BB, and OX40; preferably, the costimulatory signaling region comprises a 4-1BB and a CD3 sputum intracellular domain.
  • a costimulatory molecule selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, One or more of CD28, CD134, CD137, ICOS, CD154, 4-1BB, and OX40; preferably, the costimulatory signaling region comprises a 4-1BB and a CD3 sputum intracellular domain.
  • the chimeric antigen receptor structure NKG2D-CD8 ⁇ hinge-CD8 TM -4-1BB- CD3 ⁇ fusion protein is illustrated.
  • amino acid sequence the NKG2D-CD8 TM ⁇ hinge-CD8-4-1BB- CD3 ⁇ fusion protein as SEQ ID NO: 1 or a homologous sequence shown in FIG.
  • the homologous sequence has about 95% or more homology with the original sequence, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or Above, 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.
  • the homologous sequence has about 95% or more homology with the original sequence, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or Above, 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.
  • the NKG2D CAR-NK cells are capable of effectively killing and/or killing lung cancer cells, breast cancer cells, colon cancer cells, breast cancer cells, and/or lung cancer cells, and the like.
  • the invention also provides a preparation method of the above NKG2D CAR-NK cells, which comprises the following steps:
  • NK-92 cells were infected with the lentivirus obtained in the step (2) to obtain NKG2DCAR-NK cells.
  • step (1) the nucleotide sequence encoding the NKG2D-CD8TM ⁇ hinge-CD8-4-1BB-CD3 ⁇ fusion protein is synthesized and amplified.
  • the present invention also provides the use of the above NKG2D CAR-NK cells for the preparation of a medicament for treating and/or preventing cancer.
  • the cancer is a tumor and related disease that highly express NKG2D ligand.
  • the cancer is breast cancer, colon cancer or lung cancer or the like.
  • the invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the above NKG2D CAR-NK cells, and optionally a pharmaceutically acceptable excipient.
  • the effective target ratio of NKG2D CAR-NK cells to tumor cells is (0.5-1):1.
  • the pharmaceutical composition is in the form of a liquid.
  • the present invention also provides a method for treating and/or preventing cancer of the above NKG2D CAR-NK cells.
  • the method comprises ingesting a therapeutically effective amount of a pharmaceutical composition comprising NKG2D CAR-NK cells into a patient.
  • the cancer is a tumor and related disease that highly express NKG2D ligand.
  • the cancer is lung cancer, breast cancer or colon cancer.
  • the NKG2D CAR-NK cells are administered in an amount of (1 to 10) ⁇ 10 6 /time, preferably (2.5 to 5) ⁇ 10 6 /time.
  • the method of ingesting the NKG2D CAR-NK cells is intratumoral injection, intravenous injection, intrathoracic injection or local intervention.
  • the method of ingesting the NKG2D CAR-NK cells is intravenous.
  • the chimeric antigen receptor of the present invention is capable of specifically binding to a tumor-specific antigen NKG2D ligand and activating the NK cell through a transmembrane domain and a costimulatory signaling region.
  • the NKG2D CAR-NK cells were constructed by using NKG2D (NKG2D receptor) for CAR-NK cells.
  • the chimeric antigen receptor and NKG2D CAR-NK cells capable of expressing the chimeric antigen receptor use NKG2D ligand as a target antigen, and can specifically kill tumor cells, which can be used as a therapeutic drug for tumor diseases, and is used for NKG2D matching.
  • the treatment of high-expression tumors provides a new method for tumor prevention and treatment; and by modifying NK92 cells, it has greatly increased its targeting and increased anti-tumor target NKG2D.
  • the anti-tumor performance is greatly improved; that is to say, the NKG2D CAR-NK cells are constructed for the first time, and the NK92 cells are modified, and the targeting is not only increased after loading the CAR.
  • the safety is greatly improved, the side effects are small, and the cost is low.
  • the anti-tumor performance of the cells is greatly improved by the cooperation of CAR and NKG2D, and the anti-tumor performance of the cells is obviously superior by experiments.
  • NK92 cells in addition, the broad spectrum of treatment and prevention of tumors using CAR-NK cells is further expanded.
  • FIG. 1 is a schematic view showing the structure of a lentiviral plasmid vector PRRLSIN-NKG2D according to an embodiment of the present invention.
  • FIG. 2a-2b are graphs showing the results of NKG2D CAR-NK flow detection of CAR cell positive rate according to an embodiment of the present invention, wherein FIG. 2a is a control group; FIG. 2b is an experimental group.
  • FIG. 3a-3e are diagrams showing the results of detecting the expression of NKG2D ligand MIC-A and MIC-B molecules in different tumor cells by a conventional flow method according to an embodiment of the present invention, wherein FIG. 3a is an experiment of lung cancer cell A549. Results; Fig. 3b is the experimental result of lung cancer cell H1299; Fig. 3c is the experimental result of breast cancer cell MCF-7; Fig. 3d is the experimental result of breast cancer cell MDMB-231; Fig. 3e is the experimental result of colon cancer cell SW480.
  • FIG. 4a-4e are diagrams showing experimental results of killing different tumor cells by NKG2D CAR-NK cells according to an embodiment of the present invention, wherein FIG. 4a is an experimental result of lung cancer cell A549; FIG. 4b is an experimental result of lung cancer cell H1299; 4c is the experimental result of breast cancer cell MCF-7; Fig. 4d is the experimental result of breast cancer cell MDMB-231; Fig. 4e is the experimental result of colon cancer cell SW480.
  • FIG. 5a-5b are graphs showing tumor growth curves of NKG2D CAR-NK cells for treating NPG mice according to an embodiment of the present invention; wherein, FIG. 5a is a mouse tumor administered at a dose of 2.5 ⁇ 10 6 growth curves, FIG. 5b tumor growth curves for a dose of 5 ⁇ 10 6 th mice.
  • Figure 6 is a graph showing the anti-tumor results of NKG2D CAR-NK cells used in the treatment of NPG mice according to an embodiment of the present invention.
  • Figure 7 is a graph showing the results of NKG2D CAR-NK cells for treating tumor weight after treatment of NPG mice according to an embodiment of the present invention.
  • Figure 8 is a graph showing the results of NKG2D CAR-NK cells for treating tumor weight after treatment of NPG mice according to an embodiment of the present invention.
  • PBS is a PBS administration group
  • NK92-1 is a dose group of 2.5 ⁇ 10 6
  • NK92-1 is a dose group of 5 ⁇ 10 6
  • NKG2D CAR-NK- 1 is a 2.5 ⁇ 10 6 administration group
  • NKG2D CAR-NK-2 is a 5 ⁇ 10 6 administration group
  • * indicates P (significant difference) ⁇ 0.05.
  • NKG2D-CD8 ⁇ hinge-CD8 TM -4-1BB-CD3 ⁇ sequence of nucleotides encoding a fusion protein of the present invention is any DNA sequence capable of encoding any of the fusion protein, preferably the sequence is SEQ ID NO: 2 or its complementary sequence.
  • encoding NKG2D-CD8 ⁇ hinge-CD8 TM -4-1BB- CD3 ⁇ sequence of nucleotides may be a fusion protein with the SEQ ID NO under stringent conditions: hybridization of nucleotide sequence And a polynucleotide encoding 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.
  • 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.
  • promoter is defined as required to initiate the specific transcription of a polynucleotide sequence, which is recognized by the cell's synthetic machinery or directs the DNA sequence of the synthesizing machine.
  • the term “specifically binds” refers to the recognition of a specific antigen but does not substantially recognize or bind other molecules in the sample.
  • 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, colorectal cancer, liver cancer, 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.
  • NKG2D CAR-NK and NKG2D-CARNK have the same meaning
  • NKG2D CAR-NK and NKG2D-CARNK have the same meaning, and both represent CAR-NK cells against NKG2D molecules
  • NK- 92, NK92 NK92 cells have indicated
  • CD8 TM indicates the transmembrane domain.
  • 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), lung cancer cell A549, breast cancer MDMB-231 cells, colon cancer cell SW480, breast cancer MCF-7 cells, HCC1187 cells and lung cancer cells H1299 were purchased from the Shanghai Institute of Biotechnology, Chinese Academy of Sciences.
  • NPG mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., SPF grade, female, 5-6 weeks, body weight: 18-20 g, ⁇ 20%.
  • NKG2D-CD8 TM -4-1BB-CD3 ⁇ fusion gene sequence (which amino acid sequence is shown in SEQ ID NO: 1, the gene sequence such as SEQ ID NO: 2 below), by digestion, the NKG2D-CD8 TM -
  • the 4-1BB-CD3 ⁇ fusion gene sequence was transformed into a PRRSLIN vector, 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 cloned by restriction enzyme digestion to obtain PRRLSIN-NKG2D lentiviral transfection vector (as shown in Fig. 1).
  • 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 PRRLSIN-NKG2D (target plasmid); 39.5 ⁇ g pMD2.G (VSV-G envelop); 73 ⁇ g pCMVR8.74 (gag, pol, tat, rev); 625 ⁇ l 2M Calcium ion solution. Total volume of solution A: 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 SW28 centrifuge 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 was transferred to a sterile centrifuge tube and a layer of 20% sucrose (per 8 ml of supernatant plus 1 ml of sucrose) was carefully placed on the bottom of the tube.
  • 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 removed, 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 (llow virus vector). After cooling on ice, store at -80 °C.
  • Fig. 2a shows a control group, which is an NK92 cell which has not been transformed with a CAR molecule
  • Fig. 2b shows an experimental group which is a NK-92 cell which has a CAR molecule.
  • the ordinate SSC-H represents the flow side scatter value
  • the FSC-H represents the flow forward scatter value.
  • APC-H indicates the fluorescence intensity after staining with the antibody, and the stronger the fluorescence intensity, the greater the positive ratio of NKG2D CAR NK-92 cells compared with the control group.
  • 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 98.97%.
  • NKG2D CAR NK-92 detection To determine tumor cells for NKG2D CAR NK-92 detection, expression of NKG2D ligand MIC-A and MIC-B molecules was detected by different flow cytometry methods using conventional flow cytometry. Among them, the selected tumor cells were lung cancer cell A549, breast cancer MDMB-231 cell, colon cancer cell SW480, breast cancer MCF-7 cell and lung cancer cell H1299, and the experimental results are shown in Fig. 3a-e.
  • Fig. 3a is the experimental result of lung cancer cell A549; Fig. 3b is the experimental result of lung cancer cell H1299; Fig. 3c is the experimental result of breast cancer cell MCF-7; Fig. 3d is the experimental result of breast cancer cell MDMB-231; Fig. 3e is the colon Experimental results of cancer cell SW480.
  • lung cancer cell A549, breast cancer MDMB-231 cells and colon cancer cell SW480 are under-represented with expression rates of 0.19%, 1% and 0.12%, respectively; breast cancer MCF-7 cells are moderately expressed.
  • the positive rate was 61.99%; the lung cancer cell H1299 was highly expressed, and the expression rate was 85.16%.
  • NKG2D-CARNK92 was plated in 24 wells (20,000 and 40,000 cells, respectively) according to a ratio of 0.5:1 and 1:1, and the total system was 1 ml, and incubated at 37 degrees for 4 hours;
  • the cell killing rate was calculated by reading the plate at 450 nm.
  • Figures 4a-4e the ordinate indicates the killing efficiency and the abscissa indicates the effective target ratio.
  • Figure 4a is the experimental result of lung cancer cell A549;
  • Figure 4b is the experimental result of lung cancer cell H1299;
  • Figure 4c is the experimental result of breast cancer cell MCF-7;
  • Figure 4d is the experimental result of breast cancer cell MDMB-231;
  • Figure 4e is the experimental result of breast cancer cell MDMB-231;
  • NKG2D CAR-NK has better killing effect than normal NK-92 cells at a target ratio of 0.5:1 and 1:1.
  • the killing effect can reach 80% under the condition of 1:1 effective target ratio.
  • NKG2D CAR-NK has better killing effect than common NK-92, and the killing effect can reach 56% under 1:1 target.
  • NKG2D CAR-NK has better killing effect than ordinary NK-92 cells. That is, the killing effect of NKG2D CAR-NK is worse than that of common NK-92 cells, but the killing effect on cells with low expression of NKG2D ligand is not as effective as that of tumor cells with high expression of the ligand.
  • HCC1187 cells were continuously cultured, and cells cultured in the logarithmic growth phase were inoculated subcutaneously into the back of NPG mice by subcutaneous injection. Each spot was inoculated with about 2 x 10 6 cells, and the inoculation volume was about 100 ⁇ l.
  • Specific drug delivery design When the tumor volume reaches 40-50mm 3 , the cells are grouped, resuspended in PBS to the concentration of the drug design, and administered according to the set group.
  • the drug-administered group includes NKG2D.
  • the CAR-NK cell group, the PBS control group, and the NK-92 group were administered at a dose of 2.5 ⁇ 10 6 /times and 5 ⁇ 10 6 /times, respectively, and two sets of parallel experiments were performed.
  • the appearance and behavior of the animal NPG mice were observed after administration, and the body weight was measured, and the tumor growth inhibition rate was calculated by the following formula.
  • V (length ⁇ width 2 ) / 2
  • Tumor volume ratio (T/C,%) (Td-T0)/(Cd-C0) ⁇ 100% in the treatment group/control group
  • Td and Cd respectively indicate the tumor volume at the last measurement of the administration group and the control group
  • T0 and C0 indicate the volume of the tumor when the administration group and the control group were grouped, respectively.
  • TGI tumor growth inhibition rate
  • a indicates Mean ⁇ SD
  • b indicates the P value compared with Solution
  • V0 indicates the tumor volume before administration
  • Vt indicates the tumor volume after administration.
  • the NPG mice were euthanized, and the tumor tissues of each group of mice were taken, photographed and weighed, the weight of the tumor and the tumor weight as a percentage of body weight (% tumor weight), and the results are shown in Figure 7-8.
  • the NK92-1 group had the largest tumor weight and tumor weight percentage, PBS group, NK92-1, NKG2D CAR-NK-1 group and NKG2D CAR-NK-2 group.
  • the tumor weight and the tumor weight percentage were the largest in descending order, and the results were consistent with the results of the tumor volume measured 26 days after administration.
  • the above experimental results show that the killing and antitumor effect of the NKG2D CAR-NK cells of the present invention is significantly better than that of the NK92 group, and the NKG2D CAR-NK cells constructed by the present invention significantly enhance the targeting by increasing the binding of CAR and NKG2D receptors. , thereby greatly improving its killing and anti-tumor effect, and can better resist tumors.

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Abstract

本发明提供一种嵌合抗原受体、表达该嵌合抗原受体的NKG2D CAR-NK细胞及其制备方法和应用，所述嵌合抗原受体，包括抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原NKG2D配体，并通过跨膜结构域和共刺激信号传导区激活NK细胞。本发明提供的CAR-NK细胞，其以NKG2D配体为靶抗原，利用NKG2D CAR-NK细胞特异性地杀伤肿瘤细胞。其可作为肿瘤类疾病的治疗药物，用于NKG2D分子的配体高表达的肿瘤的治疗，为肿瘤的预防和治疗提供了新的方法。

Description

嵌合抗原受体、表达该嵌合抗原受体的NKG2D 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，具体涉及一种嵌合抗原受体、表达该嵌合抗原受体的NKG2D 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，但CAR-T细胞治疗肿瘤时会出现IL-6等细胞因子急剧升高，产生细胞因子风暴现象，存在靶向/脱靶毒性和神经毒性等问题，严重时会危及患者生命。并且，T细胞必须分离出体外(这一过程耗时长且费用高)。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。
NKG2D是NK细胞的活化型受体，可识别MHC-I类分子，在天然免疫中发挥着重要的作用。NKG2D参与病毒感染细胞的识别及NK对肿瘤细胞的杀伤。NKG2D属于C型凝集素样受体家族，由于这个基因编码的受体存在于NKG2(naturalkillergroup2)复合体中。NKG2基因复合体位于人的12号染色体上。NKG2D是II型跨膜蛋白，NKG2D需要通过跨膜区域的带电残基结合一些转接蛋白(adaptorproteins)完成信号转导。人NKG2D都能结合10kDa的DNAX活化蛋白(DAP10)。DAP10在胞浆内含有YXXM基序(Tyr-X-X-Meth)能够招募磷脂酰肌醇三羟基激酶(PI3K)和生长因子受体结合蛋白-2。所有的NK细胞、大多数NKT细胞、巨噬细胞都表达NKG2D。另外，NKG2D也存在于CD8+T细胞表面。在正常条件下，人和小鼠CD4+T细胞不表达NKG2D。但是，在患者体内，表达NKG2D的CD4+T细胞主要聚集在肿瘤组织中。NKG2D可与许多不同的配体结合，这些配体属于主要组织相容性复合体I类(MHC-I)相关蛋白。人的NKG2D配体的另一家族是MIC-A和MIC-B。MIC-A和MIC-B都具有多态性。目前，MIC-A有61个等位基因，MIC-B有30个等位基因。NKG2D分子的配体在正常细胞中不表达或者表达量非常少，但是当细胞受到感染或者发生癌变时，这些配体的表达量会大幅度上升。
目前用于治疗表达NKG2D肿瘤及相关疾病的CAR-NK细胞尚不存在，只有用于治疗表达NKG2D肿瘤及相关疾病的CAR-T细胞，但是其毒副作用大，成本高，如果能够开发研究一种NKG2D CAR-NK细胞，势必会推动肿瘤治疗领域的进展。
发明内容
有鉴于此，本发明提供了一种嵌合抗原受体、表达该嵌合抗原受体的NKG2D CAR-NK细胞及其制备方法和应用，该细胞能够特异性识别和杀伤肿瘤，具有更高效的肿瘤杀伤活性。
本发明的一方面提供一种嵌合抗原受体，所述嵌合抗原受体包含抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原NKG2D配体，并通过跨膜结构域和共刺激信号传导区激活NK细胞。
示例性地，所述的抗原结合结构域包括NKG2D，所述NKG2D与所述NKG2D配体特异性结合。
示例性地，所述NKG2D配体选自主要组织相容性复合体I类(MHC-I)分子、MIC-A和MIC-B中的一种或多种。
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；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，所述嵌合抗原受体的结构为NKG2D-CD8αhinge-CD8 TM-4-1BB-CD3ζ的融合蛋白，该融合蛋白能够特异性识别NKG2D配体。
在本发明的一个具体实施方式中，所述NKG2D-CD8αhinge-CD8 TM-4-1BB-CD3ζ融合蛋白氨基酸序列如SEQ ID NO：1所示或其同源序列等。
示例性地，所述同源序列与原序列的同源性约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。
本发明的另一方面提供一种表达上述嵌合抗原受体的核苷酸序列。
示例性地，所述核苷酸序列如SEQ ID NO：2所示或其简并序列等。
示例性地，所述简并序列与原序列的同源性约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。
本发明还提供一种NKG2D CAR-NK细胞，所述NKG2D CAR-NK细胞能够表达嵌合抗原受体，所述嵌合抗原受体包括抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原NKG2D配体，并通过跨膜结构域和共刺激信号传导区激活该NK细胞。
示例性地，所述的抗原结合结构域包括NKG2D，所述NKG2D与所述NKG2D配体特异性结合。
示例性地，所述NKG2D配体选自主要组织相容性复合体I类(MHC-I)分子、MIC-A和MIC-B。
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；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，所述嵌合抗原受体的结构为NKG2D-CD8αhinge-CD8 TM-4-1BB-CD3ζ的融合蛋白。
示例性地，所述NKG2D-CD8 TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的氨基酸的序列如SEQ ID NO：1所示或其同源序列。
示例性地，所述同源序列与原序列的同源性约95％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。
在本发明的一个优选实施方式中，所述NKG2D-CD8 TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的编码核苷酸序列如SEQ ID NO：2所示或其同源序列。
示例性地，所述同源序列与原序列的同源性约95％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。
在本发明的一个具体实施方式中，所述NKG2D CAR-NK细胞能够有效地杀伤和/或杀死肺癌细胞、乳腺癌细胞、结肠癌细胞、乳腺癌细胞和/或肺癌细胞等。
本发明还提供了上述NKG2D 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嵌合抗原受体的核苷酸序列，再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293T细胞，包装和制备慢病毒；和
(3)利用步骤(2)得到的慢病毒感染NK-92细胞，得到NKG2DCAR-NK细胞。
示例性地，在步骤(1)中，合成和扩增编码所述NKG2D-CD8TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的核苷酸序列。
本发明还提供上述NKG2D CAR-NK细胞在制备治疗和/或预防癌症的药物中的应用。
示例性地，所述癌症为高表达NKG2D配体的肿瘤及相关疾病。
示例性地，所述癌症为乳腺癌、结肠癌或肺癌等。
本发明还提供了一种药物组合物，其包括有效治疗量的上述NKG2D CAR-NK细胞，以及任选地，药学上可接受的辅料。
示例性地，所述药物组合物用于治疗或预防肿瘤时，NKG2D CAR-NK细胞与肿瘤细胞的效靶比为(0.5～1)：1。
示例性地，所述药物组合物的剂型为水剂。
本发明还提供一种上述NKG2D CAR-NK细胞用于治疗和/或预防癌症的方法。
示例性地，所述方法包括将有效治疗量的含有NKG2D CAR-NK细胞的药物组合物摄入患者体内。
示例性地，所述的癌症为高表达NKG2D配体的肿瘤及相关疾病。
示例性地，所述癌症为肺癌、乳腺癌或结肠癌。
示例性地，所述NKG2D CAR-NK细胞的给药量为(1～10)×10 6个/次，优选为(2.5～5)×10 6个/次。
示例性地，所述NKG2D 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NKG2D CAR-NK细胞的摄入方法为静脉注射。
本发明的嵌合抗原受体能够特异性结合肿瘤特异性抗原NKG2D配体，并通过跨膜结构域和共刺激信号传导区激活该NK细胞。该NKG2D CAR-NK细胞是通过将NKG2D(NKG2D受体)用于CAR-NK细胞所构建。嵌合抗原受体和能够表达该嵌合抗原受体的NKG2D CAR-NK细胞以NKG2D配体为靶抗原，能够特异性地杀伤肿瘤细胞，其可作为肿瘤类疾病的治疗药物，用于NKG2D配体高表达的肿瘤的治疗，为肿瘤的预防和治疗提供了新的方法；并且通过对NK92细胞进行改造，装上CAR后，又大大增加了它的靶向性，并增加抗肿瘤靶点NKG2D，在提高靶向性地同时，大大地提高其抗肿瘤性能；也就是说本发明首次构建了NKG2D CAR-NK细胞，其通过对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，大大提高了其安全性，毒副作用小，成本低，通过CAR和NKG2D的配合大大提高了该细胞的抗肿瘤性能，并通过实验表明其抗肿瘤性能明显优于NK92细胞；此外，进一步扩展了使用CAR-NK细胞治疗和预防肿瘤的广谱性。
附图说明
图1所示为本发明实施例提供的慢病毒质粒载体PRRLSIN-NKG2D的结构示意图。
图2a-2b所示为本发明实施例提供的NKG2D CAR-NK流式检测CAR细胞阳性率的结果图，其中，图2a为对照组；图2b为实验组。
图3a-3e所示为本发明实施例提供的利用常规流式方法检测不同肿瘤细胞中NKG2D配体MIC-A和MIC-B分子的表达的结果图，其中，图3a为肺癌细胞A549的实验结果；图3b为肺癌细胞H1299的实验结果；图3c为乳腺癌细胞MCF-7的实验结果；图3d为乳腺癌细胞MDMB-231的实验结果；图3e为结肠癌细胞SW480的实验结果。
图4a-4e所示为本发明实施例提供的NKG2D CAR-NK细胞杀伤不同肿瘤细胞的实验结果图，其中，图4a为肺癌细胞A549的实验结果；图4b为肺癌细胞H1299的实验结果；图4c为乳腺癌细胞MCF-7的实验结果；图4d为乳腺癌细胞MDMB-231的实验结果；图4e为结肠癌细胞SW480的实验结果。
图5a-5b所示为本发明实施例提供的NKG2D CAR-NK细胞的用于治疗NPG小鼠后的肿瘤生长曲线；其中，图5a为给药剂量为2.5×10 6个的小鼠肿瘤的生长曲线，图5b为给药剂量为5×10 6个的小鼠肿瘤的生长曲线。
图6所示为本发明实施例提供的NKG2D CAR-NK细胞用于治疗NPG小鼠后的抑瘤结果。
图7所示为本发明实施例提供的NKG2D CAR-NK细胞用于治疗NPG小鼠后的肿瘤重量的结果。
图8所示为本发明实施例提供的NKG2D CAR-NK细胞用于治疗NPG小鼠后的肿瘤重量的结果。
上述图5a-图8中，PBS为PBS给药组，NK92-1为剂量为2.5×10 6的给药组，NK92-1为剂量为5×10 6的给药组，NKG2D CAR-NK-1为2.5×10 6的给药组，NKG2D CAR-NK-2为5×10 6的给药组，*表示P(显著性差异)﹤0.05。
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NKG2D-CD8αhinge-CD8 TM-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：2或其互补序列。另一方面，本发明所述的编码NKG2D-CD8αhinge-CD8 TM-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：2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 Biol 215:403,1990)。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。
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“启动子”被定义为开始多核苷酸序列的特异性转录需要的，由细胞的合成机器识别或引导合成机器的DNA序列。
术语“特异性结合”指识别特异性抗原但基本上不识别或结合样本中的其他分子。
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结肠直肠癌、肝癌、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；NKG2D CAR-NK、NKG2D-CARNK表示的含义相同；NKG2D CAR-NK与NKG2D-CARNK表示相同的含义，均表示抗NKG2D分子的CAR-NK细胞；NK-92、NK92均表示NK92细胞；CD8 TM表示跨膜结构域。
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 PCTCN2019074683-appb-000001
CRL-2407)，肺癌细胞A549、乳腺癌MDMB-231细胞、结肠癌细胞SW480、乳腺癌MCF-7细胞、HCC1187细胞和肺癌细胞H1299均购自中国科学院上海生科院细胞资源中心。
NPG小鼠购自北京维通利华实验动物技术有限公司，SPF级，雌性，5-6周，体重：18-20g，±20％。
实施例1慢病毒载体的制备
基因合成NKG2D-CD8 TM-4-1BB-CD3ζ融合基因序列(其氨基酸序列如SEQ ID NO：1所示，基因序列如SEQ ID NO：2所示)，通过酶切，将NKG2D-CD8 TM-4-1BB-CD3ζ融合基因序列转化连接到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-NKG2D慢病毒转染载体(如图1所示)。
实施例2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-NKG2D(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A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 SW28 centrifuge 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(慢病毒载体)。冰上冷却后，置于-80℃保存。
实施例3 NKG2D CAR-NK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：细胞培养基＝1:5比例添加慢病毒(实施例2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NKG2D 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NKG2D CAR NK-92细胞阳性率，流式检测结果如图2a和图2b所示。图2a为对照组，为未转CAR分子的NK92细胞；图2b为实验组，为转CAR分子的NK-92细胞。图2a和图2b中，纵坐标SSC-H表示流式侧向散射数值，FSC-H表示流式前向散射数值，这两个参数主要用于圈定出两组用于分析的活细胞，如图上椭圆圈定处；APC-H表示用抗体染色后荧光强度，该荧光强度越强，表示与对照组相比NKG2D CAR NK-92细胞阳性比率越大。从图2a和图2b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98.97％。
实施例4 NKG2D CAR-NK细胞对体外肿瘤杀伤效果的评估
为了确定用于NKG2D CAR NK-92检测的肿瘤细胞，利用常规流式分析方法对不同肿瘤细胞进行NKG2D配体MIC-A和MIC-B分子的表达检测。其中，选取的肿瘤细胞分别为肺癌细胞A549、乳腺癌MDMB-231细胞、结肠癌细胞SW480、乳腺癌MCF-7细胞和肺癌细胞H1299，其实验结果如图3a-e所示。
图3a为肺癌细胞A549的实验结果；图3b为肺癌细胞H1299的实验结果；图3c为乳腺癌细胞MCF-7的实验结果；图3d为乳腺癌细胞MDMB-231的实验结果；图3e为结肠癌细胞SW480的实验结果。
从图3a-3e中可以看出，肺癌细胞A549、乳腺癌MDMB-231细胞和结肠癌细胞SW480低表达，表达率分别为0.19％、1％和0.12％；乳腺癌MCF-7细胞中度表达，阳性率61.99％；肺癌细胞H1299高表达，表达率85.16％。
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NKG2D CAR-NK细胞对上述不同肿瘤细胞系的杀伤效果。实验操作方法如下：
1)提前一天将上述五种不同的实体瘤细胞(40000个)均匀的铺于24孔中，待细胞贴壁；
2)第二天按照0.5:1和1:1的效靶比，将NKG2D-CARNK92铺在24孔中(分别为20000个和40000个细胞)，总体系为1ml，37度孵育4小时；
3)作用4小时后，将培养基舍去，加PBS轻轻地漂洗两次，加入CCK8试剂200ul，37℃作用1-4小时；
4)450nm处读板计算细胞杀伤率。
NKG2D CAR-NK细胞体外肿瘤杀伤效果评估的实验结果如下表1及图4a-4e所示。
表1 NKG2D CAR-NK细胞体外肿瘤杀伤效果
Figure PCTCN2019074683-appb-000002
图4a-4e中，纵坐标表示杀伤效率，横坐标表示效靶比。其中图4a为肺癌细胞A549的实验结果；图4b为肺癌细胞H1299的实验结果；图4c为乳腺癌细胞MCF-7的实验结果；图4d为乳腺癌细胞MDMB-231的实验结果；图4e为结肠癌细胞SW480的实验结果。
如表1及图4a-4e所示，针对高表达NKG2D配体的H1299细胞，在效靶比为0.5:1和1:1的条件下，NKG2D CAR-NK杀伤效果优于普通NK-92细胞，在1:1效靶比条件下杀伤效果可达到80％。针对中度表达NKG2D配体的MCF-7细胞，NKG2D CAR-NK杀伤效果优于普通NK-92，1:1效靶比条件下杀伤效果可达到56％。针对其它低表达NKG2D配体的肿瘤细胞，NKG2D CAR-NK杀伤效果也优于普通NK-92细胞。即NKG2D CAR-NK的杀伤效果均优于普通的NK-92细胞，但对于低表达NKG2D配体的细胞的杀伤效果不如高表达该配体的肿瘤细胞效果明显。
实施例5 NKG2D CAR-NK细胞对体内肿瘤抑制效果的评估
将HCC1187细胞连续培养，培养10代之前，收集处于对数生长期的细胞通过皮下注射接种于NPG小鼠背部皮下。每个点接种约2×10 6个细胞，接种体积为100μl左右。
1.具体给药设计：当肿瘤体积达到40-50mm 3时进行分组，使用PBS重悬至给药设计的浓度，按设定的组别以相应的给药方式给药，给药组包括NKG2D CAR-NK细胞组、PBS对照组和NK-92组，分别以2.5×10 6个/次和5×10 6个/次的剂量给药，做两组平行试验。
具体给药组如下述表2所示：
表2
Figure PCTCN2019074683-appb-000003
2.小鼠肿瘤生长曲线和抑制率的计算
给药后观察动物NPG小鼠外观和行为，并测量体重，按下式计算肿瘤生长抑制率。
肿瘤体积(V，mm 3)计算公式：V＝(长×宽 2)/2
治疗组/对照组肿瘤体积比(T/C，％)＝(Td-T0)/(Cd-C0)×100％，Td、Cd分别表示给药组与对照组最后一次测量时肿瘤的体积，T0、C0分别表示给药组与对照组分组时肿瘤的体积。
肿瘤生长抑制率(TGI，％)计算方式：TGI＝(1-T/C)×100％，具体抑制效果如下述表3，及图5a-b和图6所示。
表3
Figure PCTCN2019074683-appb-000004
备注：a表示Mean±SD；b表示与Solution比较的P值；V0表示给药前的肿瘤体积，Vt表示给药后的肿瘤体积。
肿瘤生长曲线结果如图5a-b所示，从图中可以看出，NKG2D CAR-NK用于杀伤HCC1187成瘤小鼠后，肿瘤的体积明显小于NK92组和PBS组，抑瘤率的结果如图6所示，NKG2D CAR-NK-1组和NKG2D CAR-NK-2组的抑瘤作用明显，从给药后第6天开始逐渐升高，直至试验结束；而NK92-1组和NK92-2组的抑瘤作用一直不明显。
3.肿瘤重量的变化
试验结束之后，将NPG小鼠安乐死，并取各组小鼠的肿瘤组织、拍照并称重，肿瘤的重量及肿瘤重量占体重的百分比(肿瘤重量百分比)，结果如图7-8所示，从各组的肿瘤重量及肿瘤重量占体重的百分比上看，NK92-1组的肿瘤重量及肿瘤重量百分比最大，PBS组、NK92-1、NKG2D CAR-NK-1组和NKG2D CAR-NK-2组肿瘤重量及肿瘤重量百分比最大依次递减，且与给药后26天测量的肿瘤体积的结果相比一致性较好。
上述实验结果说明，本发明的NKG2D CAR-NK细胞的杀伤和抑瘤效果明显好于NK92组，本发明构建的NKG2D CAR-NK细胞通过增加CAR和NKG2D受体的配合明显提高了其靶向性，从而大大提高了其杀伤和抑瘤效果，可以更好地抗肿瘤。
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 PCTCN2019074683-appb-000005
Figure PCTCN2019074683-appb-000006
Figure PCTCN2019074683-appb-000007

Claims (36)

  1. 一种嵌合抗原受体，其特征在于，包括抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原NKG2D配体，并通过跨膜结构域和共刺激信号传导区激活NK细胞。
  2. 根据权利要求1所述的嵌合抗原受体，其特征在于，所述的抗原结合结构域包括NKG2D，所述NKG2D与所述NKG2D配体特异性结合。
  3. 根据权利要求2所述的嵌合抗原受体，其特征在于，所述NKG2D配体选自主要组织相容性复合体I类(MHC-I)分子、MIC-A和MIC-B。
  4. 根据权利要求1至3中任一项所述的嵌合抗原受体，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；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。
  5. 根据权利要求4所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体的结构为NKG2D-CD8αhinge-CD8 TM-4-1BB-CD3ζ的融合蛋白。
  6. 根据权利要求5所述的嵌合抗原受体，其特征在于，所述NKG2D-CD8 TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的氨基酸的序列如SEQ ID NO：1所示或其同源序列。
  7. 根据权利要求6所述的嵌合抗原受体，其特征在于，所述同源序列与原序列的同源性约95％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。
  8. 一种编码权利要求1至7中任一项所述嵌合抗原受体的核苷酸序列。
  9. 根据权利要求8所述的核苷酸序列，其特征在于，所述的核苷酸序列如SEQ ID NO：2所示或其简并序列。
  10. 根据权利要求8或9所述的核苷酸序列，其特征在于，所述同源序列与原序列的同源性约95％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。
  11. 一种NKG2D CAR-NK细胞，其表达嵌合抗原受体，所述嵌合抗原受体包括抗原结合结构域、跨膜结构域和共刺激信号传导区，所述的抗原结合结构域能够特异性结合肿瘤特异性抗原NKG2D配体，并通过跨膜结构域和共刺激信号传导区激活该NK细胞。
  12. 根据权利要求11所述的NKG2D CAR-NK细胞，其特征在于，所述的抗原结合结构域包括NKG2D，所述NKG2D与所述NKG2D配体特异性结合。
  13. 根据权利要求12所述的NKG2D CAR-NK细胞，其特征在于，所述NKG2D配体选自主要组织相容性复合体I类(MHC-I)分子、MIC-A和MIC-B。
  14. 根据权利要求11至13中任一项所述的NKG2D 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；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。
  15. 根据权利要求14所述的NKG2D CAR-NK细胞，其特征在于，所述嵌合抗原受体的结构为NKG2D-CD8αhinge-CD8 TM-4-1BB-CD3ζ的融合蛋白。
  16. 根据权利要求15所述的NKG2D CAR-NK细胞，其特征在于，所述NKG2D-CD8 TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的氨基酸的序列如SEQ ID NO：1所示或其同源序列。
  17. 根据权利要求16所述的细胞，其特征在于，所述同源序列与原序列的同源性约95％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。
  18. 根据权利要求15所述的NKG2D CAR-NK细胞，其特征在于，所述NKG2D-CD8 TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的编码核苷酸序列如SEQ ID NO：2所示或其同源序列。
  19. 根据权利要求18所述的NKG2D CAR-NK细胞，其特征在于，所述同源序列与原序列的同源性约95％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。
  20. 根据权利要求11至19中任一项所述的NKG2D CAR-NK细胞，其特征在于，所述NKG2D CAR-NK细胞能够有效地杀伤和/或杀死肺癌细胞、乳腺癌细胞、结肠癌细胞、乳腺癌细胞和/或肺癌细胞。
  21. 根据权利要求11至20中任一项所述NKG2D 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7中任一项所述的嵌合抗原受体的核苷酸序列或权利要求8至10中任一项所述的核苷酸序列，并将所述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细胞，包装和制备慢病毒；和
    (3)利用步骤(2)得到的慢病毒感染NK-92细胞，得到NKG2D CAR-NK细胞。
  22. 根据权利要求21所述的NKG2D CAR-NK细胞的制备方法，其特征在于，在步骤(1)中，合成和扩增编码所述NKG2D-CD8TMαhinge-CD8-4-1BB-CD3ζ融合蛋白的核苷酸序列。
  23. 一种药物组合物，其包含有效治疗量的权利要求11至20中任一项所述的NKG2D CAR-NK细胞，和/或权利要求21或22所述的方法制备的NKG2D CAR-NK细胞。
  24. 根据权利要求23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癌症时，所述NKG2D CAR-NK细胞与肿瘤细胞的效靶比为(0.5～1)：1。
  25. 根据权利要求23或24所述的药物组合物，其特征在于，其特征在于，还包括药学可接受的辅料。
  26. 根据权利要求23至25中任一项所述的药物组合物，其特征在于，其特征在于，所述药物组合物的剂型为水剂。
  27. 根据权利要求11至20中任一项所述的NKG2D CAR-NK细胞，和/或权利要求21或22所述的方法制备的NKG2D CAR-NK细胞在制备用于治疗和/或预防癌症的药物中的应用。
  28. 根据权利要求27所述的应用，其特征在于，所述的癌症为高表达NKG2D配体的肿瘤及相关疾病。
  29. 根据权利要求28所述的应用，其特征在于，所述癌症为肺癌、乳腺癌或结肠癌。
  30. 根据权利要求11至20中任一项所述的NKG2D CAR-NK细胞，和/或权利要求21或22所述的方法制备的NKG2D CAR-NK细胞用于治疗和/或预防癌症的方法。
  31. 根据权利要求30所述的方法，其特征在于，所述方法包括将有效治疗量的含有NKG2D CAR-NK细胞的药物组合物摄入患者体内。
  32. 根据权利要求31所述的方法，其特征在于，所述的癌症为高表达NKG2D配体的肿瘤及相关疾病。
  33. 根据权利要求32所述的方法，其特征在于，所述癌症为肺癌、乳腺癌或结肠癌。
  34. 根据权利要求30至33中任一项所述的方法，其特征在于，所述NKG2D CAR-NK细胞的给药量为(1～10)×10 6个/次，优选为(2.5～5)×10 6个/次。
  35. 根据权利要求30至34中任一项所述的方法，其特征在于，所述NKG2D 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。
  36. 根据权利要求35所述的方法，其特征在于，所述NKG2D CAR-NK细胞的摄入方法为静脉注射。
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