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WO2018165913A1 - 靶向nkg2dl的特异性嵌合抗原受体及其car-t细胞以及它们的应用 - Google Patents

靶向nkg2dl的特异性嵌合抗原受体及其car-t细胞以及它们的应用 Download PDF

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Publication number
WO2018165913A1
WO2018165913A1 PCT/CN2017/076795 CN2017076795W WO2018165913A1 WO 2018165913 A1 WO2018165913 A1 WO 2018165913A1 CN 2017076795 W CN2017076795 W CN 2017076795W WO 2018165913 A1 WO2018165913 A1 WO 2018165913A1
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Prior art keywords
seq
cells
sequence
nkg2dl
human
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English (en)
French (fr)
Inventor
代红久
张征
李晓亮
周杰
赵旭东
熊波
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Nanjing Kaedi Biotech Inc
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Nanjing Kaedi Biotech Inc
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Priority to PCT/CN2017/076795 priority Critical patent/WO2018165913A1/zh
Priority to CN201880003510.0A priority patent/CN109803983B/zh
Priority to PCT/CN2018/075389 priority patent/WO2018166307A1/zh
Priority to US16/116,953 priority patent/US11090335B2/en
Publication of WO2018165913A1 publication Critical patent/WO2018165913A1/zh
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Definitions

  • the invention relates to a specific chimeric antigen receptor targeting NKG2DL and a CAR-T cell thereof, and a preparation method and application thereof, and belongs to the technical field of biomedicine.
  • Cancer immunotherapy mainly includes adoptive cell therapy, immunomodulators, tumor vaccines, and immunological junction blocking therapy. Among them, in the field of cell therapy, CAR-T therapy has undoubtedly become the star of research institutions and pharmaceutical companies vying for "pursuit”.
  • CAR-T Chimeric Antigen Receptor T-Cell, chimeric antigen receptor T cell
  • the principle is mainly through the patient's own T cells for genetic modification of chimeric antigen receptors, CAR-T cells can specifically recognize tumor-associated antigens (tumor cells) Markers) to direct T cells to target tumors.
  • CAR-T cells Compared with conventional immune cells, CAR-T cells have higher targeting, killing activity and persistence, and can overcome the local immunosuppressive microenvironment of tumors and break the host immune tolerance state.
  • the therapy has significant efficacy in the treatment of acute leukemia and non-Hodgkin's lymphoma and is considered to be one of the most promising treatments for cancer.
  • CAR-T immunotherapy to solid tumors.
  • the biggest difficulty in the application of CAR-T immunotherapy to solid tumors is that the specificity of CAR-T cells for antigen expression on tumor cells is very high, otherwise it will easily cause T cells to continue to activate and kill normal cells, or release a large number of cytokines causing serious side effect.
  • the specificity of CAR-T immunotherapy for tumor cell antigen expression is very high, tumor-specific targeting antigens are not selective, and most antigens expressed by tumors are not tumor-specific, with tumor-associated antigens as Targeted CAR-T immunotherapy has problems such as "off-target". Studying a broader spectrum, efficient and safe CAR-T immunotherapy method is the future development trend.
  • the key to the application of CAR-T technology is to identify at least one tumor-associated antigen that is highly expressed on the surface of tumor cells without expression or low expression on the surface of normal cells.
  • NKG2DL protein is an indicator of the "stress state" of cells, which is rarely expressed or only transiently expressed in healthy tissues, but usually on the surface of various tumor cells from different sources.
  • High levels of expression such as the expression of NKG2DL in myeloma cells and in more than 80% of primary ovarian cancer cells, in particular, low doses of chemotherapeutic drugs and radiation therapy can cause tumor cell DNA Damage, activate DNA
  • the damage repair pathway increases the expression of NKG2DL on the surface of tumor cells.
  • the receptor for NKG2DL protein is NKG2D. Studies have shown that the NKG2D-NKG2DL system plays an important role in the body's anti-tumor immunity.
  • NKG2D transmits activation signals and activates the immune system by recognizing NKG2DL produced on the surface of tumor cells, thereby exerting on tumor cells. Killing effect.
  • the expression of NKG2DL is a specific change in tumor cells when the tumor occurs in the body, which provides a more precise target for tumor immunotherapy and provides enlightenment for the development of related new therapies and drugs.
  • the present invention provides a specific chimeric antigen receptor targeting NKG2DL, the amino acid sequence of which is from the amino terminus to the carboxy terminus by a leader sequence, a human NKG2D sequence, a human
  • the CD8 hinge region sequence, the human CD8 transmembrane region sequence, the human 4-1BB intracellular domain sequence and the CD3 ⁇ domain sequence are sequentially connected in series;
  • the leader sequence is SEQ. ID No. 3;
  • the human NKG2D sequence is SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 or SEQ ID Any one of No. 7 is shown.
  • the human CD8 hinge region sequence is as shown in SEQ ID No. 8; the human CD8 transmembrane region sequence is as SEQ ID. No. 9; the human 4-1BB intracellular domain sequence is shown in SEQ ID No. 10; and the CD3 ⁇ domain sequence is shown in SEQ ID No. 11.
  • the invention provides a molecule encoding the above-described specific chimeric antigen receptor that targets NKG2DL.
  • the nucleotide sequence of the molecule from the 5' end to the 3' end, by a leader sequence (e.g., SEQ ID No. 12), human NKG2D sequence (such as SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15 or SEQ ID) Any of No. 16), human CD8 hinge region sequence (as shown in SEQ ID No. 17), human CD8 transmembrane region sequence (eg SEQ ID) No. 18), a human 4-1BB intracellular domain sequence (shown as SEQ ID No. 19) and a CD3 ⁇ domain sequence (shown as SEQ ID No. 20) are sequentially connected in series.
  • a leader sequence e.g., SEQ ID No. 12
  • human NKG2D sequence such as SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15 or SEQ ID
  • Any of No. 16 any of No. 16
  • human CD8 hinge region sequence as shown in SEQ ID No. 17
  • the present invention provides a recombinant vector or expression plasmid comprising the above molecule.
  • the present invention provides a recombinant cell comprising the above molecule, wherein the recombinant cell is an immune effector cell capable of expressing the above-described specific chimeric antigen receptor targeting NKG2DL; the immune effector cell is a cytotoxic T Lymphocytes, NK cells, NKT cells or helper T cells.
  • the present invention provides a recombinant virus comprising the above molecule, which is a virus capable of expressing the above-described specific chimeric antigen receptor targeting NKG2DL and capable of infecting immune effector cells;
  • the effector cells are cytotoxic T lymphocytes, NK cells, NKT cells or helper T cells, etc.; the viruses are lentiviruses, adenoviruses, adenoviruses or retroviruses.
  • the present invention provides a method for producing a specific CAR-T cell targeting NKG2DL, the preparation method comprising the steps of: first, passing the above-mentioned sequence targeting a NKG2DL-specific chimeric antigen receptor Molecular cloning was ligated into the lentiviral expression vector lentiGuide-Puro to obtain a specific CAR expression plasmid targeting NKG2DL; then, the obtained specific NKG2DL-specific CAR expression plasmid was transfected into 293T cells to obtain a virus solution. T cells are then infected with the viral solution, and T cells expressing a specific chimeric antigen receptor targeting NKG2DL are obtained from the infected cells.
  • the present invention provides the above recombinant vector or expression plasmid, the above recombinant cell, the above recombinant virus, and the specific NKG2DL-specific CAR-T cell obtained by the above preparation method for preparing a tumor-treating product.
  • the tumor comprises a tumor cell expressing an NKG2DL protein.
  • the above recombinant vector or expression plasmid, the above recombinant cell, the above recombinant virus, and the specific NKG2DL-specific CAR-T cell obtained by the above preparation method are prepared for treating gastric cancer, pancreatic cancer, lung cancer, prostate cancer, ovarian cancer, breast Use in drugs for cancer, bladder cancer, colon cancer, liver cancer, renal cell carcinoma, leukemia, lymphoma, multiple myeloma, melanoma, glioma or neuroblastoma.
  • the invention constructs a specific chimeric antigen receptor targeting NKG2DL and a CAR-T cell based on the NKG2D molecule, and the novel CAR-T cell can effectively target a plurality of tumor cells and can be used for preparing a preparation for treating tumors.
  • a preparation expressing NKG2DL positive tumor cells In particular, a preparation expressing NKG2DL positive tumor cells.
  • the preparation method of the invention has simple steps, and the specific CAR-T cells targeting NKG2DL have high killing rate against tumor cells.
  • Example 1 is a flow cytometric test result of a control group in the identification step of Example 4.
  • Example 2 is a flow cytometric test result of the experimental group in the identification step of Example 4.
  • Figure 3 is a test result of tumor cell killing rate using lymphoma cell Raji as a target cell
  • Figure 4 is a test result of tumor cell killing rate using gastric cancer cell NUGC-4 as a target cell
  • Figure 5 shows the results of tumor cell killing rate test using ovarian cancer cell line SKOV-3 as a target cell
  • Figure 6 is a test result of tumor cell killing rate using lung cancer cell A549 as a target cell
  • Figure 7 is a graph showing the results of tumor cell killing rate test using prostate cancer cell line PC-3 as a target cell.
  • Step 1) Determination of the amino acid sequence of a specific chimeric antigen receptor targeting NKG2DL
  • NKG2D the receptor for NKG2DL protein
  • Genbank database of the National Library of Medicine NCBI eg SEQ ID. 1
  • SEQ ID No. 2 the full-length amino acid sequence of NKG2D (the receptor for NKG2DL protein) was searched from the Genbank database of the National Library of Medicine NCBI (eg SEQ ID). No. 1) and the full-length cDNA sequence (shown as SEQ ID No. 2).
  • a specific chimeric antigen receptor targeting NKG2DL was constructed.
  • the molecular structure of the chimeric antigen receptor CAR
  • it mainly consists of three parts: an extramembranous antigen-binding region (extracellular single-chain antibody portion), a hinge region (transmembrane protein portion), and an intracellular signaling region.
  • the extramembranous antigen-binding region if the NKG2D full-length amino acid sequence is constructed, the effect is poor, because the NKG2D intracellular region and the like are NK cell properties and are incompatible with T cells.
  • the inventors screened the appropriate amino acid sequences targeting NKG2DL-specific CAR molecules by means of protein sequence analysis and functional domain prediction tools. Furthermore, how to choose appropriate and matching The amino acid sequence of the hinge region, the transmembrane region, and the T cell intracellular signal activation region is also a difficult technical problem for the inventors.
  • the inventors continually carried out amino acid sequence design and sequence alignment, and tested and verified the sequence of more than 10 CAR molecules (for example, constructing a virus vector infected T cell test), and then performing comparison according to the results of multiple combinations. Sequence alignment, finally screening out the best-performing sequence, and obtaining the amino acid sequence and nucleotide sequence of the specific chimeric antigen receptor targeting NKG2DL of the present invention, as follows:
  • Amino acid sequence targeting NKG2DL-specific CAR molecule from amino terminus to carboxy terminus, by leader sequence (eg SEQ ID No. 3), human NKG2D sequence (such as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 or SEQ ID) Any of No. 7), human CD8 hinge region sequence (as shown in SEQ ID No. 8), human CD8 transmembrane region sequence (eg SEQ ID) The sequence of No. 9), the human 4-1BB intracellular domain sequence (shown as SEQ ID No. 10) and the CD3 ⁇ domain sequence (shown as SEQ ID No. 11) are sequentially connected in series.
  • leader sequence eg SEQ ID No. 3
  • human NKG2D sequence such as SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 or SEQ ID
  • Any of No. 7 any of No. 7
  • human CD8 hinge region sequence as shown in SEQ ID No. 8
  • human CD8 transmembrane region sequence eg
  • Nucleotide sequence targeting NKG2DL-specific CAR molecule from the 5' end to the 3' end, by the leader sequence (eg SEQ ID No. 12), human NKG2D sequence (such as SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15 or SEQ ID) Any of No. 16), human CD8 hinge region sequence (as shown in SEQ ID No. 17), human CD8 transmembrane region sequence (eg SEQ ID) No. 18), a human 4-1BB intracellular domain sequence (shown as SEQ ID No. 19) and a CD3 ⁇ domain sequence (shown as SEQ ID No. 20) are sequentially connected in series.
  • leader sequence eg SEQ ID No. 12
  • human NKG2D sequence such as SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15 or SEQ ID
  • Any of No. 16 any of No. 16
  • human CD8 hinge region sequence as shown in SEQ ID No. 17
  • human CD8 transmembrane region sequence
  • Step 2 Construction and identification of a plasmid expressing a specific CAR molecule targeting NKG2DL
  • Ligation system 2 ⁇ l of PCR product recovered by gel, 2 ⁇ l of LentiGuide-Puro plasmid with BstEII and MluI recovered by gel; 0.5 ⁇ l of T4 ligase; 10x ligase buffer 1 ⁇ l; deionized water 4.5 ⁇ l; the reaction reaction system volume 10 ⁇ l;
  • the ligation product was transformed with competent Stbl3, and the specific transformation steps were as follows.
  • the strain of the specific CAR molecule expression plasmid targeting NKG2DL constructed in step 2) was cultured in large amount in LB medium using Qiagen Plasmid Midi Kit is highly purified and endotoxin free, and is ready for infection.
  • the specific steps are as follows:
  • the culture solution of the overnight culture is added to the centrifuge tube, centrifuged at 6000 ⁇ g for 15 minutes, and the supernatant is aspirated as much as possible (the bacterial solution can be collected into a centrifuge tube by multiple centrifugation when the bacterial solution is excessive).
  • step 7 Transfer the supernatant collected in step 5 to the adsorption column and let stand until the liquid completely enters the resin medium.
  • MPC Magnetic Particles Concentrator
  • the enriched CD3+ T cells were resuspended in culture medium (purchased from American Life) Technologies, product information for OpTmizerTM T-Cell Expansion SFM, A1048503), adjusted to a cell solubility of 1 ⁇ 106 cells/ml, and finally cultured in a 37 ° C, 5% CO 2 incubator for 2 days.
  • polyethyleneimine transfection reagent 408727, Sigma
  • the virus supernatants were collected, centrifuged at 3,000 rpm for 10 minutes at 4 ° C, filtered through a 0.45 ⁇ m filter, and finally centrifuged at 50,000 g at 4 ° C. After 3 hours, the virus was concentrated, and the collected virus concentrate was transferred to -80 ° C for storage.
  • the CD3+ T cells obtained in Example 2 were inoculated into a 24-well plate at a concentration of 1 ⁇ 105 cells/ml at 37 ° C, 5%.
  • the CO2 environment is cultured for about 24 hours (the culture time is determined according to the specific practice, and generally, the cell confluence rate is between 50-70% in the case of viral liquid infection).
  • Expression of NKG2D was obtained 48 hours after infection CAR molecule T cells (KD-025 CAR-T cells), the new CAR-T cells, can be used for the next functional experiment.
  • the cells obtained after infection are then identified, that is, the expression of the CAR molecule after infection is detected by flow cytometry analysis, and the specific operation is as follows.
  • Experimental group the cells to be detected collected during the preparation of the specific CAR-T cells targeting NKG2DL described above.
  • Control group empty vector virus-infected T cells (The lentiGuide-Puro empty vector was substituted for the specific CAR expression plasmid targeting NKG2DL of Example 1, and the empty vector virus solution was obtained according to the procedure of Example 3, and then according to the implementation. The procedure of Example 4 obtained T cells infected with empty vector virus solution).
  • PE-labeled anti-human NKG2D antibody PE anti-human-NKG2D, 320806, according to antibody instructions) Biolegend
  • PE-labeled anti-human NKG2D antibody PE anti-human-NKG2D, 320806, according to antibody instructions
  • Biolegend PE-labeled anti-human NKG2D antibody
  • flow cytometry BD FacsCanto II
  • Stained cells were obtained and the results were analyzed using FlowJo software.
  • the flow results are shown in Figures 1 and 2 (another note: in Figures 1 and 2, the abscissa PE-A represents the fluorescence intensity of the fluorescence emitted by the PE after being excited (the portion received by the PMT);
  • the coordinate SSC-A represents the granularity of the cell)
  • Figure 1 shows the results of flow cytometry in the empty vector control group. The expression of CAR molecules was almost undetectable from the graph.
  • Figure 2 shows the flow cytometry results of the experimental group. The expression rate of CAR molecules reached 28.1. %.
  • Example 4 expressed a specific chimeric antigen receptor targeting NKG2DL.
  • Test tumor cell line also known as target cell line: lymphoma cell Raji (ATCC product, product number CCL-86TM), gastric cancer cell NUGC-4 (purchased from Nanjing Kezhen Biotechnology Co., Ltd., article number: CBP60493) , ovarian cancer cell SKOV-3 (ATCC product, product number: HTB-77TM), lung cancer cell A549 (ATCC product, product number: CCL-185TM) and prostate cancer cell PC-3 (ATCC product, product number: CRL -1435TM).
  • lymphoma cell Raji ATCC product, product number CCL-86TM
  • NUGC-4 purchased from Nanjing Kezhen Biotechnology Co., Ltd., article number: CBP60493
  • ovarian cancer cell SKOV-3 ATCC product, product number: HTB-77TM
  • lung cancer cell A549 ATCC product, product number: CCL-185TM
  • prostate cancer cell PC-3 ATCC product, product number: CRL -1435TM.
  • Example 4 The specific CAR-T cell pair targeting NKG2DL obtained in Example 4 was evaluated using a 7-AAD/CFSE cytotoxicity test kit (purchased from Biovision, Cat. No. K315-100) and following the protocol of the kit. The killing of the above target cell lines.
  • each target cell line was subjected to CSFE fluorescence staining and plated in a culture plate at a seeding concentration of 2 ⁇ 10 4 /ml per well.
  • Each of the target cell lines was correspondingly provided with one experimental group and two control groups, wherein the experimental group was added with the cell suspension of the specific CAR-T cells targeting NKG2DL obtained in Example 4; T cells that are not infected with virus (ie, CD3+ T cells obtained in Example 2); control group 2 is added T cells infected with empty vector virus solution (ie, the lentiGuide-Puro empty vector replaces the targeted NKG2DL of Example 1).
  • the specific CAR expression plasmid was obtained according to the procedure of Example 3, and the empty vector virus solution was obtained, and the empty vector virus-infected T cells were obtained according to the procedure of Example 4.
  • the specific NKG2DL-targeting specific CAR-T cells of Example 4 were mixed with target cells according to three different target ratios (10:1, 5:1, and 1:1).
  • the term "effective target ratio” refers to the ratio of the number of effector cells (specific CAR-T cells targeting NKG2DL) to target cells (tumor cells).
  • control group 1 and control group 2 also mixed T cells with target cells at three different target ratios.
  • Figure 3 shows the results of tumor cell killing rate test using lymphoma cell Raji as a target cell.
  • the specific CAR-T cells targeting NKG2DL of Example 4 had a significant killing effect on lymphoma Raji (significantly higher than the two control groups), and the effective target ratio was 10:1.
  • the tumor cell killing rate reached 39.5%.
  • Figure 4 shows the results of tumor cell killing rate test using gastric cancer cell NUGC-4 as a target cell.
  • the specific CAR-T cells targeting NKG2DL of Example 4 had a significant killing effect on gastric cancer cell NUGC-4 (significantly higher than the two control groups), and the effective target ratio was 10:1.
  • the tumor cell killing rate reached 40.5%.
  • Figure 5 shows the results of tumor cell killing rate test using ovarian cancer cell line SKOV-3 as a target cell.
  • the specific CAR-T cells targeting NKG2DL of Example 4 had a significant killing effect on ovarian cancer cell SKOV-3 (significantly higher than the two control groups), and the effective target ratio was 10:1.
  • the corresponding tumor cell killing rate reached 45.5%.
  • Figure 6 shows the results of tumor cell killing rate test using lung cancer cell A549 as a target cell.
  • the specific CAR-T cells targeting NKG2DL of Example 4 have a significant killing effect on lung cancer cells A549 (significantly higher than the two control groups), and the tumors corresponding to the target ratio of 10:1 The cell killing rate reached 40.5%.
  • Figure 7 shows the results of tumor cell killing rate test using prostate cancer cell line PC-3 as a target cell.
  • the specific CAR-T cells targeting NKG2DL of Example 4 had a significant killing effect on prostate cancer cell line PC-3 (significantly higher than the two control groups), and the effective target ratio was 10:1.
  • the corresponding tumor cell killing rate reached 67%.
  • the specific NKG2DL-targeting specific CAR-T cells of Example 4 of the present invention were able to specifically recognize NKG2DL-positive tumor cells and have a targeted lethality.
  • the specific chimeric antigen receptor targeting NKG2DL constructed by the present invention and the specific CAR-T cell targeting NKG2DL infected by the viral vector thereof can be applied to treat lymphoma, gastric cancer, ovarian cancer, lung cancer and Tumor disorders such as prostate cancer.
  • Tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg Cagtgcacac gagggggctg 120

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Abstract

提供了一种靶向NKG2DL的特异性嵌合抗原受体及其CAR-T细胞，以及它们制备方法和应用。该CAR-T细胞能有效地靶向攻击多种肿瘤细胞，可用来制备用于治疗肿瘤的制剂，尤其是表达NKG2DL阳性肿瘤细胞的制剂。

Description

靶向 NKG2DL的特异性嵌合抗原受体及其CAR-T细胞以及它们的应用 技术领域
本发明涉及一种靶向NKG2DL的特异性嵌合抗原受体及其CAR-T细胞，以及它们制备方法和应用，属于生物医药技术领域。
背景技术
随着生物技术的飞速发展，免疫细胞治疗已成为癌症治疗领域的第四大疗法。
癌症免疫疗法主要包括过继性细胞治疗、免疫调节剂、肿瘤疫苗以及免疫结合点阻断治疗等。其中，在细胞治疗领域，CAR-T疗法无疑已成为研究机构和制药公司争相“追捧”的明星。
CAR-T（Chimeric Antigen Receptor T-Cell，嵌合抗原受体T细胞）免疫疗法，其原理主要是通过取病人自身T细胞进行嵌合抗原受体的基因修饰，CAR-T细胞能够特异性地识别肿瘤相关抗原（肿瘤细胞标志物），从而引导T细胞靶向肿瘤。相较于常规免疫细胞，CAR-T细胞的靶向性、杀伤活性和持久性都更高，并且可以克服肿瘤局部免疫抑制微环境并打破宿主免疫耐受状态。该疗法在急性白血病和非霍奇金淋巴瘤的治疗上有着显著的疗效，被认为是最有前景的肿瘤治疗方式之一。
然而，90%的癌症是实体瘤，更多种类的实体瘤以及更多的肿瘤表面特异性靶点抗原有待进一步确认。CAR-T免疫疗法应用于实体瘤治疗的最大难点在于CAR-T细胞对于肿瘤细胞上抗原表达的特异性要求非常高，否则容易造成T细胞持续激活而杀伤正常细胞，或释放大量细胞因子引起严重副作用。虽然CAR-T免疫疗法对于肿瘤细胞抗原表达的特异性要求非常高，但肿瘤特异的靶向抗原可选择性并不多，并且，肿瘤表达的大多数抗原不具备肿瘤特异，以肿瘤相关抗原作为靶点的CAR-T免疫疗法存在“脱靶”等问题。研究更广谱、高效、安全的CAR-T免疫治疗方法是未来的发展趋势。
因此，发挥CAR-T技术应用的关键在于确定至少一种肿瘤相关的抗原，这种抗原在肿瘤细胞表面高表达，而在正常细胞表面无表达或低表达。
近年来，研究表明，NKG2DL蛋白的表达是细胞处于“应激状态”的一个指标，其在健康组织中很少有表达或仅仅短暂地表达，而通常在不同来源的各种肿瘤细胞表面有较高水平的表达，例如在骨髓瘤细胞和超过80%的原发性卵巢癌细胞都发现了NKG2DL的表达，特别是，低剂量的化学治疗药物和放射治疗能够引起肿瘤细胞DNA 损伤，激活DNA 损伤修复途径，增加肿瘤细胞表面NKG2DL的表达。NKG2DL蛋白的受体为NKG2D，研究显示，NKG2D-NKG2DL系统在机体的抗肿瘤免疫中发挥着重要作用，NKG2D通过识别肿瘤细胞表面产生的NKG2DL来传递活化信号并激活免疫系统，从而对肿瘤细胞发挥杀伤作用。NKG2DL的表达作为机体发生肿瘤时肿瘤细胞上的一个特异性的改变，为肿瘤的免疫治疗提供了一个更为精确的靶点，为相关新疗法和药物的开发提供了启示。
 技术问题
肿瘤表达的大多数抗原不具备肿瘤特异，以肿瘤相关抗原作为靶点的CAR-T免疫疗法存在“脱靶”等问题。
技术解决方案
鉴于相关技术的上述问题和/或其他问题，本发明一方面提供了一种靶向NKG2DL的特异性嵌合抗原受体，其氨基酸序列从氨基端到羧基端由引导序列、人NKG2D序列、人CD8铰链区序列、人CD8跨膜区序列、人4-1BB胞内结构域序列和CD3ζ结构域序列依次串联而成；所述引导序列如SEQ ID No.3所示；以及，所述人NKG2D序列如SEQ ID No.4、SEQ ID No.5、SEQ ID No.6或SEQ ID No.7中任意一个所示。
优选的，所述人CD8铰链区序列如SEQ ID No.8所示；所述人CD8跨膜区序列如SEQ ID No.9所示；所述人4-1BB胞内结构域序列如SEQ ID No.10所示；所述CD3ζ结构域序列如SEQ ID No.11所示。
本发明在一方面提供了一种编码上述靶向NKG2DL的特异性嵌合抗原受体的分子。
优选的，所述分子的核苷酸序列：从5’端到3’端，由引导序列（如SEQ ID No.12所示）、人NKG2D序列（如SEQ ID No.13、SEQ ID No.14、SEQ ID No.15或SEQ ID No.16中任意一个所示）、人CD8铰链区序列（如SEQ ID No.17所示）、人CD8跨膜区序列（如SEQ ID No.18所示）、人4-1BB胞内结构域序列（如SEQ ID No.19所示）和CD3ζ结构域序列（如SEQ ID No.20所示）依次串联而成。
本发明还一方面提供了一种包含上述分子的重组载体或表达质粒。
本发明还一方面提供了一种包含上述分子的重组细胞，所述重组细胞为能够表达上述的靶向NKG2DL的特异性嵌合抗原受体的免疫效应细胞；所述免疫效应细胞为细胞毒性T淋巴细胞、NK细胞、NKT细胞或辅助性T细胞。
本发明还一方面提供了一种包含上述分子的重组病毒，所述重组病毒为能够表达上述的靶向NKG2DL的特异性嵌合抗原受体、且能够侵染免疫效应细胞的病毒；所述免疫效应细胞为细胞毒性T淋巴细胞、NK细胞、NKT细胞或辅助性T细胞等；所述病毒为慢病毒、腺病毒、腺联病毒或逆转录病毒等。
本发明还一方面提供了一种靶向NKG2DL的特异性CAR-T细胞的制备方法，所述制备方法包括以下步骤：首先，将上述的靶向NKG2DL特异性嵌合抗原受体的序列，通过分子克隆的方式连接到慢病毒表达载体lentiGuide-Puro中，获得靶向NKG2DL的特异性CAR表达质粒；然后，将获得的所述靶向NKG2DL的特异性CAR表达质粒转染293T细胞，获得病毒液；然后用所述病毒液感染T细胞，从感染后的细胞中获得表达靶向NKG2DL的特异性嵌合抗原受体的T细胞。
本发明还一方面提供了上述重组载体或表达质粒，上述重组细胞，上述重组病毒，上述制备方法获得的靶向NKG2DL的特异性CAR-T细胞在制备治疗肿瘤的产品中的应用。
优选的，所述肿瘤包含表达NKG2DL蛋白的肿瘤细胞。
优选的，上述重组载体或表达质粒，上述重组细胞，上述重组病毒，上述制备方法获得的靶向NKG2DL的特异性CAR-T细胞在制备治疗胃癌、胰腺癌、肺癌、前列腺癌、卵巢癌、乳腺癌、膀胱癌、结肠癌、肝癌、肾细胞癌、白血病、淋巴瘤、多发性骨髓瘤、黑色素瘤、神经胶质瘤或神经母细胞瘤的药物中的应用。
 有益效果
本发明基于NKG2D分子构建靶向NKG2DL的特异性嵌合抗原受体以及CAR-T细胞，该新型CAR-T细胞能有效地靶向攻击多种肿瘤细胞，可用来制备用于治疗肿瘤的制剂，尤其是表达NKG2DL阳性肿瘤细胞的制剂。本发明的制备方法步骤简单，获得的靶向NKG2DL的特异性CAR-T细胞对肿瘤细胞的杀伤率高。
附图说明
图1为实施例4的鉴定步骤中对照组的流式细胞检测结果；
图2为实施例4的鉴定步骤中实验组的流式细胞检测结果；
图3为以淋巴瘤细胞Raji为靶细胞的肿瘤细胞杀伤率测试结果；
图4为以胃癌细胞NUGC-4为靶细胞的肿瘤细胞杀伤率测试结果；
图5为以卵巢癌细胞SKOV-3为靶细胞的肿瘤细胞杀伤率测试结果；
图6为以肺癌细胞A549为靶细胞的肿瘤细胞杀伤率测试结果；
图7为以前列腺癌细胞PC-3为靶细胞的肿瘤细胞杀伤率测试结果。
 本发明的最佳实施方式
以下通过实施例对本发明作进一步的说明，但本发明并不限于这些具体实施方式。
下面实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
制备靶向NKG2DL的特异性嵌合抗原受体的表达质粒
步骤1）靶向NKG2DL的特异性嵌合抗原受体的氨基酸序列的确定
首先，从美国国立医学图书馆NCBI的Genbank数据库中搜索到NKG2D（NKG2DL蛋白的受体）的全长氨基酸序列（如SEQ ID No.1所示）和全长cDNA序列（如SEQ ID No.2所示）。
其次，构建靶向NKG2DL的特异性嵌合抗原受体。关于嵌合抗原受体（CAR）的分子结构，主要由膜外抗原结合区（胞外单链抗体部分）、铰链区（跨膜蛋白部分）和胞内信号传导区三部分构成。其中，关于膜外抗原结合区，如果以NKG2D的全长氨基酸序列来构建，效果较差，因为NKG2D胞内区等是NK细胞属性的，与T细胞不兼容。于是发明人基于NKG2D的全长氨基酸序列，通过蛋白序列分析和功能结构域预测工具等手段来筛选适宜构建靶向NKG2DL特异性CAR分子的氨基酸序列；再者，如何选择适宜的、与之匹配的铰链区、跨膜区、T细胞胞内信号激活区的氨基酸序列，对于发明人来说也是一个难解的技术问题。
发明人不断地进行氨基酸序列设计以及序列的排列组合，对10余条CAR分子的序列进行试验和验证（例如构建病毒载体感染T细胞等试验），之后根据多个组合的结果比对，再进行序列调整，最终筛选出效果最好的一段序列，获得了本发明的靶向NKG2DL的特异性嵌合抗原受体的氨基酸序列和核苷酸序列，具体如下：
靶向NKG2DL特异性CAR分子的氨基酸序列：自氨基端到羧基端，由引导序列（如SEQ ID No.3所示）、人NKG2D序列（如SEQ ID No.4、SEQ ID No.5、SEQ ID No.6或SEQ ID No.7中任意一个所示）、人CD8铰链区序列（如SEQ ID No.8所示）、人CD8跨膜区序列（如SEQ ID No.9所示）、人4-1BB胞内结构域序列（如SEQ ID No.10所示）和CD3ζ结构域序列（如SEQ ID No.11所示）依次串联而成。
靶向NKG2DL特异性CAR分子的核苷酸序列：从5’端到3’端，由引导序列（如SEQ ID No.12所示）、人NKG2D序列（如SEQ ID No.13、SEQ ID No.14、SEQ ID No.15或SEQ ID No.16中任意一个所示）、人CD8铰链区序列（如SEQ ID No.17所示）、人CD8跨膜区序列（如SEQ ID No.18所示）、人4-1BB胞内结构域序列（如SEQ ID No.19所示）和CD3ζ结构域序列（如SEQ ID No.20所示）依次串联而成。
步骤2）表达靶向NKG2DL的特异性CAR分子的质粒的构建和鉴定
全基因合成靶向NKG2DL的特异性CAR分子的核苷酸序列，通过分子克隆的方式连接到lentiGuide-Puro（Genscript），构建单一编码框的全长CAR序列表达框，利用EF1 alpha启动子（序列表SEQ ID No.21）进行表达。具体操作步骤如下：
首先将人NKG2D的氨基酸序列（如SEQ ID No.4、SEQ ID No.5、SEQ ID No.6或SEQ ID No.7中任意一个所示）进行人源密码子优化获得对应的核苷酸序列（如SEQ ID No.13、SEQ ID No.14、SEQ ID No.15或SEQ ID No.16中任意一个所示），然后全基因合成含有引导序列、人NKG2D序列、CD8铰链区、CD8跨膜区、4-1BB胞内结构域、CD3ζ结构域的靶向NKG2DL的特异性CAR分子的核苷酸序列（安徽、通用生物），引物扩增人工合成CAR分子序列，与经过限制性内切酶BstEII和MluI消化的载体lentiGuide-Puro相连接。具体连接反应的体系和条件如下：
连接体系：胶回收的PCR产物2μl，胶回收的BstEII和MluI酶切lentiGuide-Puro质粒2μl；T4连接酶0.5μl；10x连接酶buffer 1μl；去离子水4.5μl；连接反应体系体积10μl；
连接条件：PCR仪中16摄氏度连接过夜。
连接产物经感受态Stbl3转化，具体转化操作步骤如下。
将5μl连接产物加入到50μl的感受态细胞（Stbl3，购买自美国invitrogen公司）中，冰浴30min，42℃45s，冰浴2min，然后加入500μl无抗LB液体培养基后，37摄氏度，200rpm震荡培养40min，涂布氨苄抗性的LB固体平板，在37摄氏度培养箱中过夜。等待单菌落出现后，挑取5个大小适中的菌落，提取质粒，并送往商业测序公司测序（安徽、通用生物），将测序结果与拟合成的NKG2D 特异性CAR分子序列比对证实序列完全正确，证明获得了表达靶向NKG2DL的特异性CAR分子的质粒（简称KD-025 CAR 慢病毒载体）。
步骤3）靶向NKG2DL的特异性CAR表达质粒的提取和纯化
将步骤2）中构建的靶向NKG2DL的特异性CAR分子表达质粒的菌株在LB培养基中大量培养，采用Qiagen Plasmid Midi Kit进行高纯度无内毒素抽提，已备感染。具体操作步骤如下：
1. 取150 ml过夜培养的菌液，加入离心管中，6000×g离心15分钟，尽量吸除上清（菌液过多时可以通过多次离心将菌液沉淀收集到一个离心管中）。
2．向留有菌体沉淀的离心管中加入4 ml溶液P1（ 请先检查是否已加入RNase A），使用移液器或涡旋振荡器彻底悬浮细菌沉淀。
3. 向离心管中加入4 ml溶液P2， 温和地上下翻转4-6次使菌体充分裂解，室温（15-25℃）孵育5分钟。
4. 向离心管中加入4 ml预冷的溶液P3，立即温和地上下翻转4-6次，充分混匀，冰上孵育5分钟。
5. 将离心管放入4℃超速离心机，20000×g离心10分钟。
6. 柱平衡：向吸附柱加入4 ml平衡液QBT, 静置，直至液体完全流出。
7. 将步骤5中收集的上清液转移到吸附柱中，静置，直至液体完全进入树脂介质。
8. 向吸附柱中加入10 ml的漂洗液QC，静置，直至液体完全流出，重复操作1次。
9. 向吸附柱中加入5 ml的洗脱液QF，收集到15 ml离心管中。
10. 向离心管中加入3.5 ml的异丙醇，混匀，≥15000×g 4℃离心30分钟，弃去上清。
11. 向留有DNA沉淀的离心管中加入2 ml 70%的乙醇溶液, ≥15000×g离心10分钟，弃去上清。
12. 建议将留有DNA沉淀的离心管开盖，室温放置5-10分钟，加入合适体积的溶液重新溶解DNA，将含有DNA的溶液全部转移到新的1.5 ml离心管中，-20℃保存。
 本发明的实施方式
实施例2
T细胞的分离培养
取健康供者的新鲜外周血，通过密度梯度离心分离新鲜的外周血单核细胞；再利用偶联了抗-CD3抗体和抗-CD28抗体的顺磁磁珠（购买自美国Invitrogen公司，产品信息为Dynabeads® Human T-Activator CD3/CD28，货号：11161D）来富集CD3+T细胞，具体来说，将外周血单核细胞稀释至浓度为（10~30）×106个单个细胞/ml，再将磁珠与细胞以3:1的比例在培养皿中混合，室温下共孵育2-3小时，利用磁微粒收集器（Magnetic particles concentrator，简称MPC，货号：12301D，购买自美国Invitrogen公司）富集CD3+T细胞。最后将富集的CD3+T细胞重悬于培养液（购买自美国Life Technologies公司，产品信息为OpTmizer™ T-Cell Expansion SFM，A1048503），调至细胞溶度为1×106个/ml，最后在37℃、5% CO2培养箱中培养2天。
实施例3
病毒液的制备
实施例1的步骤3）获得的靶向NKG2DL的特异性CAR表达质粒，以及包装质粒psPAX2和VSVG，按照10：8：5的比例用聚乙烯亚胺转染试剂（408727，Sigma）转染试剂，共转染293T 细胞（为ATCC产品，产品编号为：CRL-3216TM）；具体的包装质粒的制备方法参见Lenti-X Packaging Single Shots（Takara）说明书；具体的转染操作过程参见Sigma转染说明书。
转染后6小时 更换为完全培养基（购买自Life Technologies公司，产品货号：11995-065），培养48小时和72小时后，分别收集病毒上清液，于4℃，3000rpm离心10分钟后经0.45μm滤膜过滤，最后于4℃，50000g超离心3小时，浓缩病毒，将收集的病毒浓缩液转入-80℃保存。
实施例4
靶向NKG2DL的特异性CAR-T细胞的制备
取实施例2获得的CD3+T细胞，接种到24孔板，接种浓度为1×105个细胞/ml，在37℃、5% CO2环境培养约24小时（培养时间依据具体实践而定，一般来说，保证在病毒液感染时细胞汇合率介于50-70%之间）。取实施例3的病毒浓缩液，第二天按照MOI=1-10的值将病毒液加入细胞培养瓶后，封好口，放入平角离心机后，低速（500g-1000g/min）离心1小时，然后放入培养箱中37℃培养。感染后48小时后获得表达NKG2D CAR分子的T细胞（KD-025 CAR-T细胞），即新型CAR-T细胞，可以进行下一步的功能实验。
鉴定
之后再对感染后获得的细胞进行鉴定，即利用流式细胞术分析检测感染后CAR分子的表达，具体操作如下。
实验组：即上述靶向NKG2DL的特异性CAR-T细胞的制备过程中所收集的待检测细胞。
对照组：空载体病毒液感染的T细胞（将lentiGuide-Puro空载体替代实施例1的靶向NKG2DL的特异性CAR表达质粒，按照实施例3的操作步骤获得空载体的病毒液，再按照实施例4的操作步骤获得空载体病毒液感染的T细胞）。
将上述实验组和对照组，PBS清洗两次后重悬于FACS液（含0.1%叠氮化钠和0.4% BSA的PBS）中。
按照抗体说明书将PE 标记的抗人NKG2D抗体（PE anti-human-NKG2D，320806， Biolegend）加入到待检测细胞组和对照组的细胞悬液中，4℃孵育60分钟。采用流式细胞仪（BD FacsCanto II）获取染色细胞，并采用FlowJo软件分析结果。流式结果如图1和图2所示（另注：图1和图2中，横坐标PE-A表示PE受激发后发射出来的荧光（被PMT接收到的那部分）的荧光强度；纵坐标SSC-A代表细胞的颗粒度）
图1是空载体对照组的流式细胞检测结果，从图上看几乎检测不到CAR分子的表达；图2是实验组的流式细胞检测结果，从图上看CAR分子的表达率达到28.1%。
从图2的流式结果可以看出，实施例4的收集的待检测细胞表达了靶向NKG2DL的特异性嵌合抗原受体
应用例以及效果数据
供试肿瘤细胞系（也称靶细胞系）：淋巴瘤细胞Raji（ATCC产品，产品编号为CCL-86™）、胃癌细胞NUGC-4（购自南京科佰生物科技有限公司，货号：CBP60493）、卵巢癌细胞SKOV-3（ATCC产品，产品编号：HTB-77™）、肺癌细胞A549（ATCC产品，产品编号：CCL-185™）和前列腺癌细胞PC-3（ATCC产品，产品编号：CRL-1435™）。
利用7-AAD/CFSE细胞毒性测试试剂盒（购买自Biovision公司，货号：K315-100），并按照该试剂盒的操作说明书来评估实施例4所得的靶向NKG2DL的特异性CAR-T细胞对上述靶细胞系的杀伤情况。
具体来说，每一种靶细胞系经过CSFE荧光染色后，以每孔2×104个/ml的接种浓度铺于培养板中。
每一种靶细胞系对应地设置一个实验组和两个对照组，其中，实验组添加的是实施例4所得的靶向NKG2DL的特异性CAR-T细胞的细胞悬液；对照组1添加的是没有感染病毒的T细胞（即实施例2获得的CD3+T细胞）；对照组2添加的是空载体病毒液感染的T细胞（即将lentiGuide-Puro空载体替代实施例1的靶向NKG2DL的特异性CAR表达质粒，按照实施例3的操作步骤获得空载体的病毒液，再按照实施例4的操作步骤获得空载体病毒液感染的T细胞）。
上述的实验组，按照三个不同的效靶比（10：1、5：1和1：1），将实施例4的靶向NKG2DL的特异性CAR-T细胞与靶细胞混合。此处，术语“效靶比”是指效应细胞（靶向NKG2DL的特异性CAR-T细胞）与靶细胞（肿瘤细胞）的数量比。
同样的，对照组1和对照组2，也各自按照三个不同的效靶比将T细胞与靶细胞混合。
培养20小时后离心去上清，将细胞沉淀物清洗之后用7AAD染色，采用流式细胞仪（BD FacsCanto II）获取染色细胞，并采用FlowJo软件分析结果。
图3所示为以淋巴瘤细胞Raji为靶细胞的肿瘤细胞杀伤率测试结果。从图3可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞对淋巴瘤细胞Raji有明显的杀伤作用（明显高于两个对照组），并且效靶比10:1对应的肿瘤细胞杀伤率达到39.5%。
图4所示为以胃癌细胞NUGC-4为靶细胞的肿瘤细胞杀伤率测试结果。从图4可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞对胃癌细胞NUGC-4有明显的杀伤作用（明显高于两个对照组），并且效靶比10:1对应的肿瘤细胞杀伤率达到40.5%。
图5所示为以卵巢癌细胞SKOV-3为靶细胞的肿瘤细胞杀伤率测试结果。从图5可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞对卵巢癌细胞SKOV-3有明显的杀伤作用（明显高于两个对照组），并且效靶比10:1对应的肿瘤细胞杀伤率达到45.5%。
图6所示为以肺癌细胞A549为靶细胞的肿瘤细胞杀伤率测试结果。从图6可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞对肺癌细胞A549有明显的杀伤作用（明显高于两个对照组），并且效靶比10:1对应的肿瘤细胞杀伤率达到40.5%。
图7所示为以前列腺癌细胞PC-3为靶细胞的肿瘤细胞杀伤率测试结果。从图7可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞对前列腺癌细胞PC-3有明显的杀伤作用（明显高于两个对照组），并且效靶比10:1对应的肿瘤细胞杀伤率达到67%。
从上述图3至图7的结果可以看出，本发明实施例4的靶向NKG2DL的特异性CAR-T细胞能够特异性识别NKG2DL阳性的肿瘤细胞，并具有靶向杀伤力。
因此，本发明所构建的靶向NKG2DL的特异性嵌合抗原受体、以及经其病毒载体感染的靶向NKG2DL的特异性CAR-T细胞可以应用于治疗淋巴瘤、胃癌、卵巢癌、肺癌和前列腺癌等肿瘤病症。
应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施方式中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。
上文所列出的一系列的详细说明仅仅是针对本发明的可行性实施方式的具体说明，它们并非用以限制本发明的保护范围，凡未脱离本发明技艺精神所作的等效实施方式或变更均应包含在本发明的保护范围之内。
 工业实用性
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<110> 南京凯地生物科技有限公司
<120> 靶向NKG2DL的特异性嵌合抗原受体及其CAR-T细胞以及它们的应用
<130> 2017-NKG2DL-CAR-T
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Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn
65 70 75 80
Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala
85 90 95
Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr
100 105 110
Ile Cys Met Gln Arg Thr Val
115
<210> 5
<211> 136
<212> PRT
<213> 人
<400> 5
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
1 5 10 15
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
20 25 30
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
35 40 45
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
50 55 60
Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile
65 70 75 80
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
85 90 95
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
100 105 110
Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr
115 120 125
Tyr Ile Cys Met Gln Arg Thr Val
130 135
<210> 6
<211> 152
<212> PRT
<213> 人
<400> 6
Ile Arg Phe Ile Ile Met Val Thr Ile Trp Ser Ala Val Phe Leu Asn
1 5 10 15
Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys
20 25 30
Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln
35 40 45
Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
50 55 60
Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp
65 70 75 80
Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile
85 90 95
Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro
100 105 110
Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr
115 120 125
Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr
130 135 140
Tyr Ile Cys Met Gln Arg Thr Val
145 150
<210> 7
<211> 170
<212> PRT
<213> 人
<400> 7
Arg Glu Asn Ala Ser Pro Phe Phe Phe Cys Cys Phe Ile Ala Val Ala
1 5 10 15
Met Gly Ile Arg Phe Ile Ile Met Val Thr Ile Trp Ser Ala Val Phe
20 25 30
Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser
35 40 45
Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys
50 55 60
Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser
65 70 75 80
Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp
85 90 95
Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val
100 105 110
His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu
115 120 125
Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala
130 135 140
Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro
145 150 155 160
Asn Thr Tyr Ile Cys Met Gln Arg Thr Val
165 170
<210> 8
<211> 45
<212> PRT
<213> 人
<400> 8
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 9
<211> 24
<212> PRT
<213> 人
<400> 9
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 10
<211> 42
<212> PRT
<213> 人
<400> 10
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 11
<211> 112
<212> PRT
<213> 人
<400> 11
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 12
<211> 63
<212> DNA
<213> 人
<400> 12
atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60
ccg 63
<210> 1 3
<211> 357
<212> DNA
<213> 人
<400> 1 3
ccctgcccaa agaactggat atgttacaaa aataattgct accagttttt cgacgagtcc 60
aagaattggt atgaatcaca agccagctgc atgtcccaaa atgcgtcatt gttgaaggta 120
tattctaagg aggaccaaga tttgttgaag ttggttaaat cctatcattg gatggggttg 180
gtccatatac ctacaaatgg ttcatggcag tgggaagatg gatctatact gagcccaaat 240
cttctgacaa taattgaaat gcaaaaaggc gattgtgccc tttacgctag tagcttcaaa 300
ggttatattg agaactgtag cacaccgaac acttatatct gtatgcagag aacggtt 357
<210> 1 4
<211> 408
<212> DNA
<213> 人
<400> 1 4
tctctgttca accaagaggt gcagatacca cttaccgaat catattgtgg cccctgccca 60
aagaactgga tatgttacaa aaataattgc taccagtttt tcgacgagtc caagaattgg 120
tatgaatcac aagccagctg catgtcccaa aatgcgtcat tgttgaaggt atattctaag 180
gaggaccaag atttgttgaa gttggttaaa tcctatcatt ggatggggtt ggtccatata 240
cctacaaatg gttcatggca gtgggaagat ggatctatac tgagcccaaa tcttctgaca 300
ataattgaaa tgcaaaaagg cgattgtgcc ctttacgcta gtagcttcaa aggttatatt 360
gagaactgta gcacaccgaa cacttatatc tgtatgcaga gaacggtt 408
<210> 15
<211> 456
<212> DNA
<213> 人
<400> 15
attaggttta ttattatggt cacaatatgg tctgccgtat ttctcaactc tctgttcaac 60
caagaggtgc agataccact taccgaatca tattgtggcc cctgcccaaa gaactggata 120
tgttacaaaa ataattgcta ccagtttttc gacgagtcca agaattggta tgaatcacaa 180
gccagctgca tgtcccaaaa tgcgtcattg ttgaaggtat attctaagga ggaccaagat 240
ttgttgaagt tggttaaatc ctatcattgg atggggttgg tccatatacc tacaaatggt 300
tcatggcagt gggaagatgg atctatactg agcccaaatc ttctgacaat aattgaaatg 360
caaaaaggcg attgtgccct ttacgctagt agcttcaaag gttatattga gaactgtagc 420
acaccgaaca cttatatctg tatgcagaga acggtt 456
<210> 16
<211> 510
<212> DNA
<213> 人
<400> 16
agagaaaatg ctagtccctt tttcttctgt tgctttattg cggtcgcaat ggggattagg 60
tttattatta tggtcacaat atggtctgcc gtatttctca actctctgtt caaccaagag 120
gtgcagatac cacttaccga atcatattgt ggcccctgcc caaagaactg gatatgttac 180
aaaaataatt gctaccagtt tttcgacgag tccaagaatt ggtatgaatc acaagccagc 240
tgcatgtccc aaaatgcgtc attgttgaag gtatattcta aggaggacca agatttgttg 300
aagttggtta aatcctatca ttggatgggg ttggtccata tacctacaaa tggttcatgg 360
cagtgggaag atggatctat actgagccca aatcttctga caataattga aatgcaaaaa 420
ggcgattgtg ccctttacgc tagtagcttc aaaggttata ttgagaactg tagcacaccg 480
aacacttata tctgtatgca gagaacggtt 510
<210> 1 7
<211> 135
<212> DNA
<213> 人
<400> 1 7
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 1 8
<211> 72
<212> DNA
<213> 人
<400> 1 8
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 1 9
<211> 126
<212> DNA
<213> 人
<400> 1 9
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 20
<211> 336
<212> DNA
<213> 人
<400> 20
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 21
<211> 1259
<212> DNA
<213> 人
<400> 21
tgcaaagatg gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg 60
tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 120
cagtccccga gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc 180
gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 240
gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 300
ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 360
gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc 420
ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc 480
gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc 540
ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg 600
ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg 660
gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt 720
cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg 780
gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa 840
ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg 900
cagggagctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac 960
aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg 1020
cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg 1080
gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc 1140
cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt 1200
tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtga 1259

Claims (10)

  1. 靶向NKG2DL的特异性嵌合抗原受体，其氨基酸序列从氨基端到羧基端由引导序列、人NKG2D序列、人CD8铰链区序列、人CD8跨膜区序列、人4-1BB胞内结构域序列和CD3ζ结构域序列依次串联而成；
    所述引导序列如SEQ ID No.3所示；以及，
    所述人NKG2D序列如SEQ ID No.4、SEQ ID No.5、SEQ ID No.6或SEQ ID No.7中任意一个所示。
  2. 如权利要求1所述的靶向NKG2DL的特异性嵌合抗原受体，其特征在于：
    所述人CD8铰链区的氨基酸序列如SEQ ID No.8所示；
    所述人CD8跨膜区的氨基酸序列如SEQ ID No.9所示；
    所述人4-1BB胞内结构域的氨基酸序列如SEQ ID No.10所示；
    所述CD3ζ结构域的氨基酸序列如SEQ ID No.11所示。
  3. 编码权利要求1或2所述的靶向NKG2DL的特异性嵌合抗原受体的分子。
  4. 如权利要求3所述的分子，其特征在于：
    所述基因的核苷酸序列：从5’端到3’端，由引导序列（如SEQ ID No.12所示）、人NKG2D序列（如SEQ ID No.13、SEQ ID No.14、SEQ ID No.15或SEQ ID No.16中任意一个所示）、人CD8铰链区序列（如SEQ ID No.17所示）、人CD8跨膜区序列（如SEQ ID No.18所示）、人4-1BB胞内结构域序列（如SEQ ID No.19所示）和CD3ζ结构域序列（如SEQ ID No.20所示）依次串联而成。
  5. 包含权利要求3或4所述基因的重组载体或表达质粒。
  6. 包含权利要求3或4所述基因的重组细胞，其特征在于：
    所述重组细胞为能够表达权利要求1或2所述的靶向NKG2DL的特异性嵌合抗原受体的免疫效应细胞；
    所述免疫效应细胞为细胞毒性T淋巴细胞、NK细胞、NKT细胞或辅助性T细胞等。
  7. 包含权利要求3或4所述基因的重组病毒，其特征在于：
    所述重组病毒为能够表达权利要求1或2所述的靶向NKG2DL的特异性嵌合抗原受体、且能够侵染免疫效应细胞的病毒；
    所述免疫效应细胞为细胞毒性T淋巴细胞、NK细胞、NKT细胞或辅助性T细胞等；
    所述病毒为慢病毒、腺病毒、腺联病毒或逆转录病毒等。
  8. 靶向NKG2DL的特异性CAR-T细胞的制备方法，其特征在于：
    所述制备方法包括以下步骤：
    首先，将权利要求3所述的靶向NKG2DL特异性嵌合抗原受体的分子，通过分子克隆的方式连接到慢病毒表达载体lentiGuide-Puro中，获得靶向NKG2DL的特异性CAR表达质粒；
    然后，将获得的所述靶向NKG2DL的特异性CAR表达质粒转染293T细胞，获得病毒液；
    然后用所述病毒液感染T细胞，从感染后的细胞中获得表达靶向NKG2DL的特异性嵌合抗原受体的T细胞。
  9. 如权利要求5所述重组载体或表达质粒，如权利要求6所述重组细胞，如权利要求7所述重组病毒，如权利要求8所述制备方法获得的靶向NKG2DL的特异性CAR-T细胞在制备治疗肿瘤的产品中的应用。
  10. 如权利要求9所述应用，其特征在于：
    所述肿瘤包含表达NKG2DL蛋白的肿瘤细胞
PCT/CN2017/076795 2017-03-15 2017-03-15 靶向nkg2dl的特异性嵌合抗原受体及其car-t细胞以及它们的应用 Ceased WO2018165913A1 (zh)

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CN201880003510.0A CN109803983B (zh) 2017-03-15 2018-02-06 靶向nkg2dl的特异性嵌合抗原受体t细胞，其制备方法和应用
PCT/CN2018/075389 WO2018166307A1 (zh) 2017-03-15 2018-02-06 靶向nkg2dl的特异性嵌合抗原受体t细胞，其制备方法和应用
US16/116,953 US11090335B2 (en) 2017-03-15 2018-08-30 Chimeric antigen receptor targeting human NKG2DL and methods of preparing said receptor and pharmaceutical composition

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CN111632135A (zh) * 2020-05-09 2020-09-08 深圳宾德生物技术有限公司 靶向nkg2d的嵌合抗原受体t细胞在治疗前列腺癌中的应用、治疗前列腺癌的药物
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CN114796470A (zh) * 2021-01-28 2022-07-29 深圳宾德生物技术有限公司 白藜芦醇类化合物与靶向nkg2d配体的car-t细胞联合制备治疗恶性肿瘤的药物
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CN115960256A (zh) * 2022-09-26 2023-04-14 东南大学 一种长效的嵌合抗原受体、载体及其构建方法和应用
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CN119569892A (zh) * 2023-09-06 2025-03-07 上海恩凯细胞技术有限公司 特异性靶向肿瘤非经典hla分子的嵌合抗原受体的应用
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