WO2021179353A1 - 靶向nkg2d的嵌合抗原受体、嵌合抗原受体t细胞及其制备方法和应用 - Google Patents

靶向nkg2d的嵌合抗原受体、嵌合抗原受体t细胞及其制备方法和应用 Download PDF

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WO2021179353A1
WO2021179353A1 PCT/CN2020/081339 CN2020081339W WO2021179353A1 WO 2021179353 A1 WO2021179353 A1 WO 2021179353A1 CN 2020081339 W CN2020081339 W CN 2020081339W WO 2021179353 A1 WO2021179353 A1 WO 2021179353A1
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nkg2d
chimeric antigen
antigen receptor
targeting
cells
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French (fr)
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刘茂玄
许晨光
黎琴子
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深圳宾德生物技术有限公司
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    • C07K16/2851—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 lectin superfamily, e.g. CD23, CD72
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/844—Liver
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/852—Pancreas
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K2319/00—Fusion polypeptide
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    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention requires the priority of the prior application filed on March 11, 2020 with the title of "Chimeric Antigen Receptor Targeting NKG2D, Chimeric Antigen Receptor T Cells, and Preparation Method and Application thereof" and the application number 202010168618.4 , The content of the above-mentioned prior application is incorporated into this text by way of introduction.
  • the invention relates to the field of biomedicine, in particular to a chimeric antigen receptor targeting NKG2D, a chimeric antigen receptor T cell, and a preparation method and application thereof.
  • CAR-T Chimeric Antigen Receptor T Cell
  • CAR-T technology is a new type of immune cell therapy, which is to infuse CAR-modified T cells into the human body to activate the body’s own immune system and continuously kill targeted tumor cells , Is considered to be one of the most effective treatments for malignant tumors.
  • NKG2D is a lectin-like type II transmembrane glycoprotein, which is expressed on the surface of NK cells, CD8+ T cells, activated macrophages and tumor-infiltrating ⁇ T cells, and the expression of NKG2D ligand is a cell in An indicator of "stress state", such as when cells are infected by viruses and malignant transformation, but rarely appears on the surface of healthy cells.
  • NKG2D ligands are expressed in varying degrees on tumor cells derived from almost all tissues of the human body, such as liver cancer, ovarian cancer, and pancreatic cancer.
  • NKG2D ligands provide a more precise target for tumor immunotherapy, there are currently few studies on CAR-T cells targeting NKG2D, and they cannot achieve long-lasting and long-lasting effects in killing tumor cells in practical applications. ability.
  • the present invention provides a chimeric antigen receptor targeting NKG2D, chimeric antigen receptor T cells and preparation thereof, a recombinant vector containing a gene encoding the chimeric antigen receptor targeting NKG2D, And their application in the prevention, diagnosis and treatment of malignant tumors with NKG2D ligand.
  • the present invention provides a chimeric antigen receptor CAR-NKG2D targeting NKG2D.
  • the amino acid sequence of the CAR-NKG2D includes a single NKG2D targeting NKG2D linked sequentially from the amino terminus to the carboxy terminus.
  • the amino acid sequence of the chain antibody, the extracellular hinge region, the transmembrane region and the intracellular signal region, wherein the NKG2D-targeted single chain antibody includes the amino acid sequence shown in SEQ ID NO:1.
  • the “sequential connection from the amino terminal to the carboxyl terminal” is specifically: the carboxyl terminal of the amino acid sequence of the single-chain antibody targeting NKG2D is connected to the amino terminal of the amino acid sequence of the extracellular hinge region, and the extracellular hinge region
  • the carboxy terminus of the amino acid sequence of the transmembrane region is connected to the amino terminus of the amino acid sequence of the transmembrane region, and the carboxy terminus of the amino acid sequence of the transmembrane region is connected to the amino terminus of the amino acid sequence of the intracellular signal region.
  • the NKG2D-targeted single-chain antibody is a humanized single-chain antibody, which can avoid causing an immune response in the human body and has high safety.
  • the encoding gene of the single-chain antibody targeting NKG2D includes the nucleotide sequence shown in SEQ ID NO: 5.
  • the extracellular hinge region is used to promote the binding of the single-chain antibody targeting NKG2D to FGFR4 expressed on tumors.
  • the extracellular hinge region includes a CD8 ⁇ hinge region, a CD28 hinge region, a CD4 hinge region, a CD5 hinge region, a CD134 hinge region, a CD137 hinge region, and a combination of one or more of the ICOS hinge region.
  • the transmembrane region is used to immobilize the NKG2D-targeting chimeric antigen receptor CAR-NKG2D.
  • the transmembrane region includes one or a combination of a CD3 transmembrane region, a CD4 transmembrane region, a CD8 transmembrane region, and a CD28 transmembrane region.
  • the intracellular signal area is used to provide signals for T cell activation, maintain the survival time of T cells and activate T cell proliferation signal pathways.
  • the intracellular signal area includes the 4-1BB signal area, the CD3 ⁇ signal area, the ICOS signal area, the CD27 signal area, the OX40 signal area, the CD28 signal area, the IL1R1 signal area, the CD70 signal area, and the TNFRSF19L signal area.
  • the intracellular signal area includes the 4-1BB signal area, the CD3 ⁇ signal area, the ICOS signal area, the CD27 signal area, the OX40 signal area, the CD28 signal area, the IL1R1 signal area, the CD70 signal area, and the TNFRSF19L signal area.
  • the extracellular hinge region is a CD8 ⁇ hinge region; the transmembrane region is a CD8 transmembrane region; and the intracellular signal region includes 4-1BB sequentially connected from the amino terminus to the carboxy terminus Signal area and CD3 ⁇ signal area. That is, the amino acid sequence of the CAR-NKG2D includes the amino acid sequences of the NKG2D-targeting single-chain antibody, the CD8 ⁇ hinge region, the CD8 transmembrane region, the 4-1BB signal region, and the CD3 ⁇ signal region that are sequentially linked from the amino terminal to the carboxy terminal. .
  • amino acid sequence of the CAR-NKG2D is shown in SEQ ID NO: 2.
  • the CAR-NKG2D encoding gene includes a nucleotide sequence as shown in SEQ ID NO: 3.
  • the CAR-NKG2D coding gene includes the coding gene of the signal peptide sequentially connected from the 5'end to the 3'end, the coding gene of the single-chain antibody targeting NKG2D, the coding gene of the extracellular hinge region, and The coding gene of the membrane region and the coding gene of the intracellular signal region.
  • the coding gene of the single-chain antibody targeting NKG2D includes a nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO:1.
  • the CAR-NKG2D encoding gene includes the nucleotide sequence shown in SEQ ID NO: 4. Compared with the nucleotide sequence shown in SEQ ID NO: 3, the nucleotide sequence shown in SEQ ID NO: 4 contains more genes encoding signal peptides.
  • the gene encoding the signal peptide can better guide the expression of the CAR-NKG2D to the cell surface, but when the chimeric antigen receptor CAR-NKG2D is expressed on the surface of the T cell, the signal peptide is used in the process of protein translation and maturation. Signal peptidase cleavage. Therefore, the amino acid sequence of the translated CAR-NKG2D (SEQ ID NO: 2) does not contain the amino acid sequence of the signal peptide.
  • the amino acid sequence of the signal peptide includes the amino acid sequence shown in SEQ ID NO:6.
  • the gene encoding the signal peptide includes the nucleotide sequence shown in SEQ ID NO:7.
  • the NKG2D-targeting chimeric antigen receptor CAR-NKG2D provided in the first aspect of the present invention can specifically target tumor cells expressing NKG2D ligand. After CAR-NKG2D is combined with NKG2D ligand, it can make T The intracellular signal area of the cell is activated, which promotes the expansion of T cells in the patient's body, and efficiently and specifically kills tumor cells, while causing almost no damage to normal cells, and can maintain its self-renewal ability and tumors for a long time Lethality.
  • the present invention provides a chimeric antigen receptor T cell targeting NKG2D, including the chimeric antigen receptor targeting NKG2D described in the first aspect.
  • the chimeric antigen receptor T cell targeting NKG2D provided in the second aspect of the present invention can specifically target tumor cells expressing NKG2D.
  • CAR-FGFR4 is combined with FGFR4, the intracellular signal region of the CAR-T cell It is activated, promotes its expansion in the patient's body, and has a long-lasting and long-lasting ability to efficiently and specifically kill tumor cells, while causing almost no damage to normal cells.
  • the present invention provides a recombinant vector comprising the gene encoding the chimeric antigen receptor CAR-NKG2D targeting FGFR4 as described in the first aspect.
  • the vector in the recombinant vector is at least one of a viral vector and a non-viral vector.
  • the viral vector includes a lentiviral vector, an adenoviral vector or a retroviral vector.
  • it is a lentiviral vector, such as at least one of pWPXLD vector, pLEX-MCS vector, pSico vector, and pCgpV vector.
  • the non-viral vectors include plasmid vectors and phage vectors.
  • the plasmid vector may be, but is not limited to, eukaryotic plasmid vector, prokaryotic plasmid vector, minicircle DNA, transposon and the like.
  • the vector is minicircle DNA
  • the recombinant minicircle DNA inserted into the gene encoding the chimeric antigen receptor targeting NKG2D can be directly transfected into CD3-positive T lymphocytes to prepare the chimeric antigen receptor targeting NKG2D T cells.
  • the recombinant vector provided by the third aspect of the present invention is safe and efficient, can stably and efficiently introduce CAR-NKG2D encoding gene into host cells or replicate, and can be used to prepare chimeric antigen receptor T cells targeting NKG2D to make the T cells continuously and steadily exert their targeting and killing effects.
  • the present invention provides a method for preparing chimeric antigen receptor T cells targeting NKG2D, including:
  • coding genes for chimeric antigen receptors targeting NKG2D including coding genes for signal peptides sequentially connected from the 5'end to 3'ends, coding genes for single-chain antibodies targeting NKG2D, and extracellular hinges Region coding gene, transmembrane region coding gene, intracellular signal region coding gene, wherein the coding gene of the single-chain antibody targeting NKG2D includes the nucleus corresponding to the amino acid sequence shown in SEQ ID NO:1 Nucleotide sequence
  • packaging the recombinant gene delivery vector and transfecting the host cell to obtain a recombinant lentivirus includes: co-transfecting the recombinant gene delivery vector with an envelope plasmid and a packaging plasmid into the host cell to obtain the recombinant lentivirus Lentivirus.
  • the packaging of the recombinant lentivirus can adopt a three-plasmid system or a four-plasmid system, and the envelope plasmid and the packaging plasmid are materials commonly used in the field.
  • the host cells may include HEK293T cells, 293 cells, 293T cells, 293FT cells, SW480 cells, u87MG cells, HOS cells or COS7 cells, etc., but are not limited thereto.
  • the gene delivery vector may be the above-mentioned plasmid vector, for example, the pWPXLd plasmid.
  • the CAR-NKG2D encoding gene is located between the BamH I restriction site and the EcoR I restriction site of the pWPXLD vector.
  • the envelope plasmid is PMD2G
  • the packaging plasmid is psPAX2
  • the host cell is a HEK293T cell.
  • CD3-positive T lymphocytes are obtained from human peripheral blood mononuclear cells.
  • the human peripheral blood mononuclear cells are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood, and the like. Further optionally, it is derived from fresh peripheral blood or bone marrow collected from a cancer patient one month after surgery and one month after radiotherapy and chemotherapy.
  • the present invention provides the NKG2D-targeting chimeric antigen receptor as described in the first aspect, and the NKG2D-targeting chimeric antigen receptor prepared by the preparation method as described in the second aspect or the preparation method as described in the fourth aspect.
  • the specific form of the application may be: a kit is provided.
  • the malignant tumors are tumors that highly express NKG2D ligand, such as liver cancer, cervical cancer, and pancreatic cancer.
  • the mode of administration can be, but not limited to, intravenous injection, tumor in situ injection, subcutaneous injection, etc.
  • the dosage, frequency, etc. selected in the specific application are selected according to actual needs, and there is no limitation on this.
  • the present invention provides a pharmaceutical composition for preventing, diagnosing and treating malignant tumors, the pharmaceutical composition comprising the chimeric antigen receptor targeting NKG2D as described in the first aspect, such as the second The chimeric antigen receptor T cell targeting NKG2D according to the aspect, or the chimeric antigen receptor T cell targeting NKG2D prepared by the preparation method according to the fourth aspect, and the recombinant vector according to the third aspect At least one of.
  • the NKG2D-targeting chimeric antigen receptor T cell provided in the second aspect is a preferred composition.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier and/or adjuvant.
  • the pharmaceutically acceptable carrier includes water, physiological saline and other non-aqueous solvents, albumin, hemoglobin, phospholipids and the like.
  • the auxiliary materials include one or more of diluents, excipients and stabilizers.
  • the form of the pharmaceutical composition includes one or more of a decoction, a powder, a tablet, a capsule, a pill, an oral agent, and a granule.
  • the form of the drug depends on the actual application.
  • the pharmaceutical composition is administered orally or by injection. Further, the injection is administered by intraperitoneal injection, subcutaneous injection, intramuscular injection or intravenous injection.
  • the pharmaceutical composition further includes other active ingredients that have or treat malignant tumors.
  • other active ingredients that have or treat malignant tumors.
  • chemotherapeutics for example, chemotherapeutics.
  • the NKG2D-targeted chimeric antigen receptor and T cells can self-replicate and multiply, have a long half-life, and form memory cells, which play a continuous targeting effect. It can specifically bind to tumor cells and produce a strong and long-lasting killing effect on tumor cells without causing damage to normal cells.
  • Figure 1 is a plasmid map of the pWPXLd-CAR-NKG2D recombinant plasmid provided by the embodiment of the present invention.
  • FIG. 2 is a flow cytometric diagram showing the expression of the chimeric antigen receptor CAR-NKG2D after the recombinant lentivirus carrying CAR-NKG2D encoding gene infects T cells according to an embodiment of the present invention; wherein the T cells are labeled with anti-CD3-FITC , Use anti-NKG2D-APC antibody to label CAR-NKG2D.
  • UTD stands for T cells that have not been infected with the virus
  • CAR-T stands for CAR-T cells expressing CAR-NKG2D.
  • Figure 3 is a diagram showing the in vitro killing effect of NKG2D-targeted chimeric antigen receptor T cells (CAR-T) and virus-infected T lymphocytes (UTD) on Huh7 liver cancer cells in vitro.
  • CAR-T NKG2D-targeted chimeric antigen receptor T cells
  • UTD virus-infected T lymphocytes
  • Fig. 4 is a diagram of the in vitro killing effect of CAR-T cells targeting NKG2D and T lymphocytes (UTD) not infected with virus (UTD) on Panc-1 pancreatic cancer cells provided by the embodiments of the present invention.
  • Figure 5 is a diagram of the in vitro killing effect of CAR-T cells targeting NKG2D and T lymphocytes (UTD) not infected with virus on Hela cervical cancer cells provided by embodiments of the present invention.
  • Fig. 6 is an effect diagram of in vivo treatment of tumor-bearing mice (mice inoculated with cervical cancer cells) on CAR-T cells targeting NKG2D provided by an embodiment of the present invention; the left picture shows CAR-T cell injection tumor-bearing mice The picture of the tumor removed 40 days after the mouse, the right picture is the tumor weight data of the mice in each treatment group.
  • the preparation of chimeric antigen receptor T cells targeting NKG2D specifically includes the following steps:
  • the coding genes of signal peptide, single chain antibody targeting NKG2D, CD8 ⁇ hinge region, CD8 transmembrane region, 4-1BB signal region and CD3 ⁇ signal region were prepared separately, and the signal peptide was targeted to the single chain of NKG2D by PCR.
  • the coding genes of antibody, CD8 ⁇ hinge region, CD8 transmembrane region, 4-1BB signal region and CD3 ⁇ signal region are linked together from 5'end to 3'end to obtain CAR-NKG2D encoding gene, which encodes CAR-NKG2D
  • the gene is shown in SEQ ID NO: 4.
  • the coding gene sequence of the signal peptide is shown in SEQ ID NO: 7
  • the coding gene sequence of the single-chain antibody targeting NKG2D is shown in SEQ ID NO: 2.
  • the CAR-NKG2D encoding gene was inserted between the BamH I and EcoR I restriction sites of the pWPXLD vector, and located after the elongation factor 1 ⁇ (EF1 ⁇ ) of the pWPXLD vector, with EF1 ⁇ as the promoter.
  • a start codon such as ATG
  • a stop codon such as TAA
  • the obtained expression plasmid, envelope plasmid and packaging plasmid (psPAX2, pMD2.G) were co-transfected into HEK293T packaging cells with lipofectamine 3000, and the recombinant lentivirus was harvested by centrifugation after 48 hours. Then, the recombinant lentivirus is infected with CD3 positive T lymphocytes stimulated by CD3/CD28 magnetic beads to obtain NKG2D CAR-T cells.
  • the pWPXLd-CAR-NKG2D recombinant plasmid obtained above and the packaging plasmid psPAX2 and the envelope plasmid pMD2G were co-transfected into the cultured HEK293T cells by lipofection reagent Lipofectamine3000.
  • the virus-containing supernatant was harvested at 48h, filtered through a 0.45 ⁇ m filter, and stored in an ultra-low temperature refrigerator at -80°C; at 72h, the virus-containing supernatant was harvested for the second time, filtered with a 0.45 ⁇ m filter, and the virus supernatant harvested at 48h After merging, add them to the ultracentrifuge tube and put them into the Beckman ultracentrifuge one by one.
  • the centrifugation parameters to 25000rpm, the centrifugation time to 2h, and the centrifugation temperature to be controlled at 4°C; after centrifugation, discard the supernatant and try to remove residues Add the virus preservation solution to the liquid on the tube wall and gently pipette to resuspend; after it is fully dissolved, centrifuge at high speed at 10,000 rpm and centrifuge for 5 minutes. Take the supernatant to determine the titer by fluorescence.
  • the virus is 100 ⁇ l, 2 ⁇ 10 8 /mL aliquoted and stored in an ultra-low temperature refrigerator at -80°C to obtain a recombinant lentivirus with CAR-NKG2D encoding gene.
  • PBMC peripheral blood mononuclear cells
  • PBMC is derived from autologous venous blood, autologous bone marrow, umbilical cord blood and placental blood. It is best from fresh peripheral blood or bone marrow collected from cancer patients one month after surgery and one month after radiotherapy and chemotherapy.
  • the patient's blood is drawn and sent to the blood separation chamber; the peripheral blood mononuclear cells are collected, Ficoll centrifugal separation, and the middle layer cells are collected; after washing with PBS, PBMCs are obtained.
  • PBMC blood pressure
  • serum-free basal medium to prepare a cell suspension
  • CD3/CD28 immunomagnetic beads according to the ratio of magnetic beads to cells of 3:1, and incubate for 1-2h at room temperature; incubate with a magnet pair.
  • the cells of the magnetic beads are screened; after washing with PBS and removing the immunomagnetic beads, CD3 positive T lymphocytes are obtained.
  • CD3 positive T lymphocytes obtained by the immunomagnetic bead separation method in b) Take the CD3 positive T lymphocytes obtained by the immunomagnetic bead separation method in b), and add the recombinant lentivirus in step (3) corresponding to the number of CD3 positive cells for culture.
  • a real-time cell analyzer (xCElligence RTCA SP) was used for tumor cell killing experiments.
  • Effective target ratio (E:T) was added to the corresponding number of two groups of effector cells (ie, NKG2D CAR-T cells and T cells stimulated only by magnetic beads without virus infection (abbreviated as UTD cells, ie, Example 1) CD3 positive T lymphocytes in step (4b)), and then co-culture for a period of time ( ⁇ 24 hours).
  • UTD cells ie, NKG2D CAR-T cells and T cells stimulated only by magnetic beads without virus infection
  • FIGs 3 to 5 are graphs showing the effects of CAR-T cells targeting NKG2D in killing Huh7 liver cancer cells, Panc-1 pancreatic cancer cells, and Hela cervical cancer cells in vitro, respectively, according to the embodiments of the present invention. It can be seen from Figure 3 to Figure 5 that the CAR-T cells targeting NKG2D provided by the present invention have very strong killing ability against liver cancer cells, pancreatic cancer cells, and cervical cancer cells in vitro, and the killing ability is much higher than that of negative
  • the control group and the blank control group show that the CAR-T cells targeting NKG2D provided by the present invention have very considerable application prospects in the preparation of drugs for preventing, diagnosing and treating malignant tumors.
  • tumor cells specifically Hela cervical cancer cells
  • NKG2D-targeted CAR-T cells provided by the present invention
  • T lymphocytes stimulated by magnetic beads and not infected with virus abbreviated as UTD group
  • physiological saline blade control group
  • the number of T cells in both the UTD group and the NKG2D CAR-T high-dose group was 3.5 ⁇ 10 6
  • the number of T cells in the NKG2D CAR-T low-dose group was 0.85 ⁇ 10 6 .
  • NKG2D CAR-T high-dose group has a significant difference (P ⁇ 0.01) compared with the blank control group and the UTD group, showing a strong tumor suppressing effect.
  • the results in Figure 6 show that the NKG2D-targeted CAR-T cells prepared by this method can better protect mice from death due to tumors.

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Abstract

一种靶向NKG2D的嵌合抗原受体，其氨基酸序列包括从氨基端到羧基端顺次连接的靶向NKG2D的单链抗体、胞外铰链区、跨膜区和胞内信号区的氨基酸序列，其中，所述靶向NKG2D的单链抗体包括如SEQ ID NO：1所示的氨基酸序列。一种包括靶向NKG2D的的嵌合抗原受体的CAR-T细胞及其制备，以及它们在制备预防、诊断、治疗肝癌、***和胰腺癌等恶性肿瘤中的应用。

Description

靶向NKG2D的嵌合抗原受体、嵌合抗原受体T细胞及其制备方法和应用
本发明要求2020年3月11日递交的发明名称为“靶向NKG2D的嵌合抗原受体、嵌合抗原受体T细胞及其制备方法和应用”、申请号为202010168618.4的在先申请优先权，上述在先申请的内容以引入的方式并入本文本中。
技术领域
本发明涉及生物医药领域，具体涉及一种靶向NKG2D的嵌合抗原受体、嵌合抗原受体T细胞及其制备方法和应用。
背景技术
CAR-T(嵌合抗原受体T细胞)技术是一种新型免疫细胞疗法，它是将经过CAR改造的T细胞回输至人体，激活人体自身免疫***，持续性对靶向肿瘤细胞进行杀伤，被认为是目前最有效的恶性肿瘤的治疗方式之一。
NKG2D是一种凝集素样的II型跨膜糖蛋白，其在NK细胞、CD8+T细胞、活化的巨噬细胞和肿瘤浸润的γδT细胞表面均有表达，且NKG2D配体的表达是细胞处于“应激状态”的一个指标，例如当细胞受到病毒感染和恶性转化时，但很少会出现在健康细胞的表面。而NKG2D配体几乎在人体所有组织来源的肿瘤细胞上有不同程度的表达，例如肝癌、卵巢癌、胰腺癌等。
尽管NKG2D配体为肿瘤的免疫治疗提供了一个更为精确的靶点，但目前靶向NKG2D的CAR-T细胞的研究较少，且在实际应用中不能长效持久地发挥高杀伤肿瘤细胞的能力。
发明内容
为解决上述技术问题，本发明提供了一种靶向NKG2D的嵌合抗原受体、嵌合抗原受 体T细胞及其制备、包含靶向NKG2D的嵌合抗原受体的编码基因的重组载体，以及它们在预防、诊断和治疗具有NKG2D配体的恶性肿瘤中的应用。
具体地，第一方面，本发明提供了一种靶向NKG2D的嵌合抗原受体CAR-NKG2D，所述CAR-NKG2D的氨基酸序列包括从氨基端到羧基端顺次连接的靶向NKG2D的单链抗体、胞外铰链区、跨膜区和胞内信号区的氨基酸序列，其中，所述靶向NKG2D的的单链抗体包括如SEQ ID NO：1所示的氨基酸序列。
所述“从氨基端到羧基端顺次连接”具体为：所述靶向NKG2D的单链抗体的氨基酸序列的羧基端与所述胞外铰链区的氨基酸序列的氨基端相连，胞外铰链区的氨基酸序列的羧基端与所述跨膜区的氨基酸序列的氨基端相连，所述跨膜区的氨基酸序列的羧基端与所述胞内信号区的氨基酸序列的氨基端相连。
其中，所述靶向NKG2D的单链抗体为人源化单链抗体，可以避免引起人机体的免疫反应，具有较高的安全性。可选地，所述靶向NKG2D的单链抗体的编码基因包括如SEQ ID NO：5所示的核苷酸序列。
本发明中，所述胞外铰链区用于促进所述靶向NKG2D的单链抗体与肿瘤上的表达的F GFR4结合。可选地，所述胞外铰链区包括CD8α铰链区、CD28铰链区、CD4铰链区、CD5铰链区、CD134铰链区、CD137铰链区、ICOS铰链区中的一种或多种的组合。
本发明中，所述跨膜区用于固定所述靶向NKG2D的嵌合抗原受体CAR-NKG2D。可选地，所述跨膜区包括CD3跨膜区、CD4跨膜区、CD8跨膜区、CD28跨膜区中的一种或多种的组合。
本发明中，所述胞内信号区用于提供T细胞活化的信号，维持T细胞的生存时间和激活T细胞增殖信号通路。可选地，所述胞内信号区包括4-1BB信号区、CD3ζ信号区、ICOS信号区、CD27信号区、OX40信号区、CD28信号区、IL1R1信号区、CD70信号区、TNFRSF19L信号区中的一种或多种的组合。
在本发明一实施方式中，所述胞外铰链区为CD8α铰链区；所述跨膜区为CD8跨膜区； 所述胞内信号区包括从氨基端到羧基端顺次连接的4-1BB信号区和CD3ζ信号区。即，所述CAR-NKG2D的氨基酸序列包括从氨基端到羧基端顺次连接的靶向NKG2D的单链抗体、CD8α铰链区、CD8跨膜区、4-1BB信号区和CD3ζ信号区的氨基酸序列。
可选地，所述CAR-NKG2D的氨基酸序列如SEQ ID NO：2所示。
进一步可选地，所述CAR-NKG2D的编码基因包括如SEQ ID NO：3所示的核苷酸序列。
进一步地，所述CAR-NKG2D的编码基因包括从5’端到3’端顺次连接的信号肽的编码基因、靶向NKG2D的单链抗体的编码基因、胞外铰链区的编码基因、跨膜区的编码基因和胞内信号区的编码基因。其中，所述靶向NKG2D的单链抗体的编码基因包括如SEQ ID NO：1所示的氨基酸序列所对应的核苷酸序列。
可选地，所述CAR-NKG2D的编码基因包括如SEQ ID NO：4所示的核苷酸序列。与SEQ ID NO：3所示的核苷酸序列相比，SEQ ID NO：4所示的核苷酸序列中多了信号肽的编码基因。
其中，所述信号肽的编码基因可较好地指导所述CAR-NKG2D表达到细胞表面，但在嵌合抗原受体CAR-NKG2D表达到T细胞表面时，信号肽在蛋白翻译成熟过程中被信号肽酶切割。因此，在翻译成的CAR-NKG2D的氨基酸序列(SEQ ID NO：2)中并未带有信号肽的氨基酸序列。
可选地，所述信号肽的氨基酸序列包括如SEQ ID NO：6所示的氨基酸序列。可选地，所述信号肽的编码基因包括如SEQ ID NO：7所示的核苷酸序列。
本发明第一方面提供的所述靶向NKG2D的嵌合抗原受体CAR-NKG2D可以专一性地靶向表达NKG2D配体的肿瘤细胞，在CAR-NKG2D与NKG2D配体结合后，可以使T细胞的胞内信号区被激活，促进T细胞在患者体内的扩增，并高效且特异性地杀伤肿瘤细胞，而对正常细胞几乎不会造成损伤，且能够持久地维持其自我更新能力和肿瘤杀伤力。
第二方面，本发明提供了一种靶向NKG2D的嵌合抗原受体T细胞，包括第一方面所述的靶向NKG2D的嵌合抗原受体。
本发明第二方面提供的靶向NKG2D的嵌合抗原受体T细胞可以专一性地靶向表达NKG2D的肿瘤细胞，在CAR-FGFR4与FGFR4结合后，该CAR-T细胞的胞内信号区被激活，促进其在患者体内的扩增，长效持久地发挥高效且特异性杀伤肿瘤细胞的能力，而对正常细胞几乎不会造成损伤。
第三方面，本发明提供了一种重组载体，所述重组载体包括如第一方面所述的靶向FGFR4的嵌合抗原受体CAR-NKG2D的编码基因。
可选地，所述重组载体中的所述载体为病毒载体和非病毒载体中的至少一种。
其中，所述病毒载体包括慢病毒载体、腺病毒载体或逆转录病毒载体。优选为慢病毒载体，例如pWPXLD载体、pLEX-MCS载体、pSico载体和pCgpV载体中的至少一个。
其中，所述非病毒载体包括质粒载体和噬菌体载体。具体地，所述质粒载体可以但不限于为真核质粒载体、原核质粒载体、微环DNA、转座子等。当所述载体为微环DNA时，可以将***靶向NKG2D的嵌合抗原受体的编码基因的重组微环DNA直接转染CD3阳性T淋巴细胞，制得靶向NKG2D的嵌合抗原受体T细胞。
本发明第三方面提供的重组载体安全高效，可以稳定高效地将CAR-NKG2D的编码基因导入宿主细胞中或复制，并可以用于制备靶向NKG2D的嵌合抗原受体T细胞，使所述T细胞持续、稳定地发挥靶向、杀伤效力。
第四方面，本发明提供了一种靶向NKG2D的嵌合抗原受体T细胞的制备方法，包括：
(1)提供靶向NKG2D的嵌合抗原受体的编码基因，包括从5’端到3’端顺次连接的信号肽的编码基因、靶向NKG2D的单链抗体的编码基因、胞外铰链区的编码基因、跨膜区的编码基因、胞内信号区的编码基因，其中，所述靶向NKG2D的单链抗体的编码基因包括如SEQ ID NO：1所示的氨基酸序列所对应的核苷酸序列；
(2)将所述靶向NKG2D的嵌合抗原受体的编码基因***到基因传递载体中，得到重组基因传递载体；
(3)将所述重组基因传递载体进行包装并转染宿主细胞，得到重组慢病毒；
(4)将所述重组慢病毒转染CD3阳性T淋巴细胞，获得靶向NKG2D的嵌合抗原受体T细胞。
可选地，将所述重组基因传递载体进行包装并转染宿主细胞，得到重组慢病毒，包括：将所述重组基因传递载体与包膜质粒和包装质粒共同转染宿主细胞，得到所述重组慢病毒。所述重组慢病毒的包装可采用三质粒***或四质粒***，包膜质粒、包装质粒为本领域常用的物质。
可选地，所述宿主细胞可以包括HEK293T细胞、293细胞、293T细胞、293FT细胞、SW480细胞、u87MG细胞、HOS细胞或COS7细胞等，但不限于此。
在本发明一实施方式中，所述基因传递载体可以为上述质粒载体，例如为pWPXLd质粒。此时，所得重组基因传递载体(可称为“重组质粒载体”)中，CAR-NKG2D的编码基因位于pWPXLD载体的BamH Ⅰ酶切位点和EcoR Ⅰ酶切位点之间。
在本发明一实施方式中，所述包膜质粒为PMD2G，所述包装质粒为psPAX2，所述宿主细胞为HEK293T细胞。
可选地，步骤(4)中，CD3阳性T淋巴细胞是从人源外周血单个核细胞中分离获得。
其中，所述人源外周血单个核细胞来源于自体静脉血、自体骨髓、脐带血和胎盘血等。进一步可选地，来源于癌症患者手术一个月后、放化疗一个月后采集的新鲜外周血或骨髓。
第五方面，本发明提供了如第一方面所述的靶向NKG2D的嵌合抗原受体、如第二方面所述的或如第四方面所述的制备方法制得的靶向NKG2D的嵌合抗原受体T细胞、如第三方面所述的重组载体在制备预防、诊断和治疗恶性肿瘤的药物中的应用。
在本发明一实施方式中，所述应用的具体形式可以为：提供了一种试剂盒。
其中，所述恶性肿瘤为高表达NKG2D配体的肿瘤，如肝癌、***和胰腺癌等。
在应用中，给药方式可以但不限于为静脉注射、肿瘤原位注射、皮下注射等。具体应用时选择的剂量、次数等根据实际需要进行选择，对此不做限定。
第六方面，本发明提供了一种药物组合物，用于预防、诊断和治疗恶性肿瘤，所述药 物组合物包括如第一方面所述的靶向NKG2D的嵌合抗原受体、如第二方面所述的靶向NKG2D的嵌合抗原受体T细胞或如第四方面所述的制备方法制得的靶向NKG2D的嵌合抗原受体T细胞、如第三方面所述的重组载体中的至少一种。
其中，第二方面提供的所述靶向NKG2D的嵌合抗原受体T细胞为优选组成。
可选地，所述药物组合物还包括药学上可接受的载体和/或辅料。
其中，所述药学上可接受的载体包括水、生理盐水及其他非水性溶剂、白蛋白、血红蛋白、磷脂等。
其中，所述辅料包括稀释剂、赋形剂和稳定剂中的一种或多种。
可选地，所述药物组合物的形式包括汤剂、散剂、片剂、胶囊剂、丸剂、口服剂和颗粒剂中的一种或多种。所述药物的形式取决于在实际中的应用。
可选地，所述药物组合物通过口服或注射的方式施用。进一步地，所述注射通过腹膜内注射、皮下注射、肌肉注射或静脉注射的方式施用。
可选地，所述药物组合物还包括其他具有或治疗恶性肿瘤的活性成分。例如，化疗剂。
本发明第六方面提供的所述药物组合物中，所述靶向NKG2D的嵌合抗原受体及T细胞能够自我复制繁殖，半衰期长，并形成记忆细胞，起到持续的靶向作用。可以特异性地结合肿瘤细胞，并对肿瘤细胞产生持久的强杀伤效果，且对正常细胞不会造成损伤。
附图说明
图1为本发明实施例提供的pWPXLd-CAR-NKG2D重组质粒的质粒图谱。
图2为本发明实施例提供的带CAR-NKG2D编码基因的重组慢病毒感染T细胞后表达嵌合抗原受体CAR-NKG2D的流式检测结果图；其中，使用anti-CD3-FITC标记T细胞，用anti-NKG2D-APC抗体标记CAR-NKG2D，图中UTD代表未经病毒感染的T细胞，CAR-T代表表达CAR-NKG2D的CAR-T细胞。
图3为本发明实施例提供的靶向NKG2D的嵌合抗原受体T细胞(CAR-T)和未经病毒感 染的T淋巴细胞(UTD)的体外杀伤Huh7肝癌细胞的效果图。
图4为本发明实施例提供的靶向NKG2D的CAR-T细胞和未经病毒感染的T淋巴细胞(UTD)的体外杀伤Panc-1胰腺癌细胞的效果图。
图5为本发明实施例提供的靶向NKG2D的CAR-T细胞和未经病毒感染的T淋巴细胞(UTD)的体外杀伤Hela***细胞的效果图。
图6为本发明实施例提供的靶向NKG2D的CAR-T细胞的体内治疗荷瘤小鼠(接种了***细胞的小鼠)的效果图；其中，左图为CAR-T细胞注射荷瘤鼠40天后取出的肿瘤的图片，右图为对各处理组小鼠的瘤重数据。
具体实施方式
以下所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明的保护范围。
实施例1
靶向NKG2D的嵌合抗原受体T细胞的制备，具体包括以下步骤：
(1)制备靶向NKG2D的嵌合抗原受体CAR-NKG2D的基因序列
分别制备信号肽、靶向NKG2D的单链抗体、CD8α铰链区、CD8跨膜区、4-1BB信号区和CD3ζ信号区的编码基因，通过PCR的方法将上述信号肽、靶向NKG2D的单链抗体、CD8α铰链区、CD8跨膜区、4-1BB信号区和CD3ζ信号区的编码基因依次从5’端到3’端连接到一起，得到CAR-NKG2D的编码基因，该CAR-NKG2D的编码基因如SEQ ID NO：4所示。其中，所述信号肽的编码基因序列如SEQ ID NO：7所示，靶向NKG2D的单链抗体的编码基因序列如SEQ ID NO：2所示。
(2)构建pWPXLd-CAR-NKG2D重组质粒
将上述CAR-NKG2D的编码基因***到pWPXLD载体的BamH Ⅰ和EcoR Ⅰ酶切位点 之间，且位于pWPXLD载体的延伸因子1α(EF1α)之后，以EF1α为启动子。所述CAR-NKG2D的编码基因***到pWPXLD载体时，在所述CAR-NKG2D的编码基因的5’端可加入起始密码子(如ATG)与pWPXLD载体中BamH Ⅰ酶切位点相连，3’端可加入终止密码子(如TAA)与pWPXLD载体中EcoR Ⅰ酶切位点相连。然后转入大肠杆菌感受态细胞DH5α，进行阳性克隆PCR鉴定和测序鉴定。经过PCR产物凝胶电泳检测和测序鉴定符合目的片段大小和序列，成功构建如图1所示的pWPXLd-CAR-NKG2D重组质粒。
将所获得的表达质粒与包膜质粒和包装质粒(psPAX2，pMD2.G)通过脂质体转染试剂Lipofectamine3000共转染HEK293T包装细胞中，48小时后离心收获重组慢病毒。然后将所述重组慢病毒感染经CD3/CD28磁珠刺激的CD3阳性T淋巴细胞，获得NKG2D CAR-T细胞。
(3)重组慢病毒构建
将上述获得的pWPXLd-CAR-NKG2D重组质粒与包装质粒psPAX2、包膜质粒pMD2G三者通过脂质体转染试剂Lipofectamine3000共转染培养好的HEK293T细胞。第48h收获含病毒的上清，经0.45μm滤膜过滤，于-80℃超低温冰箱中保存；第72h二次收获含病毒的上清，0.45μm滤膜过滤，与第48h收获的病毒上清合并后一起加入超速离心管中，逐一放入至Beck man超速离心机内，设置离心参数为25000rpm，离心时间为2h，离心温度控制在4℃；离心结束后，弃去上清，尽量去除残留在管壁上的液体，加入病毒保存液，轻轻反复吹打重悬；经充分溶解后，高速离心10000rpm，离心5min后，取上清荧光法测定滴度，病毒按照100μl，2×10 8个/mL分装，保存于-80℃超低温冰箱，得到带CAR-NKG2D编码基因的重组慢病毒。
(4)靶向NKG2D的嵌合抗原受体T细胞的制备
a)PBMC(外周血单个核细胞)的分离
PBMC来源于自体静脉血、自体骨髓、脐带血和胎盘血等。最好是来源于癌症患者手术一个月后、放化疗一个月后采集的新鲜外周血或骨髓。
抽取病人血液，送样至血液分离室；采集外周血单个核细胞，Ficoll离心分离后取中间 层细胞；经PBS洗涤后，得到PBMC。
b)免疫磁珠法分离抗原特异性T淋巴细胞
取上述PBMC，加入不含血清的基础培养基，配成细胞悬液；按磁珠与细胞的比例为3:1，加入CD3/CD28免疫磁珠，室温孵1-2h；采用磁铁对孵育好磁珠的细胞进行筛选；PBS洗涤，去除免疫磁珠后，得到CD3阳性T淋巴细胞。
c)病毒转染法制备抗原特异性T淋巴细胞
取b)中经免疫磁珠分离法得到的CD3阳性T淋巴细胞，加入与CD3阳性细胞数相应的病毒滴度的步骤(3)中的所述重组慢病毒进行培养。
培养的第3天，进行细胞计数和换液，调整细胞浓度为1×10 6个/mL，接种，培养；培养的第5天，观察细胞状态，如果细胞密度增大，则稀释细胞浓度为1×10 6个/mL，检测细胞活性，继续培养。扩增培养到第9-11天，收集细胞，同时经过流式细胞仪检测靶向NKG2D的嵌合抗原受体CAR-NKG2D的表达，结果如图2所示。经检测，经上述重组慢病毒感染的T细胞中，CAR-NKG2D的阳性率约为70％。这表明成功制得得到靶向NKG2D的CAR-T细胞，并保存在回输专用的细胞冻存液中，以供患者回输用。
效果实施例
为了评估本发明所描述的上述方法制备的靶向NKG2D的CAR-T细胞在杀伤肿瘤细胞方面的效果，进行如下效果实施例。
体外评价：
采用实时细胞分析仪(xCElligence RTCA SP)进行肿瘤细胞杀伤实验。首先在该仪器配套的E-Plate 96孔板中加入5000个肿瘤靶细胞(Huh7肝癌细胞、Panc-1胰腺癌细胞、Hela***细胞)的完全培养基进行培养，培养24小时后，按照不同效靶比(E:T)分别加入相应数量的两组效应细胞(即，NKG2D CAR-T细胞和只经磁珠刺激、未加病毒感染的T细胞(简写为UTD细胞，即，实施例1步骤(4b)中的CD3阳性T淋巴细胞)，随后共培养一段时间(≥24小时)。使用实时细胞分析仪的细胞指数(cell index)值对细胞杀伤 效果进行分析。
图3-图5分别为本发明实施例提供的靶向NKG2D的CAR-T细胞体外杀伤Huh7肝癌细胞、Panc-1胰腺癌细胞、Hela***细胞的效果图。从图3-图5可以看出，本发明提供的靶向NKG2D的CAR-T细胞对体外肝癌细胞、胰腺癌细胞、***细胞均具有非常强的杀伤能力，且杀伤能力远远高于阴性对照组和空白对照组，这表明本发明提供的靶向NKG2D的CAR-T细胞在制备预防、诊断和治疗恶性肿瘤的药物中具有非常可观的应用前景。
体内评价：
向每只小鼠尾静脉注射1×10 6个肿瘤细胞(具体为Hela***细胞)，得到荷瘤小鼠模型。然后向荷瘤小鼠体内分别注射本发明提供的靶向NKG2D的CAR-T细胞、只经磁珠刺激、未加病毒感染的T淋巴细胞(简写为UTD组)及以及生理盐水(空白对照组)，注射40天后，取出各处理组的小鼠内的肿瘤进行观察，并进行瘤重统计，结果如图6所示。其中，UTD组和NKG2D CAR-T高剂量组的T细胞个数均为3.5×10 6个，NKG2D CAR-T低剂量组的T细胞个数为0.85×10 6个。
从图6可以看出，NKG2D CAR-T高剂量组与空白对照组和UTD组相比，具有显著性的差异(P<0.01)，显示出较强的抑瘤效果。图6的结果表明，本方法制备的靶向NKG2D的CAR-T细胞能够更好地保护小鼠免于因肿瘤导致的死亡。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (15)

  1. 一种靶向NKG2D的嵌合抗原受体，其特征在于，所述靶向NKG2D的嵌合抗原受体的氨基酸序列包括从氨基端到羧基端顺次连接的靶向NKG2D的单链抗体、胞外铰链区、跨膜区和胞内信号区的氨基酸序列，其中，所述靶向NKG2D的的单链抗体包括如SEQ ID NO：1所示的氨基酸序列。
  2. 如权利要求1所述的靶向NKG2D的嵌合抗原受体，其特征在于，所述胞外铰链区为CD8α铰链区，所述跨膜区为CD8跨膜区，所述胞内信号区包括从氨基端到羧基端顺次连接的4-1BB信号区和CD3ζ信号区。
  3. 如权利要求2所述的靶向NKG2D的嵌合抗原受体，其特征在于，所述靶向NKG2D的嵌合抗原受体的氨基酸序列包括如SEQ ID NO：2所示的氨基酸序列。
  4. 如权利要求3所述的靶向NKG2D的嵌合抗原受体，其特征在于，所述靶向NKG2D的嵌合抗原受体的编码基因包括如SEQ ID NO：3所示的核苷酸序列。
  5. 如权利要求4所述的靶向NKG2D的嵌合抗原受体，其特征在于，所述靶向NKG2D的嵌合抗原受体的编码基因包括如SEQ ID NO：4所示的核苷酸序列。
  6. 一种靶向NKG2D嵌合抗原受体T细胞，其特征在于，其表面表达如权利要求1-5任一项所述的靶向NKG2D的嵌合抗原受体。
  7. 一种重组载体，其特征在于，包括如权利要求1-5任一项所述的靶向NKG2D的嵌合抗原受体的编码基因。
  8. 一种靶向NKG2D的嵌合抗原受体T细胞的制备方法，其特征在于，包括：
    (1)提供靶向NKG2D的嵌合抗原受体的编码基因，包括从5’端到3’端顺次连接的信号肽的编码基因、靶向NKG2D的单链抗体的编码基因、胞外铰链区的编码基因、跨膜区的编码基因、胞内信号区的编码基因，其中，所述靶向NKG2D的单链抗体的编码基因包括如SEQ ID NO：1所示的氨基酸序列所对应的核苷酸序列；
    (2)将所述靶向NKG2D的嵌合抗原受体的编码基因***到基因传递载体中，得到重组基因传递载体；
    (3)将所述重组基因传递载体进行包装并转染宿主细胞，得到重组慢病毒；
    (4)将所述重组慢病毒转染CD3阳性T淋巴细胞，获得靶向NKG2D的嵌合抗原受体T细胞。
  9. 如权利要求8所述的制备方法，其特征在于，所述靶向NKG2D的嵌合抗原受体的编码基因包括如SEQ ID NO：4所示的核苷酸序列。
  10. 如权利要求1-5任一项所述的靶向NKG2D的嵌合抗原受体、如权利要求6所述的或如权利要求8-9任一项所述的制备方法制得的靶向NKG2D的嵌合抗原受体T细胞、如权利要求7所述的重组载体在制备预防、诊断和治疗恶性肿瘤的药物中的应用。
  11. 如权利要求10所述的应用，其特征在于，所述恶性肿瘤为高表达NKG2D配体的肿瘤，包括肝癌、***和胰腺癌。
  12. 一种预防、诊断和治疗恶性肿瘤的药物组合物，其特征在于，所述药物组合物包括如权利要求1-5任一项所述的靶向NKG2D的嵌合抗原受体、如权利要求6所述的或如权利要求8-9任一项所述的制备方法制得的靶向NKG2D的嵌合抗原受体T细胞、如权利要求7所述的重组载体。
  13. 如权利要求12所述的药物组合物，其特征在于，所述药物组合物还包括药学上可接受的载体和/或辅料。
  14. 如权利要求13所述的药物组合物，其特征在于，所述载体包括水、生理盐水及其他非水性溶剂、白蛋白、血红蛋白、磷脂中的至少一种，所述辅料包括稀释剂、赋形剂和稳定剂中的至少一种。
  15. 如权利要求12所述的药物组合物，其特征在于，所述药物组合物通过口服或注射的方式施用。
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