WO2018166307A1 - 靶向nkg2dl的特异性嵌合抗原受体t细胞，其制备方法和应用 - Google Patents

靶向nkg2dl的特异性嵌合抗原受体t细胞，其制备方法和应用 Download PDF

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WO2018166307A1
WO2018166307A1 PCT/CN2018/075389 CN2018075389W WO2018166307A1 WO 2018166307 A1 WO2018166307 A1 WO 2018166307A1 CN 2018075389 W CN2018075389 W CN 2018075389W WO 2018166307 A1 WO2018166307 A1 WO 2018166307A1
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seq
human
amino acid
sequence
nkg2dl
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PCT/CN2018/075389
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French (fr)
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代红久
孙彬
赵旭东
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南京凯地生物科技有限公司
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Priority to CN201880003510.0A priority Critical patent/CN109803983B/zh
Priority to US16/116,953 priority patent/US11090335B2/en
Publication of WO2018166307A1 publication Critical patent/WO2018166307A1/zh

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Definitions

  • the invention belongs to the field of tumor immunotherapy biomedical technology, and relates to a specific chimeric antigen receptor T cell, in particular to a targeted NKG2DL.
  • Cancer immunotherapy mainly includes adoptive cell therapy, immunomodulators, tumor vaccines, and immunoassay block therapy.
  • chimeric antigen receptor-modified immune cells especially Chimeric
  • Antigen Receptor T-Cell, CAR-T has undoubtedly become the star of research institutions and pharmaceutical companies vying for "seeking".
  • CAR-T Chimeric Antigen Receptor T-Cell, a chimeric antigen receptor-modified T cell
  • the principle is mainly to genetically modify a patient's self-extracted T cells to form a CAR-T cell by modifying a chimeric antigen receptor.
  • T cells can specifically recognize tumor surface-associated antigens (tumor cell markers) to target killing of tumors.
  • CAR-T cells Compared with conventional immune cells, CAR-T cells have higher targeting, killing activity and persistence, and can overcome the tumor local immunosuppressive microenvironment and break the host immune tolerance state.
  • the modified immune cell therapy represented by CAR-T cells has a remarkable therapeutic effect on the treatment of acute leukemia and non-Hodgkin's lymphoma, and is considered to be one of the most promising treatment methods for tumors.
  • 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 an urgent problem to be solved.
  • the key to the application of immune response cell technology that exerts chimeric antigen receptor modification 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 object of the present invention is to overcome the problem that the specificity of T cell binding and killing tumor cells in the tumor internal environment faced by the existing tumor clinical technology is not strong, and the killing efficiency is low.
  • the engineered NKG2DL-targeted immune response cells of the present invention are capable of increasing the killing efficiency of tumor cells, thereby providing a novel approach for tumor therapy with promising applications.
  • the application provides a human NKG2D protein ligand or a functional variant thereof that binds to human NKG2DL, comprising a sequence selected from the group consisting of:
  • SEQ ID No. 4 SEQ ID No. 5, SEQ ID No. 6, SEQ ID The amino acid sequence shown in No. 7, SEQ ID No. 8, or SEQ ID No. 9, or (2) (1) a functional variant produced by modification of one or more amino acids.
  • amino acid modifications include, but are not limited to, substitutions, deletions, and additions of amino acids including, but not limited to, derived polypeptides produced by substitutions, deletions, and additions of one or more amino acids or Peptide analogs.
  • the amino acid sequence of the human NKG2D protein ligand or a functional variant thereof that binds to human NKG2DL is SEQ It is represented by ID No. 4 or homologous to the amino acid sequence shown by SEQ ID No. 4.
  • the amino acid sequence of the human NKG2D protein ligand or a functional variant thereof that binds to human NKG2DL is SEQ It is represented by ID No. 5 or homologous to the amino acid sequence shown by SEQ ID No. 5.
  • the amino acid sequence of the human NKG2D protein ligand or a functional variant thereof that binds to human NKG2DL is SEQ It is represented by ID No. 6 or homologous to the amino acid sequence shown by SEQ ID No. 6.
  • the amino acid sequence of the human NKG2D protein ligand or a functional variant thereof that binds to human NKG2DL is SEQ It is represented by ID No. 7 or homologous to the amino acid sequence shown by SEQ ID No. 7.
  • the amino acid sequence of the human NKG2D protein ligand or a functional variant thereof that binds to human NKG2DL is SEQ It is represented by ID No. 8 or homologous to the amino acid sequence shown by SEQ ID No. 8.
  • the amino acid sequence of the human NKG2D protein ligand or a functional variant thereof that binds to human NKG2DL is SEQ It is represented by ID No. 9 or homologous to the amino acid sequence shown by SEQ ID No. 9.
  • the present application provides a specific chimeric antigen receptor that targets human NKG2DL, comprising an amino acid sequence of a leader sequence that is ligated from the amino terminus to the carboxy terminus, and an extracellular targeting of human NKG2DL.
  • Identifying a domain amino acid sequence, a transmembrane domain amino acid sequence, and an intracellular signal domain amino acid sequence, the amino acid sequence of the extracellular recognition domain that binds to human NKG2DL comprising the targeted binding human of the first aspect of the present application
  • the target ratio is 10:1
  • the killing efficiency against tumor cells is 40% ⁇ 70%.
  • the specific chimeric antigen receptor that targets binding to human NKG2DL further comprises a hinge region.
  • the transmembrane domain comprises a transmembrane region.
  • the intracellular signaling domain comprises an immunoreceptor tyrosine activation motif and a costimulatory signal domain;
  • the transmembrane region comprises a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 ⁇ transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, 2B4 transmembrane domain, BTLA transmembrane domain, synthetic peptide (not based on immune response) Any of the related proteins).
  • the immunoreceptor tyrosine activation motif comprises an intracellular signal domain of an CD3 ⁇ chain or an Fc ⁇ RI ⁇ intracellular signal structure
  • the costimulatory signal domain comprises at least one of a CD28 intracellular signaling domain, a CD137/4-1BB intracellular signaling domain, a CD134/OX40 intracellular signaling domain, and an ICOS intracellular signaling domain.
  • the hinge region is selected from the group consisting of a CD8 alpha hinge region, an IgG hinge region, or a hinge region or hinge region comprising all or part of an immunoglobulin that has been modified by one or more amino acids.
  • the leader sequence is selected from the group consisting of: (1) having the amino acid sequence set forth in SEQ ID No. 3, or (2) The amino acid-modified functional variant of (1); wherein the amino acid-modified functional variant is a polypeptide having homology to the amino acid sequence of SEQ ID No. 3.
  • the transmembrane region is a human CD8 polypeptide.
  • the hinge region is a human CD8 polypeptide.
  • the amino acid sequence of the human CD8 polypeptide of the hinge region is selected from the group consisting of SEQ ID A polypeptide represented by No. 10 or an amino acid-modified functional variant of (2) (1), wherein the amino acid-modified functional variant is the same as SEQ ID A polypeptide having homology to the amino acid sequence shown in No. 10.
  • amino acid sequence of human CD8 of the transmembrane region is selected from the group consisting of SEQ ID A polypeptide represented by No. 11 or an amino acid-modified functional variant of (2) (1), wherein the amino acid-modified functional variant is the same as SEQ ID A polypeptide having homology to the amino acid sequence shown in No. 11.
  • the human 4-1BB intracellular domain is selected from:
  • (1) a polypeptide having the amino acid sequence of SEQ ID NO. 12; or (2) The amino acid-modified functional variant of (1), wherein the amino acid-modified functional variant is a polypeptide having homology to the amino acid sequence of SEQ ID No. 12.
  • the human CD28 ⁇ intracellular domain is selected from the group consisting of: (1) having the SEQ ID A polypeptide of the amino acid sequence shown in No. 13; or 2) an amino acid-modified functional variant of (1), wherein the amino acid-modified functional variant is the same as SEQ ID A polypeptide having homology to the amino acid sequence shown in No. 13.
  • the CD3 ⁇ intracellular domain is selected from the group consisting of: (1) having the SEQ ID A polypeptide of the amino acid sequence shown in No. 14; or (2) an amino acid-modified functional variant of (1). Where the amino acid modified functional variant is the same as SEQ ID A polypeptide having homology to the amino acid sequence shown in No. 14.
  • the CD3 ⁇ intracellular domain is selected from the group consisting of: (1) having the SEQ ID A polypeptide of the amino acid sequence shown in No. 15; or (2) an amino acid-modified functional variant of (1). Where the amino acid modified functional variant is SEQ ID A polypeptide having homology to the amino acid sequence shown in No. 15.
  • the specific chimeric antigen receptor that targets binding to human NKG2DL is recombinantly expressed or expressed by a vector.
  • the present application provides a nucleic acid molecule encoding a specific chimeric antigen receptor that binds to human NKG2DL according to the second aspect, the nucleic acid molecule comprising a coding sequenced in series from 5' to 3' A nucleotide sequence of a leader sequence, a nucleotide sequence encoding human NKG2D, a nucleotide sequence encoding a transmembrane domain, and a nucleotide sequence encoding an intracellular signal domain.
  • the intracellular signal domain comprises an immunoreceptor tyrosine activation motif and a costimulatory signal domain.
  • the nucleic acid molecule that targets a specific chimeric antigen receptor that binds to human NKG2DL further comprises a nucleotide sequence that encodes a hinge region.
  • the transmembrane domain comprises a transmembrane region.
  • the transmembrane region comprises a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 ⁇ transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, ICOS transmembrane domain, CTLA-4 transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, 2B4 transmembrane domain, BTLA transmembrane domain, synthetic peptide (not based on immune response) Any of the related proteins),
  • the immunoreceptor tyrosine activation motif comprises an intracellular signal domain of an CD3 ⁇ chain or an Fc ⁇ RI ⁇ intracellular signal structure.
  • the costimulatory signal domain comprises at least one of a CD28 intracellular signaling domain, a CD137/4-1BB intracellular signaling domain, a CD134/OX40 intracellular signaling domain, and an ICOS intracellular signaling domain.
  • the hinge region is selected from the group consisting of a CD8 alpha hinge region, an IgG hinge region, or a modification comprising one or more amino acid modifications in the hinge region or hinge region of the entire or portion of the immunoglobulin.
  • the nucleotide encoding the leader sequence is as set forth in SEQ ID No. 16 or with SEQ ID The nucleotide sequence shown in No. 16 is homologous.
  • nucleotide sequence encoding human NKG2D is set forth in SEQ ID No. 17 or with SEQ ID The nucleotide sequence shown in No. 17 is homologous.
  • nucleotide sequence encoding human NKG2D is set forth in SEQ ID No. 18 or with SEQ ID The nucleotide sequence shown in No. 18 is homologous.
  • nucleotide sequence encoding human NKG2D is set forth in SEQ ID No. 19 or with SEQ ID
  • the nucleotide sequence shown in No. 19 is homologous.
  • nucleotide sequence encoding human NKG2D is set forth in SEQ ID No. 20 or with SEQ ID The nucleotide sequence shown in No. 20 is homologous.
  • nucleotide sequence encoding human NKG2D is set forth in SEQ ID No. 21 or with SEQ ID The nucleotide sequence shown in No. 21 is homologous.
  • nucleotide sequence encoding human NKG2D is set forth in SEQ ID No 22 or with SEQ ID
  • the nucleotide sequence shown in No. 22 is homologous.
  • nucleotide sequence of human CD8 encoding the hinge region is set forth in SEQ ID No. 23 or with SEQ ID The nucleotide sequence shown in No. 23 is homologous.
  • the nucleotide sequence of the human CD8 encoding the transmembrane region is set forth in SEQ ID No. 24 or with SEQ ID The nucleotide sequence shown in No. 24 is homologous.
  • the nucleotide sequence encoding the human 4-1BB intracellular domain is set forth in SEQ ID No. 25 or with SEQ The nucleotide sequence shown by ID No. 25 is homologous.
  • nucleotide sequence encoding the human CD28 intracellular domain is set forth in SEQ ID No. 26 or with SEQ The nucleotide sequence shown by ID No. 26 is homologous.
  • the nucleotide sequence encoding the human CD3 sputum intracellular domain is set forth in SEQ ID No. 27 or with SEQ The nucleotide sequence shown by ID No. 27 is homologous.
  • the nucleotide sequence encoding the human CD3 sputum intracellular domain is SEQ ID It is shown by No. 28 or homologous to the nucleotide sequence shown by SEQ ID No. 28.
  • the present application provides a recombinant vector or expression plasmid comprising the chimeric antigen receptor of the second aspect of the application or the nucleic acid of the third aspect of the application.
  • the present application provides a promoter for constructing the recombinant vector of the fourth aspect of the present application and expressing the specific chimeric antigen receptor targeted for binding to human NKG2DL according to the second aspect of the present application
  • the promoter includes, but is not limited to, a nucleotide sequence such as SEQ The EF1 alpha promoter shown in No. 29 and the EFS promoter shown in SEQ No. 30.
  • the promoter is a nucleotide sequence such as the EFS promoter set forth in SEQ No. 30.
  • the promoter is a nucleotide sequence such as the EF1 alpha promoter of SEQ No. 29.
  • the present application provides a recombinant virus which is capable of expressing a specific chimeric antigen receptor targeting NKG2DL according to the second aspect of the present invention and capable of infecting an immune response cell.
  • the present application provides an isolated modified immune response cell comprising the chimeric antigen receptor targeting human NKG2DL as described in the second aspect of the invention, which is recombined by the fourth aspect of the invention
  • the vector or expression plasmid is transformed.
  • the immune response cell comprises a cytotoxic T lymphocyte, an NK cell, an NKT cell, or a helper T cell.
  • the immune response cell further comprises at least one exogenous co-stimulatory ligand.
  • the present application provides a method for preparing an isolated chimeric antigen receptor-modified immune response cell of the seventh aspect of the present application, comprising the steps of:
  • the nucleic acid molecule of the third aspect is ligated into an expression vector by molecular cloning to obtain an expression vector targeting a specific chimeric antigen receptor of NKG2DL;
  • the obtained CAR expression vector targeting NKG2DL is then transfected into 293T cells to obtain a virus solution;
  • the immune response cells are infected with the virus solution, and modified immune response cells expressing the seventh aspect of the specific chimeric antigen receptor targeting NKG2DL of the second aspect are obtained from the infected cells.
  • the immune response cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a human embryonic stem cell, and a pluripotent that can differentiate into a lymphoid cell.
  • a T cell preferably T cells or natural killer (NK) cells.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of a human NKG2D protein ligand or a functional variant thereof, which binds to human NKG2DL, as described in one aspect of the invention, or a functional variant thereof
  • the present application provides a reagent for treating or preventing a neoplasia, a pathogen infection, an autoimmune disease, an allograft, or a transplant rejection or anti-aging agent in a subject suffering from a disease, discomfort or health disorder.
  • a cassette comprising the human NKG2D protein targeting human NKG2DL and a functional variant thereof according to the first aspect of the invention, the second chimeric antigen receptor capable of binding to human NKG2DL, and the invention
  • the kit further comprises written instructions for treating or preventing a disease, discomfort, or health disorder in the immune response cell.
  • the present application provides the human NKG2D protein targeting human NKG2DL and the variant thereof according to the first aspect of the present invention, and the specific chimeric antigen receptor targeting the binding of human NKG2DL according to the second aspect
  • An effective amount of a chimeric antigen receptor T cell (CAR-T cell) comprising NKG2DL-targeting NKG2D as described in the seventh aspect of the present application.
  • uncomfortable or healthy disorders are associated with specific interactions of NKG2DL and NKG2D
  • the disease, discomfort, or health disorder includes neoplasia, infection, autoimmune disease, allograft, transplant rejection, aging.
  • the invention constructs a specific chimeric antigen receptor (CAR) targeting human NKG2DL and the CAR modified T cell (CAR-T cell) based on the human NKG2D molecule, and the novel CAR-T cell can effectively target multiple attacks.
  • Tumor cells can be used to prepare a preparation for treating a tumor, especially a drug preparation for a tumor positively expressing NKG2DL.
  • the inventors of the present invention unexpectedly discovered in the study that the chimeric antigen receptor-modified T cells comprising the NKG2D specifically recognizing human NKG2DL constructed by the present invention have simple preparation steps, and when the effective target ratio is 10:1, It has a tumor cell killing rate of up to 40% ⁇ 70%, which can significantly prolong the survival time of immune cells in patients, and enhance the ability of immune cells to target tumor cells, especially tumors expressing NKG2DL, and strengthen the tumor or tumor formation.
  • modified immune response cells of the invention targeting NKG2DL chimeric human NKG2D provide a new solution for treating tumors, and have good industrial application prospects.
  • Figure 1 is a schematic illustration of the structure of a lentiviral vector used in the present invention as an example.
  • Figure 2 is a schematic illustration showing the sequence of ligation of the various portions of the chimeric antigen receptor in Examples 1 and 2.
  • A. EF1 alpha promoter (SEQ ID NO: SEQ ID NO: ID No. 29), B. EFS promoter (SEQ ID NO: 30); wherein 1 is the amino acid sequence of the human CD3 ⁇ domain, 2 is the amino acid sequence of the human 4-1BB intracellular domain, 3 is the amino acid sequence of the human CD8 transmembrane domain, 4 is the amino acid sequence of the CD8 hinge region, 5 is the human NKG2D amino acid sequence, 6 is the leader sequence, and 7 is the EF1 The alpha promoter, 8 is the EFS promoter.
  • Figure 3 is a graph showing the results of T cell purity flow cytometry in Example 2.
  • Figure 4 is a flow cytometric test result of CAR-T cell activity in Example 5, wherein 4-A was a blank control group: T cells without virus solution; 4-B was KD-019 control group: specific CAR-T cells targeting CD19; 4-C was KD-025: Specific CAR-T cells that target NKG2DL.
  • Figure 5 is a comparison of KD-025 in Example 5.
  • Flow cytometry results of CAR molecular expression 5-A. blank control group: no T cells infected with viral fluid; 5-B. KD-019 control group: specific CAR-T cells targeting CD19; 5-C.KD-025: specific CAR-T cells targeting NKG2DL.
  • Figure 6 Major subtypes of NKG2DL MicA, MicB, ULBP1, Detection of expression of ULBP2 and ULBP3 in tumor cell lines, liver cancer, ovarian cancer, gastric cancer, prostate cancer, lung cancer, breast cancer, glioma (Fig.
  • 6-A is control: isotype control (R&D) Systems, IC003A); 6-B is a liver cancer cell SMMC7721, 6-C is a glioma cell U251, 6-D is an ovarian cancer cell SKOV3, 6-E is a gastric cancer cell NUGC4, 6-F is a parental cell tumor HTB186, 6-G is a pancreas
  • the cancer cells BXPC3 and 6-H are lung cancer cells A549, 6-I are prostate cancer cells PC3, and 6-J are breast cancer cells MCF7).
  • Fig. 7 is a graph showing the results of testing the specific cell-killing rate of the specific NKG2DL-targeting CAR-T cells of the present application using the liver cancer cell SMMC7721 as a target cell.
  • Figure 8 is a graph showing the results of tumor cell killing rate test of specific NKG2DL-targeting specific CAR-T cells of the present application using glioma cell U251 as a target cell.
  • Fig. 9 is a graph showing the results of measuring the tumor cell killing rate of the specific NKG2DL-targeting specific CAR-T cells of the present application using ovarian cancer cell line SKOV3 as a target cell.
  • Fig. 10 shows the results of measuring the tumor cell killing rate of the specific NKG2DL-targeting specific CAR-T cells of the present application using the gastric cancer cell NUGC4 as a target cell.
  • Figure 11 is a graph showing the results of tumor cell killing rate test of specific NKG2DL-targeting specific CAR-T cells of the present application by using the parental cell tumor cell HTB186 as a target cell.
  • Figure 12 is a graph showing the results of tumor cell killing rate test of specific NKG2DL-targeting specific CAR-T cells of the present application using pancreatic cancer cell BXPC3 as a target cell.
  • Figure 13 is a graph showing the results of tumor cell killing rate test of specific NKG2DL-targeting specific CAR-T cells of the present application using lung cancer cell A549 as a target cell.
  • Figure 14 is a graph showing the results of tumor cell killing rate test of specific NKG2DL-targeting specific CAR-T cells of the present application using prostate cancer cell line PC3 as a target cell.
  • Figure 15 is a graph showing the results of tumor cell killing rate test of specific NKG2DL-targeting specific CAR-T cells of the present application using breast cancer cell MCF7 as a target cell.
  • Figure 16 is a graph showing the killing test results of a tumor animal model of a specific CAR-T cell targeting NKG2DL of the present application using liver cancer SMMC7721 as a target cell, wherein 16-A shows the growth of xenografts in nude mice; 16-B shows tumor fluorescence intensity values.
  • Figure 17 is a graph showing the results of a tumor animal model killing test for the specific NKG2DL-targeting specific CAR-T cells of the present application using glioma U251 as a target cell. among them 17-A showed growth of xenografts in nude mice; 17-B showed tumor fluorescence intensity values.
  • Figure 18 is a graph showing the killing test results of a tumor animal model of a specific CAR-T cell targeting NKG2DL of the present application using ovarian cancer SKOV3 as a target cell.
  • 18-A showed growth of transplanted tumors in nude mice; 18-B showed tumor fluorescence intensity values.
  • Figure 19 is a graph showing the killing test results of a tumor animal model of a specific CAR-T cell targeting NKG2DL of the present application using gastric cancer NUGC4 as a target cell, wherein 19-A showed growth of transplanted tumors in nude mice; 19-B showed tumor fluorescence intensity values.
  • Figure 20 shows KD-025 with B-NSG animals Test results of CAR toxicology and safe dose experiments, 20-A shows the test results of toxicological experiments; 20-B shows the test results of safe dose experiments.
  • Figure 21 is a test result of the KD-025 CAR body retention experiment performed with B-NSG animals.
  • Figure 22 shows the results of detection of related oncogene expression after T cell infection by KD-025 CAR virus.
  • the term "functional variant” is a modification of the parent structure.
  • the term “functional variant” refers to a variant of a structure having the same or similar biological functions and properties as the parent, such as the same targeting binding function as the parent. .
  • a “functional variant” can be obtained by performing one or more conservative substitutions in the parent.
  • a functional variant in the present application is a structure that is modified to bind to the NKG2DL target based on the amino acid sequence of the ligand NKG2D of NKG2DL.
  • analog refers to a structure-related polypeptide that has the function of a reference polypeptide molecule. In the present application, it refers to a polyamino acid structure which is related to the amino acid sequence structure of the ligand NKG2D and which has a function of targeting NKG2DL of NKG2D.
  • amino acid modification refers to a conservative amino acid modification that does not significantly affect or alter the binding characteristics of a CAR (eg, an extracellular recognition domain) of the disclosure comprising an amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions.
  • conservative amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid within the same group.
  • homology refers to a high degree of amino acid or nucleotide match of a target amino sequence or a nucleotide sequence of interest as compared to a reference sequence. Homology herein can be determined using standard software such as BLAST or FASTA
  • CAR chimeric antigen receptor
  • Chimeric antigen receptors include a leader peptide portion, an extracellular target recognition domain, a transmembrane domain, and an intracellular domain.
  • a specific chimeric antigen receptor is an artificially designed specific antibody or ligand that binds to a target antigen, and in this application is a specific chimeric antigen receptor (CAR) that targets human NKG2DL.
  • CAR can be used to transfer the specificity of a monoclonal antibody to a T cell by promoting the transfer of its coding sequence by a retroviral vector.
  • CAR can both bind antigen and transduce the function of T cell activation, which is independent of MHC restriction.
  • CAR is a "universal" immune antigen receptor that can treat a population of patients with antigen-positive tumors regardless of their HLA genotype.
  • Adoptive immunotherapy with T lymphocytes expressing tumor-specific CAR can be a powerful therapeutic strategy for treating cancer.
  • the term "antigen” refers to a target molecule that is specifically expressed on the cell surface of a tumor cell and that specifically recognizes and binds to the ligand. In the present invention, it refers to human NKG2DL.
  • the expression of the NKG2DL protein is an indicator of the "stress state" of the cells, which is rarely expressed or only transiently expressed in healthy tissues, but is usually expressed at a higher level on the surface of various tumor cells from different sources.
  • the extracellular antigen binding domain is NKG2D.
  • any sequence of NKG2D molecules can be included in a fusion protein having a heterologous sequence to form an extracellular antigen binding domain.
  • NKG2D The receptor for NKG2DL protein is NKG2D.
  • the NKG2D-NKG2DL system plays an important role in the body's anti-tumor immunity.
  • NKG2D plays a role in killing tumor cells by recognizing NKG2DL produced on the surface of tumor cells to transmit activation signals and activate the immune system.
  • recognition refers to the selective binding of a target.
  • specifically binds or “specifically binds” or “specifically targets” as used herein, refers to a polypeptide or fragment thereof that recognizes and binds to a biomolecule of interest (eg, a polypeptide), but which does not substantially recognize the bound sample.
  • biomolecule of interest eg, a polypeptide
  • Other molecules such as other molecules in a biological sample that naturally include the polypeptide of the invention.
  • the term "specifically binds” refers to a bond between two molecules (eg, a ligand and a receptor) characterized by one molecule (ligand) and another specific molecule in the presence of many other different molecules ( The ability of a receptor to bind, ie, the ability to display a preferential binding of one molecule to another in a heterogeneous mixture of molecules.
  • the specific binding of the ligand to the receptor is also demonstrated as follows: when an excess of unlabeled ligand is present, the binding of the detectably labeled ligand to the receptor is reduced (ie Combine competition experiments).
  • receptor is a molecular structure that specifically binds to a target sequence.
  • the receptor binds to a ligand on another cell, allowing for cell-to-cell recognition and/or interaction.
  • a receptor refers to human NKG2D that targets a human NKG2DL target.
  • costimulatory molecule refers to a cell surface molecule other than an antigen receptor or its ligand required for an effective response of a lymphocyte to an antigen.
  • vector refers to any genetic element, such as a plasmid, bacteriophage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replicating when associated with appropriate control elements, and which can transfer the gene sequence to In the cell.
  • vector includes both cloning and expression vectors, as well as viral vectors and plasmid vectors.
  • expression vector refers to a recombinant nucleic acid sequence, ie, a recombinant DNA molecule containing the desired coding sequence and the appropriate nucleic acid sequence necessary for expression of an operably linked coding sequence in a particular host organism.
  • Nucleic acid sequences necessary for expression in prokaryotes typically include a promoter, an operon (optional), and a ribosome binding site, usually accompanied by other sequences.
  • Eukaryotic cells are known to utilize promoters, enhancers and terminators and polyadenylation signals.
  • immune response cell refers to a cell that functions in an immune response, or a progenitor cell thereof, or a progeny thereof.
  • isolated cell refers to an immune cell that is separated from the molecules and/or cellular components of a naturally accompanying cell.
  • modulation refers to a positive or negative change.
  • pathogen refers to a virus, bacterium, fungus, parasite or protozoa that is capable of causing a disease.
  • treating, treatment refers to a clinical intervention that attempts to alter the disease process of the individual or cell being treated, and can be used for prevention, or during a clinical pathological procedure.
  • Therapeutic effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of progression of the disease, mitigation or alleviation of the disease state, and alleviation or improvement. The prognosis.
  • treatment can prevent the deterioration caused by a condition in an invaded or diagnosed subject or a subject suspected of having a condition, and the treatment can prevent a subject having a risk of the condition or suspected of having the condition The onset of symptoms of a condition or disorder.
  • the term "killing efficiency" is the proportion of swollen cell inactivation observed in an in vitro test system in which a certain proportion of tumor antagonists and tumor cells are mixed.
  • the proportion of tumor cell death caused by chimeric antigen receptor-modified immune response cells is 10:1, the killing ratio of the NKG2D chimeric antigen receptor-modified T cells to the NKG2DL-expressing tumor cells.
  • disease refers to any condition or disorder that disrupts or interferes with the normal function of a cell, tissue or organ. Examples of diseases include neoplasia or pathogen infection of cells.
  • an “effective amount” refers to an amount sufficient to have a therapeutic effect. In one embodiment, an “effective amount” is an amount sufficient to prevent, ameliorate or inhibit the continued proliferation, growth or metastasis (eg, invasion or migration) of neoplasia.
  • exogenous refers to a nucleic acid molecule or polypeptide that is not present endogenously in a cell or that is present at a level sufficient to achieve a functional effect obtained upon overexpression.
  • exogenous will include any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as exogenous, heterologous and overexpressed nucleic acid molecules and polypeptides.
  • exogenous nucleic acid molecule or polypeptide refers to a nucleic acid molecule (eg, a cDNA, DNA or RNA molecule) or polypeptide that is not normally present in or obtained from a cell.
  • the nucleic acid may be from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in a cell or sample.
  • treating, treatment refers to a clinical intervention that attempts to alter the disease process of the individual or cell being treated, and can be used for prevention, or during a clinical pathological procedure.
  • Therapeutic effects of treatment include, but are not limited to, prevention of the occurrence or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of progression of the disease, mitigation or alleviation of the disease state, and alleviation or improvement. The prognosis.
  • treatment can prevent the deterioration caused by a condition in an invaded or diagnosed subject or a subject suspected of having a condition, and the treatment can prevent a subject having a risk of the condition or suspected of having the condition The onset of symptoms of a condition or disorder.
  • killing efficiency is the proportion of swollen cell inactivation observed in an in vitro test system in which a certain proportion of tumor antagonists and tumor cells are mixed.
  • proportion of tumor cell death caused by chimeric antigen receptor-modified immune response cells is 10:1, the killing ratio of the NKG2D chimeric antigen receptor-modified T cells to the NKG2DL-expressing tumor cells.
  • an “effective amount” means an amount sufficient to have a therapeutic effect. In one embodiment, an “effective amount” is an amount sufficient to prevent, ameliorate or inhibit the continued proliferation, growth or metastasis (eg, invasion or migration) of neoplasia.
  • a costimulatory ligand is a protein expressed on the surface of a cell that, when bound to its receptor, produces a costimulatory response, i.e., an intracellular response that provides stimulation when the antigen binds to its CAR molecule.
  • exogenous refers to a nucleic acid molecule or polypeptide that is not present endogenously in a cell or that is present at a level sufficient to achieve a functional effect obtained upon overexpression.
  • exogenous will include any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as exogenous, heterologous and overexpressed nucleic acid molecules and polypeptides.
  • the present application provides a human NKG2D protein and a functional variant thereof that bind to human NKG2DL, Contains a sequence selected from the group consisting of:
  • SEQ ID No. 4 SEQ ID No. 5, SEQ ID No. 6, SEQ ID The amino acid sequence shown in No. 7, SEQ ID No. 8, or SEQ ID No. 9, or (2) (1) a variant produced by one or more amino acid modifications.
  • the peptide human NKG2D protein that targets human NKG2DL is based on the amino acid sequence of the ligand human NKG2D protein.
  • the CAR is constructed with the full-length amino acid sequence of NKG2D
  • the effect is poor, because the functional domains such as the NKG2D intracellular region are natural killer cell properties, and are not necessarily compatible with T cells. Therefore, based on the full-length amino acid sequence of NKG2D, the inventors screened and analyzed the target human NKG2DL target and NKG2D fragment by means of protein sequence analysis and functional domain optimization prediction tools, and further designed and constructed a specific target human NKG2DL containing improved NKG2D.
  • Chimeric antigen receptor Chimeric Antigen Receptor, CAR
  • CAR Chimeric Antigen Receptor
  • the inventors continually carry out amino acid sequence design and sequence alignment and screening, and perform random screening tests and targeted functional verification on the sequences of more than 10 CAR molecules (for example, constructing viral vectors and further infecting T cells, Obtaining modified T cells, and detecting the in vitro killing activity of the obtained modified T cells, and the like, and then performing sequence adjustment according to the results of multiple random combinations, and finally screening the best performing sequences, and obtaining the present invention.
  • the high titer targets the human NKG2D amino acid sequence of human NKG2DL and its functional variants.
  • the amino acid modifications include, but are not limited to, substitutions, deletions, and additions of amino acids including, but not limited to, those that target human NKG2DL by substitution, deletion, and addition of amino acids.
  • the same amino acid modifications include, but are not limited to, chemical modifications of amino acid side chains, natural or unnatural amino acid substitutions, mutations, deletions, insertions, or post-translational modifications.
  • the one or more amino acid modified variant of the human NKG2D amino acid sequence that targets human NKG2DL is SEQ ID A polypeptide having homology to the amino acid sequence shown in No. 5.
  • the human NKG2D amino acid sequence that targets human NKG2DL undergoes one or more amino acid modification variants with SEQ ID A polypeptide having homology to the amino acid sequence shown in No. 6.
  • the human NKG2D amino acid sequence that targets human NKG2DL undergoes one or more amino acid modification variants with SEQ ID A polypeptide having homology to the amino acid sequence shown in No. 7.
  • the human NKG2D amino acid sequence that targets human NKG2DL undergoes one or more amino acid modification variants with SEQ ID A polypeptide having homology to the amino acid sequence shown in No. 8. In some non-limiting embodiments, the human NKG2D amino acid sequence that targets human NKG2DL undergoes one or more amino acid modification variants with SEQ ID A polypeptide having homology to the amino acid sequence shown in No. 9.
  • the homologous polypeptide is relative to a reference polypeptide, and herein refers to 70 to 99%, preferably 80 to 99%, more preferably 90 to 99%, and most preferably 95 to 99% of NKG2D targeting a human NKG2DL target. Derived polypeptide.
  • the polypeptide having the homology of the amino acid sequence shown in No. 6, SEQ ID No. 7, SEQ ID No. 8, or SEQ ID No. 9 is SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 or SEQ ID Derivation of the amino acid sequence shown in any one of No. 9 after 1 to 10 amino acid residues, preferably 1 to 5 amino acid residues or more preferably 1 to 3 amino acid residues, substitutions or deletions Peptide.
  • the subject matter disclosed in the first aspect of the present application also provides a fragment of any of the polypeptide or peptide domains of the subject matter disclosed herein.
  • the fragment can be at least 5-15 amino acids.
  • the fragment is at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids.
  • the fragment is at least 60 to 80, 100, 200, 300 or more contiguous amino acids.
  • Amino acid fragments of the subject matter disclosed in the first aspect of the present application can be produced by methods known to those of ordinary skill in the art, or can produce non-protein analogs from normal protein processing strategies (eg, removal of amino acids not required for biological activity from nascent polypeptides) Or removing amino acids by alternative mRNA splicing or alternative protein processing events).
  • Non-protein analogs can have chemical structures designed to mimic the functional activity of the proteins of the invention. Such analogs are administered in accordance with the methods of the presently disclosed subject matter. Such analogs may exceed the physiological activity of the original polypeptide.
  • Assays for measuring the functional activity of the peptides described in the first aspect or functional variants thereof include, but are not limited to, the test methods described in the Examples below.
  • the present application provides a specific chimeric antigen receptor that targets human NKG2DL, comprising a leader sequence amino acid sequence that is ligated from the amino terminus to the carboxy terminus, and an extracellular recognition that targets human NKG2DL.
  • Extracellular recognition domain (Also referred to as the extracellular domain or simply by the recognition element contained therein) comprises an identifying element that specifically binds to a molecule present on the surface of the cell of the target cell.
  • the function of the transmembrane domain is to anchor the binding cell membrane.
  • the intracellular signaling domain comprises an immunoreceptor activation motif and optionally one or more costimulatory signaling domains.
  • the domain of the extracellular recognition antigen may also comprise a leader sequence or signal peptide that introduces the nascent protein into the endoplasmic reticulum.
  • a signal peptide or leader sequence may be necessary if the CAR will be glycosylated and anchored in the cell membrane.
  • the signal sequence or leader sequence can be a peptide sequence (about 5, about 10, about 15, about 20, about 25 or about 30 amino acids in length) present at the N-terminus of the newly synthesized protein, which directs the protein into the secretory pathway.
  • the leader sequence is covalently linked to the 5' end of the extracellular antigen binding domain.
  • the specific chimeric antigen receptor that targets binding to human NKG2DL comprises a hinge region.
  • the transmembrane domain comprises a transmembrane region.
  • the intracellular signal domain comprises an immunoreceptor tyrosine activation motif and a costimulatory signal domain
  • the transmembrane domain of the CAR comprises a hydrophobic alpha helix spanning at least a portion of the membrane. Different transmembrane domains produce different receptor stability. After antigen recognition, receptor clusters and signals are delivered to the cells.
  • the The transmembrane domain of CAR contains CD8 transmembrane domain, CD28 transmembrane domain, CD3 ⁇ transmembrane domain, CD4 transmembrane domain, 4-1BB transmembrane domain, OX40 transmembrane domain, ICOS transmembrane domain , CTLA-4 transmembrane domain, PD-1 transmembrane domain, LAG-3 transmembrane domain, 2B4 transmembrane domain, BTLA transmembrane domain, synthetic peptide (not based on proteins associated with immune response) Any of them.
  • the immunoreceptor tyrosine activation motif comprises an intracellular signal domain of an CD3 ⁇ chain or an Fc ⁇ RI ⁇ intracellular signal structure.
  • the costimulatory signal domain comprises a CD28 intracellular signaling domain, a CD137/4-1BB intracellular signaling domain, a CD134/OX40 intracellular signaling domain, and an ICOS intracellular signaling domain At least one of them.
  • the hinge region is selected from the group consisting of a CD8 alpha hinge region, an IgG hinge region, or a hinge region or hinge region comprising all or a portion of an immunoglobulin that has been modified by one or more amino acids.
  • the amino acid sequence of the chimeric antigen receptor is from the amino terminus to the carboxy terminus by a leader sequence, the amino acid sequence of human NKG2D targeted to bind human NKG2DL, and the human CD8 hinge region described in the first aspect of the present application.
  • the amino acid sequence, the amino acid sequence of the human CD8 transmembrane region, the amino acid sequence of the human CD28 intracellular domain, the amino acid sequence of the human 4-1BB intracellular domain, and the amino acid sequence of the human CD3 cell intracellular domain are sequentially connected in series;
  • the amino acid sequence of the chimeric antigen receptor is from the amino terminus to the carboxy terminus by the amino acid sequence of the leader peptide, the amino acid sequence of human NKG2D targeted to bind human NKG2DL, human CD8, as described in the first aspect of the application.
  • the amino acid sequence of the transmembrane region, the amino acid sequence of the human CD8 hinge region sequence, the amino acid sequence of the human CD28 intracellular domain, and the amino acid sequence of the CD3 intracellular domain are sequentially connected in series.
  • the amino acid sequence of the chimeric antigen receptor is directed from the amino terminus to the carboxy terminus by a leader sequence, a human NKG2D amino acid sequence targeted to bind human NKG2DL, a human CD8 transmembrane region, as described in the first aspect of the present application.
  • the amino acid sequence, the amino acid sequence of the intracellular domain of 4-1BB, and the amino acid sequence of the intracellular domain of CD3 are sequentially connected in series;
  • the amino acid sequence of the chimeric antigen receptor is from the amino terminus to the carboxy terminus by a leader sequence, the human NKG2D amino acid sequence targeting human NKG2DL, and the human CD8 transmembrane region, as described in the first aspect of the present application.
  • the amino acid sequence, the amino acid sequence of the intracellular domain of human CD28 and the amino acid sequence of the intracellular domain of the human CD3 ⁇ sequence are sequentially connected in series.
  • the amino acid sequence of the chimeric antigen receptor is amino acid sequence from the amino terminus to the carboxy terminus of the leader peptide sequence, and the amino acid sequence of human NKG2D targeting human NKG2DL, as described in the first aspect of the present application,
  • the amino acid sequence of the human CD8 transmembrane region, the amino acid sequence of the human CD8 hinge region, the amino acid sequence of the human CD28 intracellular domain, and the amino acid sequence of human CD3 ⁇ are sequentially connected in series.
  • the chimeric antigen receptor comprises the amino acid sequence of a leader peptide sequentially linked from the amino terminus to the carboxy terminus, and the human NKG2D targeted to bind human NKG2DL as described in the first aspect of the present application.
  • the chimeric antigen receptor comprises the amino acid sequence of a leader peptide sequentially linked from the amino terminus to the carboxy terminus, and the human NKG2D targeted to bind human NKG2DL as described in the first aspect of the present application.
  • the chimeric antigen receptor comprises the amino acid sequence of a leader peptide sequentially linked from the amino terminus to the carboxy terminus, and the human NKG2D targeted to bind human NKG2DL as described in the first aspect of the present application.
  • the chimeric antigen receptor comprises the amino acid sequence of a leader peptide sequentially linked from the amino terminus to the carboxy terminus, and the human NKG2D targeted to bind human NKG2DL as described in the first aspect of the present application.
  • the leader sequence is selected from the group consisting of: (1) having SEQ ID No. 3 a polypeptide of the indicated amino acid sequence, or (2) an amino acid modified functional variant of (1); wherein the amino acid modified functional variant is SEQ ID
  • the amino acid sequence shown in No. 3 has a polypeptide having 70 to 99% homology, preferably 80 to 99%, more preferably 90 to 99%, and most preferably 95 to 99% homology.
  • the CAR of the subject matter disclosed in the second aspect of the present application comprises a spacer spacer, also referred to as a hinge region, that links the antigen binding domain to a transmembrane domain.
  • a spacer region can be sufficiently flexible to allow the antigen binding domain to be oriented in different directions to facilitate antigen recognition.
  • the spacer region can be a hinge region from IgG1, or a CH2CH3 region of immunoglobulin and a portion of CD3. Some spacer regions comprise an immunoglobulin CH3 domain or both a CH3 domain and a CH2 domain.
  • spacer regions comprise all or part of the immunoglobulin (eg, IgG1, IgG2, IgG3, IgG4) hinge region, ie, fall in immunoglobulin A sequence between the CH1 and CH2 domains, for example, an IgG4Fc hinge or a CD8 hinge.
  • the immunoglobulin-derived sequence may comprise one or more amino acid modifications, for example 1, 2, 3, 4 or 5 substitutions.
  • the spacer region is a hinge region.
  • the CAR of the subject matter disclosed in the second aspect of the present application comprises a transmembrane domain comprising a CD28 polypeptide and a costimulatory signaling region comprising a CD28 polypeptide.
  • the hinge region comprises a human CD8 polypeptide.
  • the hinge region CD8 polypeptide is selected from the group consisting of (1) having SEQ ID No. 10 a polypeptide of the indicated amino acid sequence, or (2) an amino acid modified functional variant of (1); wherein the amino acid modified functional variant is SEQ ID
  • the amino acid sequence shown in No. 310 has an amino acid sequence of 70 to 99% homology, preferably 80 to 99%, more preferably 90 to 99%, most preferably 95 to 99% homology, or may optionally contain at most one Or at most two or at most three conservative amino acid substitutions.
  • the hinge region human CD8 amino acid sequence has SEQ ID NO: The amino acid sequence of a contiguous portion of 11 having a length of at least 20, or at least 30, or at least 40, or at least 50, and at most 164 amino acids.
  • the transmembrane region comprises a human CD8 polypeptide.
  • the transmembrane region CD8 polypeptide is selected from the group consisting of (1) having SEQ ID No. 11 a polypeptide of the indicated amino acid sequence, or (2) an amino acid modified functional variant of (1); wherein the amino acid modified functional variant is SEQ ID
  • the amino acid sequence shown in No. 11 has an amino acid sequence of 70 to 99% homology, preferably 80 to 99%, more preferably 90 to 99%, most preferably 95 to 99% homology, or may optionally contain at most one Or at most two or at most three conservative amino acid substitutions.
  • the human transmembrane region CD8 amino acid sequence has SEQ ID NO: The amino acid sequence of a contiguous portion of 11 having a length of at least 20, or at least 30, or at least 40, or at least 50, and at most 164 amino acids.
  • amino acid sequence of the human 4-1BB intracellular domain is selected from:
  • polypeptide having the amino acid sequence of SEQ ID NO: 12 or (2) an amino acid-modified variant of (1); wherein the amino acid-modified variant is the same as SEQ ID
  • the amino acid sequence represented by NO: 12 has a polypeptide having 70 to 99% homology, preferably 80 to 99%, more preferably 90 to 99%, most preferably 95 to 99% homology, or may optionally contain at most one or Up to two or up to three conservative amino acid substitutions.
  • the human 4-1BB intracellular domain amino acid sequence has SEQ ID NO: The amino acid sequence of a contiguous portion of 12 having a length of at least 20, or at least 30, or at least 40, or at least 50, and at most 164 amino acids.
  • the intracellular domain of a CAR can comprise a CD3 ⁇ polypeptide that can activate or stimulate cells (eg, cells of the lymphoid lineage, such as T cells).
  • CD3 ⁇ contains 3 immunoreceptor tyrosine activation motifs (Immunoreceptor) Tyrosine-based activation motif (ITAM), and transmits an activation signal to cells (eg, cells of the lymphoid lineage, such as T cells) upon binding to the antigen.
  • Immunoreceptor Immunoreceptor Tyrosine-based activation motif
  • the CD3 ⁇ intracellular domain is selected from the group consisting of: (1) having the SEQ ID a polypeptide of the amino acid sequence shown in No. 14; or (2) an amino acid-modified variant of (1); wherein the amino acid-modified variant is the same as SEQ ID
  • the amino acid sequence shown in No. 14 has 70 to 99% homology, preferably 80 to 99%, more preferably 80 to 99%, most preferably 95 to 99% homologous polypeptide, or may optionally contain at most one Or at most two or at most three conservative amino acid substitutions.
  • the human CD3 sputum intracellular domain amino acid sequence has SEQ ID NO: The amino acid sequence of a contiguous portion of 14 having a length of at least 20, or at least 30, or at least 40, or at least 50, and at most 164 amino acids.
  • the amino acid sequence of the human CD3 sputum intracellular domain is selected from the group consisting of (1) having SEQ. An amino acid sequence of ID No. 15; or (2) an amino acid modified variant of (1); wherein the amino acid modified variant is SEQ ID The amino acid sequence shown in No.
  • the human CD3 sputum intracellular domain amino acid sequence has SEQ ID NO: The amino acid sequence of a contiguous portion of 13 having a length of at least 20, or at least 30, or at least 40, or at least 50, and at most 164 amino acids.
  • the human CD28 cell intracellular domain is selected from:
  • the amino acid sequence shown in No. 13 has 70 to 99% homology, preferably 80 to 99%, more preferably 80 to 99%, most preferably 95 to 99% homologous polypeptide, or may optionally contain at most one Or at most two or at most three conservative amino acid substitutions.
  • the human CD28 sputum intracellular domain polypeptide has SEQ ID NO: The amino acid sequence of a contiguous portion of 13 having a length of at least 20, or at least 30, or at least 40, or at least 50, and at most 255 amino acids.
  • the leader sequence is as set forth in SEQ ID No. 3.
  • the amino acid sequence of the human NKG2D is SEQ ID No.4 is shown. In an exemplary embodiment, the amino acid sequence of the human NKG2D is set forth in SEQ ID No. 5. In an exemplary embodiment, the amino acid sequence of the human NKG2D is SEQ ID No.6 is shown. In an exemplary embodiment, the amino acid sequence of the human NKG2D is set forth in SEQ ID No. 7. In an exemplary embodiment, the amino acid sequence of the human NKG2D is SEQ ID No.8 is shown. In an exemplary embodiment, the amino acid sequence of the human NKG2D is set forth in SEQ ID No. 9.
  • amino acid sequence of the human CD8 hinge region is set forth in SEQ ID No. 10;
  • amino acid sequence of the human CD8 transmembrane region is set forth in SEQ ID No. 11;
  • amino acid sequence of the human 4-1BB intracellular domain is SEQ ID No.12;
  • amino acid sequence of the CD28 domain is set forth in SEQ ID No. 13;
  • amino acid sequence of the CD3 ⁇ domain is set forth in SEQ ID No. 14.
  • amino acid sequence of the CD3 ⁇ domain is set forth in SEQ ID No. 15.
  • a specific chimeric antigen receptor (CAR) that targets binding to human NKG2DL is recombinantly expressed or expressed by a vector.
  • the intracellular domain of a CAR of the present application that binds to human NKG2DL further comprises at least one costimulatory signaling region comprising at least one costimulatory stimulus that provides optimal lymphocyte activation Ligand molecule. It is combined with CAR signaling to induce effector cell function of CAR+ T cells.
  • the costimulatory ligands include, but are not limited to, CD80, CD86, CD70, OX40L, 4-1BBL, CD48, TNFRSF14, and PD-L1.
  • the intracellular domain of a CAR that targets human NKG2DL comprises a costimulatory signaling region comprising two costimulatory molecules: CD28 and 4-1BB or CD28 And OX40.
  • the CAR intracellular domain of the present application that targets human NKG2DL comprises a 4-1BB polypeptide.
  • 4-1BB acts as a tumor necrosis factor (TNF) ligand and has stimulatory activity.
  • 4-1BBL can be covalently linked to the 5' end of the extracellular antigen binding domain.
  • 4-1BBL can be covalently linked to the 3' end of the intracellular domain.
  • the 4-1BB polypeptide is selected from the group consisting of (1) having the SEQ ID NO: 12 amino acid sequence or (2) (1) amino acid modified functional variant, trans amino acid modified variant with SEQ ID
  • the amino acid sequence shown in No. 12 has 70 to 99% homology, preferably 80 to 99%, more preferably 80 to 99%, most preferably 95 to 99% homology of the polypeptide / or optionally contain at most one Or at most two or at most three conservative amino acid substitutions.
  • the 4-1BB polypeptide has the SEQ ID NO: The amino acid sequence of a contiguous portion of 12 having a length of at least 20, or at least 30, or at least 40, or at least 50, and at most 255 amino acids.
  • a CAR can further comprise a spacer that joins the antigen binding domain to a transmembrane domain.
  • the spacers can be sufficiently flexible to allow the antigen binding domains to be oriented in different directions to facilitate antigen recognition.
  • the spacer may be a hinge region from IgGl, or a CH2CH3 region of immunoglobulin and a portion of CD3.
  • the intracellular domain of a CAR can comprise a human CD3 ⁇ polypeptide that can activate or stimulate cells (eg, cells of the lymphoid lineage, such as T cells).
  • CD3 ⁇ contains 3 ITAMs and transmits an activation signal to cells (eg, cells of the lymphoid lineage, such as T cells) upon binding to the antigen.
  • the intracellular domain of the CAR further comprises at least one costimulatory signaling region comprising at least one costimulatory molecule that provides optimal lymphocyte activation.
  • costimulatory molecule refers to a cell surface molecule other than an antigen receptor or its ligand required for an effective response of a lymphocyte to an antigen.
  • the at least one costimulatory signaling region can comprise a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide (not based on a protein associated with an immune response), or a combination thereof.
  • a costimulatory molecule can bind to a costimulatory ligand, which is a protein expressed on the cell surface that, when bound to its receptor, produces a costimulatory response, ie, a cell that provides stimulation when the antigen binds to its CAR molecule. Should be inside.
  • Costimulatory ligands include, but are not limited to, CD80, CD86, CD70, OX40L, 4-1BBL, CD48, TNFRSF14, and PD-L1.
  • a 4-1BB ligand ie, 4-1BBL
  • 4-1BB also known as "CD137”
  • the costimulatory signaling region of the intracellular domain of CAR comprises two costimulatory molecules: CD28 and 4-1BB (see Sadelain et al., Cancer) Discovery, OF1-11, (2013)), or CD28 and OX40.
  • 4-1BB acts as a tumor necrosis factor (TNF) ligand and has stimulatory activity.
  • 4-1BB acts as a tumor necrosis factor (TNF) ligand and has stimulatory activity.
  • 4-1BB polypeptide has SEQ ID Amino acid sequence of at least about 85%, about 90%, about 5%, about 96%, about 97%, about 98%, about 99% or 100% homology of the sequence of NO: 12 or a fragment thereof, and/or Optionally, at most one or at most two or at most three conservative amino acid substitutions are included.
  • the 4-1BB polypeptide has SEQ ID NO: The amino acid sequence of a contiguous portion of 12 having a length of at least 20, or at least 30, or at least 40, or at least 50, and at most 255 amino acids.
  • the amino acid sequence of the 4-1BB polypeptide is SEQ ID NO: 12 amino acids.
  • a CAR intracellular domain of the presently disclosed subject matter comprises a 4-1BB polypeptide.
  • the present application provides a nucleic acid molecule encoding a specific chimeric antigen receptor that binds to human NKG2DL according to the second aspect, the nucleic acid molecule comprising a coding sequenced in series from 5' to 3' A nucleotide sequence of the leader sequence, a nucleotide sequence encoding human NKG2D that binds to human NKG2DL, a nucleotide sequence encoding a transmembrane domain, and a nucleotide sequence encoding an intracellular signal domain.
  • the nucleic acid molecule further comprises a nucleotide sequence encoding a hinge region.
  • the intracellular signal domain comprises an immunoreceptor tyrosine activation motif
  • the nucleic acid molecule comprises a sequence encoding a leader sequence, sequentially linked from 5' to 3', a human NKG2D sequence, a human CD8 hinge region sequence, a human CD8 transmembrane, as described in the first aspect of the present application.
  • the nucleotide sequence of the region sequence, the human CD28 intracellular domain sequence, the human 4-1BB intracellular domain sequence, and the human CD3 cell intracellular domain are examples of the region sequence, sequentially linked from 5' to 3', a human NKG2D sequence, a human CD8 hinge region sequence, a human CD8 transmembrane, as described in the first aspect of the present application.
  • the nucleic acid molecule comprises a sequence encoding a leader sequence, sequentially linked from 5' to 3', a human NKG2D sequence, a human CD8 transmembrane region sequence, a human 4-, as described in the first aspect of the present application.
  • the nucleotide sequence of the 1BB intracellular domain sequence and the human CD3 cell intracellular domain sequence are described in the first aspect of the present application.
  • the nucleic acid molecule comprises a nucleotide sequence encoding a leader sequence, a human NKG2D, and a human CD8 hinge region, respectively, linked in series from 5' to 3'.
  • Polynucleotides encoding an extracellular recognition domain that binds to human NKG2DL can be modified by codon optimization. Codon optimization can alter naturally occurring and recombinant gene sequences to achieve the highest possible level of productivity in any given expression system. Factors involved in different stages of protein expression include codon adaptation, mRNA structure, and various cis elements in transcription and translation.
  • the nucleotide sequence encoding human NKG2D that targets an extracellular recognition domain that binds to human NKG2DL is SEQ.
  • nucleotide sequence encoding human NKG2D that binds to an extracellular recognition domain that binds to human NKG2DL is set forth in SEQ ID No. 18 or with SEQ
  • the nucleotide sequence shown by ID No. 18 is 99%, 95%, 90%, 85% or 80% homologous.
  • the nucleotide sequence encoding human NKG2D encoding an extracellular recognition domain that binds to human NKG2DL is SEQ.
  • the nucleotide sequence encoding human NKG2D encoding an extracellular recognition domain that binds to human NKG2DL is SEQ.
  • the nucleotide sequence encoding human NKG2D encoding an extracellular recognition domain that binds to human NKG2DL is SEQ.
  • the nucleotide sequence encoding human NKG2D encoding an extracellular recognition domain that binds to human NKG2DL is SEQ.
  • the nucleotide sequence encoding the human CD8 hinge region is set forth in SEQ ID No. 23 or with SEQ ID
  • the nucleotide sequence shown in No. 23 is 99%, 95%, 90%, 85% or 80% homologous.
  • the nucleotide sequence encoding the human CD8 transmembrane region is set forth in SEQ ID No. 24 or with SEQ ID
  • the nucleotide sequence shown in No. 24 is 99%, 95%, 90%, 85% or 80% homologous.
  • the nucleotide sequence encoding the human 4-1BB intracellular domain is set forth in SEQ ID No. 25 or with SEQ The nucleotide sequence shown by ID No. 25 is 99%, 95%, 90%, 85% or 80% homologous.
  • the nucleotide sequence encoding the intracellular domain of CD28 is set forth in SEQ ID No. 26 or with SEQ The nucleotide sequence shown by ID No. 26 is 99%, 95%, 90%, 85% or 80% homologous.
  • the nucleotide sequence encoding the CD3 ⁇ intracellular domain is set forth in SEQ ID No. 27 or with SEQ The nucleotide sequence shown by ID No. 27 is 99%, 95%, 90%, 85% or 80% homologous.
  • the nucleotide sequence encoding the CD3 ⁇ intracellular domain is set forth in SEQ ID No. 28 or with SEQ ID
  • the nucleotide sequence shown in No. 28 is 99%, 95%, 90%, 85% or 80% homologous.
  • the present application provides a recombinant vector or expression plasmid comprising the chimeric antigen receptor of the second aspect of the application or the nucleic acid of the third aspect of the application.
  • the vector is a retroviral vector (eg, a gamma retrovirus or a lentivirus) that can introduce a DNA or RNA construct into the host cell genome.
  • a retroviral vector eg, a gamma retrovirus or a lentivirus
  • a polynucleotide encoding NKG2D that targets a human NKG2DL-specific CAR can be cloned into a retroviral vector and can be driven from its endogenous promoter, retroviral long terminal repeat or driven by an alternative internal promoter.
  • Non-viral vectors or RNA can also be used. Random chromosomal integration or targeted integration can be used (eg, using nucleases, transcriptional activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and/or regular clustering intervals, short palindromic repeats (CRISPR) or transgenes Expression (eg, using natural or chemically modified RNA).
  • TALENs transcriptional activator-like effector nucleases
  • ZFNs zinc finger nucleases
  • CRISPR short palindromic repeats
  • transgenes Expression eg, using natural or chemically modified RNA.
  • the vector is selected from the group consisting of a ⁇ -retroviral vector, a lentiviral vector, an adenoviral vector, and an adenoviral vector.
  • the vector is a ⁇ -retroviral vector.
  • the present application provides a promoter for constructing the recombinant vector of the fourth aspect of the present application and expressing the specific chimeric antigen receptor targeted for binding to human NKG2DL according to the second aspect of the present application
  • the promoter includes, but is not limited to, a nucleotide sequence such as SEQ The EF1 alpha promoter shown in No. 29 and the EFS promoter shown in SEQ No. 30.
  • Promoter is like SEQ The EFS promoter shown in No.30.
  • the promoter is as SEQ The EF1 alpha promoter shown in No. 29.
  • the present application provides a recombinant virus which is capable of expressing a specific chimeric antigen receptor targeting NKG2DL of the second aspect of the invention and capable of infecting an immune response cell.
  • the immune response cell is a cytotoxic T lymphocyte, an NK cell, an NKT cell, or a helper T cell.
  • the immune response cell is a cytotoxic T lymphocyte.
  • the virus is a lentivirus, an adenovirus, an adenovirus or a retrovirus, and the like.
  • the virus is a lentivirus.
  • the virus is a retrovirus.
  • the present application provides an isolated modified immune response cell comprising the chimeric antigen receptor of the second aspect of the present invention, which is transformed with the recombinant vector or expression plasmid of the third aspect of the present application. .
  • the initial genetic modification of the cells to provide targeting of human NKG2DL-specific immune response cells is typically performed using a retroviral vector, although any other suitable viral vector or non-viral delivery system can be used.
  • Retroviral gene transfer has also proven to be effective for subsequent genetic modification of cells to provide cells comprising an antigen-presenting complex comprising at least two costimulatory ligands.
  • Combinations of retroviral vectors and suitable assembly lines are also suitable, wherein the capsid protein is functional for infecting human cells.
  • the immune response cell further comprises at least one exogenous co-stimulatory ligand.
  • Transduction methods also include direct co-culture of cells with producer cells.
  • Transducing viral vectors can be used to express costimulatory ligands (e.g., 4-1BBL and IL-12) in immune response cells.
  • the selected vector exhibits high infection efficiency and stable integration and expression.
  • the at least one costimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF 14, and combinations thereof, or more preferably, the costimulatory ligand It is 4-1BBL.
  • the immune response cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a human embryonic stem cell, and a pluripotent that can differentiate into a lymphoid cell.
  • a T cell preferably T cells and natural killer (NK) cells, more preferably T cells.
  • T cell subsets isolated from a patient can be transduced with a vector for CAR expression.
  • the modified immune response cells disclosed in the seventh aspect can express an extracellular target recognition domain that specifically binds to human NKG2DL for treating or preventing neoplasia, immune diseases, or anti-aging.
  • the modified immune response cell is a CAR-T cell.
  • Modified genetically modified central memory T cells can be prepared using the human NKG2D chimeric antigen receptor targeting human NKG2DL and then cryopreserved.
  • the application provides a method for preparing an isolated chimeric antigen receptor-modified immune response cell of the sixth aspect of the present application, comprising the steps of:
  • the nucleic acid molecule of the third aspect is ligated into an expression vector by molecular cloning to obtain an expression vector targeting a specific chimeric antigen receptor of NKG2DL;
  • the obtained CAR expression vector targeting NKG2DL is transfected into 293T cells to obtain a virus solution;
  • the immune response cells are infected with the virus solution, and immune response cells expressing a specific chimeric antigen receptor-targeted NKG2DL-expressing cell are obtained from the infected cells.
  • the modified immune response cells of the invention may be cells of the lymphoid lineage.
  • the lymphoid lineage cells are selected from the group consisting of B, T and natural killer (NK) cells, and provide functions such as production of antibodies, regulation of the cellular immune system, detection of foreign substances in the blood, and detection of foreign cells of the host.
  • Non-limiting examples of cells of the lymphoid lineage include T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, embryonic stem cells, and pluripotent stem cells (eg, can differentiate into lymphoid cells) Pluripotent stem cells).
  • the immune response cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a human embryonic stem cell, and a pluripotent that can differentiate into a lymphoid cell.
  • a T cell preferably T cells or natural killer (NK) cells.
  • the T cell is a lymphocyte that matures in the thymus and is primarily responsible for cell-mediated immunity. T cells are involved in the acquired immune system.
  • T cells include, but are not limited to, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell memory T cells (or dry memory T cells), and both Types of effector memory T cells (eg, TEM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated constant T cells, and ⁇ T cells.
  • T helper cells include, but are not limited to, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell memory T cells (or dry memory T cells), and both Types of effector memory T cells (eg, TEM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated constant T cells, and ⁇ T cells.
  • CAR-expressing T cells express Foxp3 to achieve and maintain a T-regulated phenotype.
  • the modified immune response cell of the present application may further comprise at least one exogenous co-stimulatory ligand such that the immune response cell is co-expressed exogenously or induced to exogenously co-express based on NKG2D, targeting human NKG2DL a specific chimeric antibody and at least one exogenous co-stimulatory ligand.
  • the interaction between a specific CAR that targets human NKG2DL and at least one costimulatory ligand provides a non-antigen-specific signal that is important for the complete activation of immune response cells (eg, T cells).
  • the at least one costimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, and combinations thereof.
  • the costimulatory ligand is 4-1BBL.
  • the isolated modified immune response cell is a T cell.
  • the isolated modified immune response cell is a natural killer (NK) cell.
  • NK natural killer
  • the isolated modified immune response cells can be autologous, non-autologous (eg, allogeneic), or derived from engineered progenitor cells in vitro or stem cell.
  • the present application provides a pharmaceutical composition comprising an effective amount of a modified immune response cell isolated as described in the sixth aspect of the invention and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition disclosed in the present application comprises an isolated modified immune response cell expressing a specific chimeric antigen receptor that targets human NKG2DL and a pharmaceutically acceptable carrier.
  • Administration of the pharmaceutical composition can be autologous or non-autologous.
  • an immune response cell expressing a specific CAR that targets human NKG2DL and a composition comprising the same can be obtained from one subject and administered to the same subject or to a different compatible subject.
  • Peripheral blood-derived T cells or progeny thereof of the presently disclosed subject matter can be administered by topical injection, including catheter administration, systemic injection, topical injection, intravenous injection or parenteral administration (eg, in vivo, ex vivo or Derived in vitro).
  • a pharmaceutical composition of the presently disclosed subject matter eg, a pharmaceutical composition comprising an immune response cell expressing an NKG2D-specific CAR
  • a pharmaceutical composition of the presently disclosed subject matter is administered, it is typically formulated in a unit dosage injectable form (solution, suspension, emulsion) .
  • the composition of the present application may be a formulation.
  • the immune response cells expressing the specific chimeric antigen receptor (CAR) targeting human NKG2DL and compositions comprising the same disclosed herein can be conveniently provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, milks A turbid, dispersion or viscous composition that can be buffered to a selected pH.
  • Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions.
  • liquid compositions are more convenient to administer, especially by injection.
  • the viscous composition can be formulated in a suitable viscosity range to provide longer contact times with a particular tissue.
  • the liquid or viscous composition may comprise a carrier which may be a solvent or dispersion medium comprising, for example, water, physiological saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • a carrier which may be a solvent or dispersion medium comprising, for example, water, physiological saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers.
  • any vector, diluent or additive used must be compatible with an immune response cell that expresses a human chimeric NKG2D-specific chimeric antigen receptor (CAR) that is a subject of the presently disclosed subject matter.
  • CAR human chimeric NKG2D-specific chimeric antigen receptor
  • the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent.
  • a suitable carrier and other additives will depend on the exact route of administration and the nature of the particular dosage form, such as a liquid dosage form (for example, whether the composition is formulated as a solution, suspension, gel or another liquid, such as time) Release form or liquid fill form).
  • the present application provides a kit for treating or preventing neoplasia, pathogen infection, autoimmune disease, allogeneic transplantation, or transplant rejection or anti-aging, comprising the sixth aspect of the present invention An immune response cell or a nucleic acid of the third aspect of the invention.
  • the immune response cells are provided with instructions for administering the cells to a subject having or at risk of developing neoplasia, pathogen infection, immune disorder, or allograft or aging.
  • Instructions generally include information regarding the use of the compositions for the treatment or prevention of neoplasia, pathogen infection, immune disease or allogeneic transplantation.
  • the instructions include at least one of: a description of a therapeutic agent; a dosage regimen and method of administration for treating or preventing neoplasia, pathogen infection, immune disease or allograft or a symptom thereof; Contraindications; indications; non-indications; excessive information; adverse reactions; animal pharmacology; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or attached to the container as a label, or as a separate page, booklet, card or folded print, in or with the container.
  • the present application provides the human NKG2D targeted to bind human NKG2DL according to the first aspect of the present invention.
  • Protein and variants thereof, specific chimeric antigen receptors targeting the binding of human NKG2DL as described in the second aspect The recombinant vector or expression plasmid according to the fourth aspect, the recombinant virus according to the fifth aspect, the isolated CAR-modified immune response cell obtained by the preparation method of the seventh aspect, the composition of the eighth aspect or the reagent of the ninth aspect Box in governance
  • the use of a product for treating or preventing disease, including treatment with NKG2DL and NKG2D A patient with a disease caused by a specific interaction of proteins is administered a CAR-T cell comprising NKG2D targeting NKG2DL as described in the second aspect of the present application.
  • the disease comprises neoplasia, pathogen infection, autoimmune disease, allograft, transplant rejection And aging.
  • the neoplasia, pathogen infection, autoimmune disease, inflammatory disease, allograft, or transplant rejection Or senescence is associated with specific interactions between NKG2DL and NKG2D proteins.
  • the treating or preventing neoplasia comprises reducing tumor burden in the subject, Increasing or prolonging the survival of a subject with neoplasia or increasing the immune-activated cytokine production in response to cancer cells or pathogens in the subject.
  • the tumor or neoplasia is selected from the group consisting of the tumor or neoplasia selected from the group consisting of liver cancer, glioma, Ovarian cancer, gastric cancer, blastoma, pancreatic cancer, lung cancer, prostate cancer cells, breast cancer or neuroblastoma, bladder cancer, colon cancer, renal cell carcinoma, leukemia, lymphoma, multiple myeloma, melanoma, And its combination. (corresponding to the test target cells of the examples)
  • the tumor or neoplasm is formed as liver cancer.
  • the tumor or neoplasia is formed into a glioma.
  • the tumor or neoplasia is ovarian cancer.
  • the tumor or neoplasia is gastric cancer.
  • the tumor or neoplasia is formed as a parental cell tumor.
  • the tumor or neoplasia is pancreatic cancer.
  • the tumor or neoplasia is formed into lung cancer.
  • the tumor or neoplasia is formed as a prostate cancer.
  • the tumor or neoplasia is breast cancer.
  • the present invention is based on the construction of human NKG2D molecules targeting human NKG2DL Specific chimeric antigen receptor (CAR) and the CAR-modified T cell (CAR-T cell), the novel CAR-T cell can effectively target a variety of tumor cells and can be used for the preparation of tumors for treating tumors. Preparation, especially expression A pharmaceutical preparation of a NKG2DL positive tumor.
  • CAR CAR-modified T cell
  • the inventors of the present invention unexpectedly discovered in the study that the isolated human-based construct comprising the specific recognition human NKG2DL constructed using the present invention
  • the chimeric antigen receptor-modified T cells of NKG2D have simple preparation steps, and have a tumor cell killing rate of 40% to 70% at an effective target ratio of 10:1. It significantly prolongs the survival time of immune cells in patients, enhances the ability of immune cells to target tumor cells, especially tumors expressing NKG2DL, and enhances the specific killing activity against lung cancer cells.
  • the invention Human NKG2D-based immunoreactive cells targeting chimeric antigen receptors of NKG2DL provide a new option for the treatment of tumors with good industrial application prospects.
  • the present application provides methods of treating diseases such as those requiring an enhanced immune response using such cells.
  • 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 nucleotide sequence
  • Amino acid sequence targeting a NKG2DL-specific CAR molecule from the amino terminus to the carboxy terminus, the amino acid sequence of the leader peptide (eg SEQ ID) No. 3), human NKG2D amino acid sequence (as shown in SEQ ID No. 4), amino acid sequence of human CD8 hinge region (such as SEQ ID The amino acid sequence of the human CD8 transmembrane region (shown as SEQ ID No. 11) and the amino acid sequence of the human 4-1BB intracellular domain (shown as SEQ ID) The amino acid sequence of No. 12) and the human CD3 ⁇ domain (shown as SEQ ID No. 14) are sequentially connected in series.
  • the amino acid sequence of the leader peptide eg SEQ ID) No. 3
  • human NKG2D amino acid sequence as shown in SEQ ID No. 4
  • amino acid sequence of human CD8 hinge region such as SEQ ID
  • the amino acid sequence of the human CD8 transmembrane region shown as SEQ ID No. 11
  • a nucleotide sequence that targets a NKG2DL-specific CAR molecule from the 5' end to the 3' end, a nucleotide sequence encoding a leader sequence (such as SEQ a nucleotide sequence encoding the human NKG2D sequence (as shown in SEQ ID No. 17,), a nucleotide sequence encoding the human CD8 hinge region (shown as SEQ ID). No. 23), a nucleotide sequence encoding the human CD8 transmembrane region (shown as SEQ ID No. 24), a nucleotide sequence encoding the human 4-1BB intracellular domain (eg SEQ ID)
  • the nucleotide sequence (shown as SEQ ID No. 27) encoding the CD3 ⁇ domain is sequentially connected in series with No. 25).
  • Step 2 Construction and identification of a plasmid expressing a specific CAR molecule targeting NKG2DL
  • Primer 5'-tccagaggttgattgtcgacttaacgcgtttagcgagggggcagggcctgcatgtgaag-3' (SEQ ID NO: SEQ ID No.33) PCR amplification of synthetic CAR molecular sequence, recovered by Axygen gel recovery kit (Hangzhou Zeheng), homologous to the vector lentiGuide-Puro (Nanjing Biosystems), which was digested with restriction endonucleases SmaI and MluI Reorganize the connection.
  • the system and conditions for the specific recombination ligation reaction are as follows:
  • Recombination ligation conditions The above reaction system was placed in a 50 ° C water bath, and the reaction was carried out for 15 min and then placed on ice for 1 min.
  • the Stbl3 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.
  • the Midi Kit (Qiagen, Germany) 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.
  • the human NKG2D sequence (eg SEQ ID) in the amino acid sequence of the NKG2DL-specific CAR molecule is targeted
  • the amino acid sequence of the human CD3 ⁇ domain is shown in SEQ ID No. 15, A nucleotide sequence encoding a human NKG2D sequence in a nucleotide sequence targeting a NKG2DL-specific CAR molecule (as shown in SEQ ID No. 18),
  • step 2 construction and identification of a plasmid expressing a specific CAR molecule targeting NKG2DL
  • the human NKG2D sequence eg SEQ ID
  • the amino acid sequence of the NKG2DL-specific CAR molecule is targeted No. 6
  • the nucleotide sequence encoding the human NKG2D sequence in the nucleotide sequence of the NKG2DL-specific CAR molecule is the same as in Example 1.
  • the human NKG2D sequence eg SEQ ID
  • the amino acid sequence of the human CD3 ⁇ domain is SEQ ID
  • the nucleotide sequence encoding the human NKG2D sequence in the nucleotide sequence targeting the NKG2DL-specific CAR molecule is the same as in Example 1.
  • the human NKG2D sequence eg SEQ ID
  • the amino acid sequence of the NKG2DL-specific CAR molecule is targeted No. 8
  • the nucleotide sequence encoding the human NKG2D sequence in the nucleotide sequence of the NKG2DL-specific CAR molecule is the same as in Example 1.
  • the human NKG2D sequence eg SEQ ID
  • the amino acid sequence of the human CD3 ⁇ domain is SEQ ID
  • the nucleotide sequence encoding the human NKG2D sequence in the nucleotide sequence targeting the NKG2DL-specific CAR molecule is the same as in Example 1.
  • MPC magnetic particle collector
  • the enriched CD3+ T cells were resuspended in culture medium (purchased from Life Technologies, USA, product information is OpTmizerT-Cell Expansion SFM, A1048503), adjusted to a cell solubility of 1 ⁇ 10 6 /ml, and finally Incubate for 2 days in a 37 ° C, 5% CO 2 incubator. T cell purity was measured by flow cytometry using anti-PE anti-human CD3 antibody (purchased from BioLegend, USA, Cat. No. 300408), and the results showed that the purity of T cells after magnetic bead enrichment exceeded 97% (Fig. 3). .
  • polyethyleneimine transfection reagent 408727, Sigma
  • the virus supernatants were collected and centrifuged at 4 ° C, 3000 rpm for 10-15 minutes, filtered through a 0.45 ⁇ m filter, and finally at 4 ° C, 25000 rpm. After ultracentrifugation for 2 to 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 ⁇ 10 5 cells/ml, and cultured at 37° C. in a 5% CO 2 atmosphere for about 24 hours (culture time was determined according to specific practice). In general, the cell confluence rate is between 50-70% in the case of viral infection.
  • the T-cell KD-025 CAR-T cells of CAR molecule, ie, the novel CAR-T cells, the molecular structure of CAR is shown in Figure 2, and the next functional experiment can be performed.
  • the prepared CAR-T cells were then tested for cell viability using a 7-AAD/CFSE cytotoxicity test kit (purchased from Biovision, Cat. No. K315-100) according to the kit's instructions.
  • Flow cytometry results show KD-025 CAR-T cell activity was greater than 95% ( Figure 4).
  • Blank control group T cells without viral infection.
  • KD-019 control group specific CAR-T cells targeting CD19.
  • Group KD-025 specific CAR-T cells targeting NKG2DL.
  • the cells to be detected collected during the preparation of the cells.
  • APC-labeled anti-human NKG2D antibody (APC Mouse Anti-Human NKG2D, according to antibody instructions) 558071, BD) was added to the cell suspension of the cell group to be tested and the control group, and incubated at 4 ° C for 60 minutes. Using flow cytometry (BD FacsCanto II) Stained cells were obtained and the results were analyzed using FlowJo software. The flow results are shown in Figure 5.
  • the abscissa PE-A indicates the fluorescence intensity of the fluorescence emitted by the APC after excitation (the portion received by the PMT); the ordinate SSC-A represents the cell The particle size), the expression of CAR molecule was barely detected in the control group, and the expression rate of CAR molecule in the KD-025 group reached 46.7%.
  • Example 4 expressed a specific chimeric antigen receptor that targets NKG2DL.
  • Tumor cell line also called target cell line: liver cancer cell SMMC7721 (purchased from Nanjing Kezhen Biotechnology Co., Ltd.), glioma cell U251 (purchased from the Center for Cell Resources, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) ), ovarian cancer cell SKOV3 (purchased from Kunming Institute of Zoology, Chinese Academy of Sciences), gastric cancer cell NUGC4 (purchased from Nanjing Kezhen Biotechnology Co., Ltd.), parental cell tumor HTB186 (purchased from Guangzhou Jiniou), pancreatic cancer cell BXPC3 (purchased from Guangzhou Ginny) Ou), lung cancer cell A549 (purchased from Kunming Institute of Zoology, Chinese Academy of Sciences), prostate cancer cell PC3 (purchased from Kunming Institute of Zoology, Chinese Academy of Sciences), breast cancer cell MCF7 (purchased from Kunming Institute of Zoology, Chinese Academy of Sciences).
  • liver cancer cell SMMC7721 purchasedd from Nanjing Kezhen Biotechnology Co., Ltd.
  • mice were collected by trypsinization, washed twice with PBS, resuspended in FACS solution (PBS containing 0.1% sodium azide and 0.4% BSA), counted, adjusted to a cell concentration of 1 ⁇ 10 6 /ml; respectively added MicA Antibodies (R&D Systems, FAB1300A-025), MicB Antibodies (R&D Systems, FAB1599A-025) and Isotype Controls (R&D Systems, IC0041A), and ULBP1 Antibodies (R&D Systems, FAB1380A), ULBP2 Antibodies (R&D Systems, FAB1298A), ULBP3
  • the antibody (R&D Systems, FAB1517A) and the isotype control (R&D Systems, IC003A) were incubated at 4 °C for 60 minutes
  • Example 4 the specific CAR-T targeting NKG2DL obtained in Example 4 was evaluated using the 7-AAD/CFSE Cytotoxicity Test Kit (purchased from Biovision, Cat. No. K315-100) and following the operating instructions of the kit. The killing of cells by 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; There were no T cells infected with the virus (i.e., CD3+ T cells obtained in Example 2); the KD-019 control group was added with unrelated CAR-T cells targeting CD19.
  • 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).
  • the blank control group and the KD-019 control group also mixed T cells with target cells according to three different target ratios.
  • Figure 7 shows the results of tumor cell killing rate test using liver cancer cell SMMC7721 as a target cell.
  • the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 has a significant killing effect on liver cancer cell SMMC7721 (significantly higher than the two control groups), and the effective target ratio is 10:1.
  • the corresponding tumor cell killing rate exceeds 60%.
  • Figure 8 shows the results of tumor cell killing rate test using glioma cell U251 as a target cell.
  • the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 has a significant killing effect on glioma cell U251 (significantly higher than the two control groups), and the effective target ratio is 10
  • the tumor cell killing rate corresponding to :1 is over 70%.
  • Figure 9 shows the results of tumor cell killing rate test using ovarian cancer cell line SKOV3 as a target cell.
  • the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 has a significant killing effect on ovarian cancer cell SKOV3 (significantly higher than the two control groups), and the effect ratio is 10: 1 The corresponding tumor cell killing rate exceeds 50%.
  • Figure 10 shows the results of tumor cell killing rate test using gastric cancer cell NUGC4 as a target cell.
  • the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 has a significant killing effect on gastric cancer cell NUGC4 (significantly higher than the two control groups), and the effective target ratio is 10:1.
  • the corresponding tumor cell killing rate exceeds 40%.
  • Figure 11 shows the results of tumor cell killing rate test with the parental cell tumor cell HTB186 as the target cell.
  • the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 has a significant killing effect on the parental cell tumor cell HTB186 (significantly higher than the two control groups), and the target ratio is The tumor cell killing rate corresponding to 10:1 is over 70%.
  • Figure 12 shows the results of tumor cell killing rate test using pancreatic cancer cell line BXPC3 as a target cell.
  • the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 had a significant killing effect on pancreatic cancer cell line BXPC3 (significantly higher than the two control groups), and the target ratio was 10: 1 The corresponding tumor cell killing rate exceeds 70%.
  • Figure 13 shows the results of tumor cell killing rate test using lung cancer cell A549 as a target cell.
  • the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 has a significant killing effect on lung cancer cell A549 (significantly higher than the two control groups), and the effective target ratio is 10:1.
  • the corresponding tumor cell killing rate exceeds 40%.
  • Figure 14 shows the results of tumor cell killing rate test using prostate cancer cell line PC3 as a target cell.
  • the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 had a significant killing effect on prostate cancer cell PC3 (significantly higher than the two control groups), and the target ratio was 10: 1 The corresponding tumor cell killing rate exceeds 70%.
  • Figure 15 shows the results of tumor cell killing rate test using breast cancer cell MCF7 as a target cell.
  • the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 had a significant killing effect on breast cancer cells MCF7 (significantly higher than the two control groups), and the target ratio was 10: 1 The corresponding tumor cell killing rate exceeds 50%.
  • Figure 16 shows the results of tumor suppression test of CAR-T cells on nude mice bearing liver cancer SMMC7721.
  • the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 has a significant inhibitory effect on the transplanted tumor of liver cancer SMMC7721, and the tumor completely disappeared after 14 days of inoculation (Fig. 14A), tumor.
  • the fluorescence intensity was significantly lower than the three control groups (Fig. 14B).
  • Figure 17 shows the results of tumor suppression test of CAR-T cells on glioma U251 xenograft nude mice.
  • the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 had a significant inhibitory effect on glioma U251 xenografts, and the tumor disappeared completely after 14 days of inoculation (Fig. 17A).
  • the tumor fluorescence intensity was significantly lower than that of the three control groups (Fig. 17B).
  • Figure 18 shows the results of tumor suppression test of CAR-T cells on ovarian cancer SKOV3 xenograft nude mice.
  • the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 had a significant inhibitory effect on ovarian cancer SKOV3 xenografts, and tumor growth was significantly inhibited 14 days after inoculation (Fig. 18A).
  • the tumor fluorescence intensity was significantly lower than that of the three control groups (Fig. 18B).
  • Figure 19 shows the results of tumor suppression test of CAR-T cells on nude mice bearing gastric cancer NUGC4.
  • the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 had a significant inhibitory effect on gastric cancer NUGC4 xenografts, and tumor growth was inhibited 14 days after inoculation (Fig. 19A), tumor The fluorescence intensity was lower than the three control groups (Fig. 19B).
  • Figure 20 shows different doses of KD-025 The effect of CAR-T cells on the major organs and survival cycle of mice. As can be seen from Fig. 17, the specific CAR-T cell KD-025 targeting NKG2DL of Example 4 did not cause inflammation, edema and necrosis in the main organs of the heart, liver, lung, kidney, etc. (Fig. 20A). And did not have any negative impact on the survival cycle of the mice (Fig. 20B).
  • Figure 21 shows KD-025 The retention time of CAR-T in mice. As can be seen from Fig. 21, the control and KD-025 CAR-T cells decreased to about 50% after 10 days of injection, and decreased to about 25% after 17 days. After 31 days, no residual CAR-T was detected. Cells ( Figure 21).
  • Figure 22 shows KD-025 Analysis of the effect of CAR molecular virus on the expression of protooncogene after transfection of T cells.
  • the control and KD-025CAR virus-transfected T cells were in the oncogene cMyc, kras Real-time quantitative PCR analysis of BM11 and TP53 expression revealed that KD-025CAR virus transfection of T cells did not change the expression level of intracellular oncogenes (Fig. 22).
  • the specific CAR-T cells targeting NKG2DL of Example 4 of the present invention are capable of specifically recognizing NKG2DL-positive tumor cells with targeted killing power.
  • the specific chimeric antigen receptor targeting NKG2DL and the specific CAR-T cell targeting NKG2DL infected by the viral vector of the present invention can be applied to treat liver cancer, ovarian cancer, gastric cancer, lung cancer and prostate. Cancer, breast cancer, glioma or neuroblastoma, pancreatic cancer, bladder cancer, colon cancer, renal cell carcinoma, leukemia, lymphoma, multiple myeloma, melanoma, and combinations thereof.

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Abstract

一种靶向NKG2DL的特异性嵌合抗原受体、其编码序列、及其修饰的免疫应答细胞，以及它们的制备方法和应用。所述修饰的免疫应答细胞能有效地靶向攻击多种肿瘤细胞，尤其是表达NKG2DL阳性肿瘤细胞，可用来制备用于治疗肿瘤的制剂。

Description

靶向NKG2DL的特异性嵌合抗原受体T细胞，其制备方法和应用 技术领域
相关申请的引用
本申请要求于2017年3月15日提交的申请号为PCT/CN2017/076795 的 PCT专利申请的优先权，通过引用的方式将该专利文献的内容整体并入本文。
本发明属于肿瘤免疫治疗生物医药技术领域，涉及特异性嵌合抗原受体T细胞，具体地，涉及一种靶向 NKG2DL 的、包含 NKG2D 蛋白或其变体的特异性嵌合抗原受体（ chimeric antigen receptor,CAR ）及其修饰的免疫应答细胞，以及它们的制备方法和应用。
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、肿瘤疫苗以及免疫检验点阻断治疗等。其中，在细胞治疗领域，嵌合抗原受体修饰的免疫细胞（特别是Chimeric Antigen Receptor T-Cell ，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，嵌合抗原受体修饰的T细胞）为代表的免疫疗法，其原理主要是通过基因工程手段对病人自身提取的T细胞进行嵌合抗原受体的修饰形成CAR-T细胞，该T细胞能特异性地识别肿瘤表面相关抗原（肿瘤细胞标志物），从而靶向杀伤肿瘤。相对于常规免疫细胞，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。该以CAR-T细胞为代表的的修饰免疫细胞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免疫治疗方法是时急需解决的问题。
发挥嵌合抗原受体修饰的免疫应答细胞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
鉴于相关技术的上述问题和/或其他问题，本发明的目的是为了克服现有肿瘤临床技术中面临的肿瘤内环境中T细胞结合并杀死肿瘤细胞的特异性不强，杀伤效率低的问题，提供靶向人NKG2DL的嵌合抗原受体、及其基因和重组表达载体、工程化的NKG2DL靶向性嵌合抗原受体修饰的免疫应答细胞及其应用。本发明的工程化的NKG2DL靶向性的免疫应答细胞能够地提高肿瘤细胞的杀伤效率，从而提供了一种具有应用前景的肿瘤治疗的新手段。
技术方案
第一方面，本申请提供了一种靶向结合人NKG2DL的人NKG2D蛋白配体或其功能性变体，其包含选自下组的序列：
(1)SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8 ，或SEQ ID No.9所示的氨基酸序列，或 (2)（1）在一处或多处氨基酸修饰产生的功能性变体。
在一些实施方式中，所述氨基酸修饰包括但不限于氨基酸的取代、缺失和添加，所述功能性变体包括但不限于通过一处或多处氨基酸的取代、缺失和添加产生的衍生多肽或肽类似物。
在一些实施方式中，所述靶向结合人NKG2DL的人NKG2D蛋白配体或其功能性变体的氨基酸序列为SEQ ID No.4所示或与与SEQ ID No.4所示的氨基酸序列同源。
在一些实施方式中，所述靶向结合人NKG2DL的人NKG2D蛋白配体或其功能性变体的氨基酸序列为SEQ ID No.5所示或与与SEQ ID No.5所示的氨基酸序列同源。
在一些实施方式中，所述靶向结合人NKG2DL的人NKG2D蛋白配体或其功能性变体的氨基酸序列为SEQ ID No.6所示或与与SEQ ID No.6所示的氨基酸序列同源。
在一些实施方式中，所述靶向结合人NKG2DL的人NKG2D蛋白配体或其功能性变体的氨基酸序列为SEQ ID No.7所示或与与SEQ ID No.7所示的氨基酸序列同源。
在一些实施方式中，所述靶向结合人NKG2DL的人NKG2D蛋白配体或其功能性变体的氨基酸序列为SEQ ID No.8所示或与与SEQ ID No.8所示的氨基酸序列同源。
在一些实施方式中，所述靶向结合人NKG2DL的人NKG2D蛋白配体或其功能性变体的氨基酸序列为SEQ ID No.9所示或与与SEQ ID No.9所示的氨基酸序列同源。
第二方面，本申请提供了一种靶向结合人NKG2DL的特异性嵌合抗原受体，其包含从氨基端到羧基端顺次连接的引导序列的氨基酸序列、靶向结合人NKG2DL的胞外识别结构域氨基酸序列、跨膜结构域氨基酸序列和胞内信号结构域氨基酸序列，所述靶向结合人NKG2DL的胞外识别结构域的氨基酸序列包含本申请第一方面所述的靶向结合人NKG2DL的人NKG2D蛋白或其功能性变体，所述靶向NKG2DL的人NKG2D蛋白优选人NKG2DL的受体，所述靶向结合人NKG2DL的特异性嵌合抗原受体修饰的免疫应答细胞在效靶比为10:1时对肿瘤细胞杀伤效率达40%~70%。
在一些实施方式中，所述靶向结合人NKG2DL的特异性嵌合抗原受体还包括铰链区。
在一些实施方式中，所述跨膜结构域包括跨膜区。
在一些实施方式中，所述胞内信号结构域包括免疫受体酪氨酸活化基序和共刺激信号域；
在一些实施方式中，所述跨膜区包含CD8跨膜结构区、CD28跨膜结构区、CD3ζ跨膜结构区、CD4跨膜结构区、4-1BB跨膜结构区、OX40跨膜结构区、ICOS跨膜结构区、CTLA-4跨膜结构区、PD-1跨膜结构区、LAG-3跨膜结构区、2B4跨膜结构区、BTLA跨膜结构区、合成肽(不基于与免疫应答相关的蛋白质)中的任一种。
在一些实施方式中，所述免疫受体酪氨酸活化基序包含CD3ζ链的胞内信号结构域或FcεRIγ胞内信号结构，
在一些实施方式中，所述共刺激信号域包含CD28胞内信号结构域、CD137/4-1BB胞内信号结构域、CD134/OX40胞内信号结构域、ICOS胞内信号结构域中的至少一种。
在一实施方式中，所述铰链区选自CD8α铰链区、IgG铰链区，或包含整个或部分的免疫球蛋白的铰链区或铰链区经过一个或多个氨基酸修饰的变体。
在一实施方式中，所述引导序列选自 (1)具有SEQ ID No.3 所示氨基酸序列，或 （2）（1）的经过氨基酸修饰的功能性变体；其中所述经过氨基酸修饰的功能性变体为与SEQ ID No.3所示氨基酸序列具有同源性的多肽。
在一些实施方式中，所述跨膜区为人CD8多肽。在一些实施方式中，所述铰链区为人CD8多肽。
在一些实施方式中，所述铰链区的人CD8多肽的氨基酸序列选自SEQ ID No.10所示的多肽或（2）（1）的经过氨基酸修饰的功能性变体，其中所述经过氨基酸修饰的功能性变体为与SEQ ID No.10所示氨基酸序列具有同源性的多肽。
在本一实施例中，所述跨膜区的人CD8的氨基酸序列选自SEQ ID No.11所示的多肽或（2）（1）的经过氨基酸修饰的功能性变体，其中所述经过氨基酸修饰的功能性变体为与SEQ ID No.11所示氨基酸序列具有同源性的多肽。
在一些实施方式中，所述人4-1BB胞内结构域选自：
（1）具有SEQ ID NO.12所示氨基酸序列的多肽；或 （2）（1）的经过氨基酸修饰的功能性变体，其中所述经过氨基酸修饰的功能性变体为与SEQ ID No.12所示氨基酸序列具有同源性的多肽。
在一些实施方式中，所述人CD28ζ胞内结构域选自：(1)具有如SEQ ID No.13所示氨基酸序列的多肽；或 2）（1）的经过氨基酸修饰的功能性变体，其中所述经过氨基酸修饰的功能性变体为与SEQ ID No.13所示氨基酸序列具有同源性的多肽。
在一些实施方式中，所述CD3ζ胞内结构域选自：(1)具有如SEQ ID No.14所示氨基酸序列的多肽；或 （2）（1）的经过氨基酸修饰的功能性变体。其中所述经过氨基酸修饰的功能性变体为与SEQ ID No.14所示氨基酸序列具有同源性的多肽。
在一些实施方式中，所述CD3ζ胞内结构域选自：(1)具有如SEQ ID No.15所示氨基酸序列的多肽；或 （2）（1）的经过氨基酸修饰的功能性变体。，其中所述经过氨基酸修饰的功能性变体为与SEQ ID No.15所示氨基酸序列具有同源性的多肽。
在一些实施方式中，所述靶向结合人NKG2DL的特异性嵌合抗原受体是重组表达或由载体表达。
第三方面，本申请提供了一种编码第二方面所述的靶向结合人NKG2DL的特异性嵌合抗原受体的核酸分子，所述核酸分子包含从5‘到3‘依次串联连接的编码引导序列的核苷酸序列、编码人NKG2D的核苷酸序列、编码跨膜结构域的核苷酸序列和编码胞内信号结构域的核苷酸序列。
在一些实施方式中，所述胞内信号结构域包括免疫受体酪氨酸活化基序和共刺激信号域。
在一些实施方式中，所述靶向结合人NKG2DL的特异性嵌合抗原受体的核酸分子还包括编码铰链区的核苷酸序列。
在一些实施方式中，所述跨膜结构域包括跨膜区。
在一些实施方式中，所述跨膜区包含CD8跨膜结构区、CD28跨膜结构区、CD3ζ跨膜结构区、CD4跨膜结构区、4-1BB跨膜结构区、OX40跨膜结构区、ICOS跨膜结构区、CTLA-4跨膜结构区、PD-1跨膜结构区、LAG-3跨膜结构区、2B4跨膜结构区、BTLA跨膜结构区、合成肽(不基于与免疫应答相关的蛋白质)中的任一种，
在一些实施方式中，所述免疫受体酪氨酸活化基序包含CD3ζ链的胞内信号结构域或FcεRIγ胞内信号结构。
在一些实施方式中，所述共刺激信号域包含CD28胞内信号结构域、CD137/4-1BB胞内信号结构域、CD134/OX40胞内信号结构域、ICOS胞内信号结构域中的至少一种。
在一些实施方式中，所述铰链区选自CD8α铰链区、IgG铰链区，或包含整个或部分的免疫球蛋白的铰链区或铰链区经过一个或多个氨基酸修饰的变体。
在一些实施方式中，所述编码引导序列的核苷酸如SEQ ID No.16所示或与SEQ ID No.16所示核苷酸序列同源。
在一些实施方式中，所述编码人NKG2D的核苷酸序列如SEQ ID No.17所示或与SEQ ID No.17所示核苷酸序列同源。
在一实施例中，所述编码人NKG2D的核苷酸序列如SEQ ID No.18所示或与SEQ ID No.18所示的核苷酸序列同源。
在一实施例中，所述编码人NKG2D的核苷酸序列如SEQ ID No.19所示或与SEQ ID No.19所示的核苷酸序列同源。
在一实施例中，所述编码人NKG2D的核苷酸序列如SEQ ID No.20所示或与SEQ ID No.20所示的核苷酸序列同源。
在一实施例中，所述编码人NKG2D的核苷酸序列如SEQ ID No.21所示或与SEQ ID No.21所示的核苷酸序列同源。
在一实施例中，所述编码人NKG2D的核苷酸序列如SEQ ID No22所示或与SEQ ID No.22所示的核苷酸序列同源。
在一些实施方式中，所述编码铰链区的人CD8的核苷酸序列如SEQ ID No.23所示或与SEQ ID No.23所示核苷酸序列同源。
在一些实施方式中，所述编码跨膜区的人CD8的核苷酸序列如SEQ ID No.24所示或与SEQ ID No.24所示核苷酸序列同源，
在一些实施方式中，所述编码人4-1BB胞内结构域的核苷酸序列如SEQ ID No.25所示或与SEQ ID No.25所示核苷酸序列同源。
在一些实施方式中，所述编码人CD28胞内结构域的核苷酸序列如SEQ ID No.26所示或与SEQ ID No.26所示核苷酸序列同源。
在一些实施方式中，所述编码人CD3ζ胞内结构域的核苷酸序列如SEQ ID No.27所示或与SEQ ID No.27所示核苷酸序列同源。
在一些实施方式中，所述编码人CD3ζ胞内结构域的核苷酸序列如SEQ ID No.28所示或与SEQID No.28所示核苷酸序列同源。
第四方面，本申请提供了一种包含本申请第二方面的嵌合抗原受体或本申请第三方面的核酸的重组载体或表达质粒。
第五方面，本申请提供了一种启动子，用于构建本申请第四方面所述的重组载体和表达本申请第二方面所述的靶向结合人NKG2DL的特异性嵌合抗原受体，所述启动子包括但不限于核苷酸序列如SEQ No.29所示的EF1 alpha启动子和如SEQ No.30 所示的EFS启动子。
在一些实施方式中，所述启动子为核苷酸序列如SEQ No.30 所示 EFS启动子。
在一些实施方式中，所述启动子为核苷酸序列如SEQ No.29所EF1 alpha启动子。
第六方面，本申请提供了一种重组病毒，其是能够表达本发明第二方面所述的靶向NKG2DL的特异性嵌合抗原受体、且能够侵染免疫应答细胞的病毒。
第七方面，本申请提供了一种分离的修饰的免疫应答细胞，其包含本申请第二方面所述的靶向结合人NKG2DL的嵌合抗原受体，其由本发明第四方面所述的重组载体或表达质粒转化所得。
在一些实施方式中，所述免疫应答细胞包括细胞毒性T淋巴细胞、NK细胞、NKT细胞或辅助性T细胞。
在一些实施方式中，所述免疫应答细胞还包含至少一种外源的共刺激配体。
第八方面，本申请提供了本申请的第七方面的分离的嵌合抗原受体修饰的免疫应答细胞的制备方法，包括以下步骤：
首先，将第三方面所述的核酸分子，通过分子克隆的方式连接到表达载体中，获得靶向NKG2DL的特异性嵌合抗原受体的表达载体；
然后将获得的所述靶向NKG2DL的特异性的CAR表达载体转染293T细胞，获得病毒液；
最后用所述病毒液感染免疫应答细胞，从感染后的细胞中获得表达第二方面的靶向NKG2DL的特异性嵌合抗原受体的第七方面的修饰的免疫应答细胞。
在一些实施方式中，所述免疫应答细胞选自T细胞、自然杀伤(NK)细胞、细胞毒性T淋巴细胞(CTL)、调节性T细胞、人胚胎干细胞和可分化成淋巴样细胞的多能干细胞，优选T细胞或自然杀伤(NK)细胞。
第九方面，本申请提供了一种药物组合物，其包含有效量的如本发明一方面所述的靶向结合人NKG2DL的人NKG2D蛋白配体或其功能性变体、第二方面所述的靶向结合人NKG2DL的嵌合抗原受体、本发明第三方面所述的核酸或第七方面的所述的分离的修饰的免疫应答细胞和药学上可接受的赋形剂。
第十方面，本申请提供了一种用于治疗或预防疾病、不适或健康失调的受试者的瘤形成、病原体感染、自身免疫性疾病、同种异体移植、或移植排斥或抗衰老的试剂盒，其包含本发明第一方面所述的靶向结合人NKG2DL的人NKG2D蛋白及其功能性变体、第二方能所述的靶向结合人NKG2DL的嵌合抗原受体、本发明第三方面所述的核酸、或第七方面所述的分离的修饰的免疫应答细胞。
在一些实施方式中，所述试剂盒还包含使用免疫应答细胞治疗或预防疾病、不适或健康失调的受试者的书面说明书。
第十一方面，本申请提供了本发明第一方面所述的靶向结合人NKG2DL的人NKG2D蛋白及其变体、第二方面所述的靶向结合人NKG2DL的特异性嵌合抗原受体、第四方面所述重组载体或表达质粒，第六方面所述重组病毒、第七方面所述的分离的修饰的免疫应答细胞，第九方面的组合物或第十方面的试剂盒在制备治疗，或预防疾病、不适或健康失调的产品中的应用，其中，所述治疗或预防包括步骤向患有与NKG2DL和NKG2D配体蛋白的特异性相互作用引起的疾病的患者施用对治疗或预防为有效量的本申请第七方面所述的包含靶向NKG2DL的NKG2D的嵌合抗原受体T细胞（CAR-T细胞）。所述、不适或健康失调与NKG2DL和NKG2D配体蛋白的特异性相互作用有关。
在一些实施方式中，所述疾病、不适或健康失调包括瘤形成、感染、自身免疫性疾病、同种异体移植、移植排斥、衰老。
本发明基于人NKG2D分子构建靶向人NKG2DL的特异性嵌合抗原受体（CAR）以及该CAR修饰的T细胞（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，尤其是阳性表达NKG2DL的肿瘤的药物制剂。
 有益效果
本发明的发明人在研究中意外发现，采用本发明构建的包含特异性识别人NKG2DL的NKG2D的嵌合抗原受体修饰的T细胞，制备方法步骤简单，在效靶比为10:1时，具有高达40%~70%的肿瘤细胞杀伤率，可明显延长免疫细胞在患者体内的存活时间，增强免疫细胞靶向识别肿瘤细胞特别是表达NKG2DL的肿瘤的能力，加强对所述肿瘤或瘤形成选自肝癌、胶质瘤、卵巢癌、胃癌、随母细胞瘤、胰腺癌、肺癌、前列腺癌细胞、乳腺癌 或神经母细胞瘤、膀胱癌、结肠癌、肾细胞癌、白血病、淋巴瘤、多发性骨髓瘤、黑色素瘤、及其组合的特异杀伤活性。本发明的靶向NKG2DL嵌合人NKG2D的的修饰的免疫应答细胞为治疗肿瘤提供了一种新的方案选择，具有良好的产业应用前景。
 附图描述
图1作为示例的本发明使用的慢病毒载体结构示意图。
图2为实施例1和2中嵌合抗原受体各部分的连接顺序的示意图。A. EF1 alpha启动子（序列表SEQ ID No.29所示）, B. EFS启动子（序列表SEQ ID No.,30）; 其中 1为人CD3ζ结构域的氨基酸序列 , 2为人4-1BB胞内结构域的氨基酸序列，3为人CD8跨膜区的氨基酸序列，4为CD8铰链区的氨基酸序列，5为人NKG2D氨基酸序列，6为引导序列，7为EF1 alpha启动子，8为EFS启动子。
图3为实施例2中T细胞纯度流式细胞检测结果。
图4为实施例5中对CAR-T细胞活性的流式细胞检测结果，其中 4-A为空白对照组：没有感染病毒液的T细胞; 4-B为KD-019对照组:靶向CD19的特异性CAR-T细胞；4-C为KD-025: 靶向NKG2DL的特异性CAR-T细胞。
图5为实施例5中对KD-025 CAR分子表达的流式细胞检测结果，其中5-A.空白对照组：没有感染病毒液的T细胞; 5-B. KD-019对照组:靶向CD19的特异性CAR-T细胞；5-C.KD-025:靶向NKG2DL的特异性CAR-T细胞。
图6 NKG2DL的主要亚型 MicA，MicB，ULBP1， ULBP2和ULBP3在肿瘤细胞系中表达的检测，肝癌、卵巢癌、胃癌、前列腺癌、肺癌、乳腺癌、胶质瘤（图6-A为对照：同型对照（R&D Systems，IC003A）； 6-B为肝癌细胞SMMC7721、6-C为胶质瘤细胞U251、6-D为卵巢癌细胞SKOV3、6-E为胃癌细胞NUGC4、6-F为随母细胞瘤HTB186、6-G为胰腺癌细胞BXPC3、6-H为肺癌细胞A549、6-I为前列腺癌细胞PC3、6-J为乳腺癌细胞MCF7）。
图7为以肝癌细胞SMMC7721为靶细胞测定本申请的靶向NKG2DL的特异性CAR-T细胞对肿瘤细胞杀伤率测试结果。
图8为以胶质瘤细胞U251为靶细胞测定本申请的靶向NKG2DL的特异性CAR-T细胞的肿瘤细胞杀伤率测试结果。
图9为以卵巢癌细胞SKOV3为靶细胞测定本申请的靶向NKG2DL的特异性CAR-T细胞的肿瘤细胞杀伤率测试结果。
图10为以胃癌细胞NUGC4为靶细胞测定本申请的靶向NKG2DL的特异性CAR-T细胞的肿瘤细胞杀伤率测试结果。
图11为以随母细胞瘤细胞HTB186为靶细胞测定本申请的靶向NKG2DL的特异性CAR-T细胞的肿瘤细胞杀伤率测试结果。
图12为以胰腺癌细胞BXPC3为靶细胞测定本申请的靶向NKG2DL的特异性CAR-T细胞的肿瘤细胞杀伤率测试结果。
图13为以肺癌细胞A549为靶细胞测定本申请的靶向NKG2DL的特异性CAR-T细胞的肿瘤细胞杀伤率测试结果。
图14为以前列腺癌细胞PC3为靶细胞测定本申请的靶向NKG2DL的特异性CAR-T细胞的肿瘤细胞杀伤率测试结果。
图15为以乳腺癌细胞MCF7为靶细胞测定本申请的靶向NKG2DL的特异性CAR-T细胞的肿瘤细胞杀伤率测试结果。
图16为以肝癌SMMC7721为靶细胞测定本申请的靶向NKG2DL的特异性CAR-T细胞的肿瘤动物模型杀伤测试结果，其中 16-A显示移植瘤在裸鼠中的生长; 16-B显示肿瘤荧光强度值。
图17为以胶质瘤U251为靶细胞测定本申请的靶向NKG2DL的特异性CAR-T细胞的肿瘤动物模型杀伤测试结果。其中 17-A显示移植瘤在裸鼠中的生长; 17-B显示肿瘤荧光强度值。
图18为以卵巢癌SKOV3为靶细胞测定本申请的靶向NKG2DL的特异性CAR-T细胞的肿瘤动物模型杀伤测试结果其中 18-A显示移植瘤在裸鼠中的生长; 18-B显示肿瘤荧光强度值。
图19为以胃癌NUGC4为靶细胞测定本申请的靶向NKG2DL的特异性CAR-T细胞的肿瘤动物模型杀伤测试结果，其中 19-A显示移植瘤在裸鼠中的生长; 19-B显示肿瘤荧光强度值。
图20为用B-NSG动物进行KD-025 CAR毒理及安全剂量实验的测试结果，其中20-A显示毒理实验的测试结果；20-B显示安全剂量实验的测试结果。
图21为用B-NSG动物进行KD-025 CAR体内存留实验的测试结果。
图22为KD-025 CAR病毒感染的T细胞后相关癌基因表达检测结果。
最佳实施方式
除非另有定义，本文使用的所有技术和科学术语具有本发明所属领域的技术员通常理解的含义。
术语“功能性变体”，是母体结构经过修饰得到的术语“功能性变体”是指与母体具有相同或相近的生物学功能和性质如与母体具有相同靶向结合功能的结构的变体。作为非限制性的实例，“功能性变体”可以通过在母体中进行一处或多处保守型取代获得。本申请中功能性变体是，在NKG2DL的配体NKG2D氨基酸序列的基础上修饰产生的与NKG2DL靶标结合的结构。
术语“类似物”是指结构相关的多肽，其具有参照多肽分子的功能。在本申请中，指与配体NKG2D氨基酸序列结构相关的，并具有NKG2D的靶向结合NKG2DL的功能的多聚氨基酸结构。
术语“氨基酸修饰”是指不显著影响或改变包含氨基酸序列的本公开的CAR(例如，细胞外识别结构域)的结合特征的保守氨基酸修饰。这种保守修饰包括氨基酸取代、添加和缺失。
术语“保守性氨基酸取代”是其中氨基酸残基被相同组内的氨基酸替代的取代。
术语“同源性”：指目标氨基序列或目标核苷酸序列与参考序列比较显示的氨基酸或核苷酸的高比例匹配性。本文中的同源性可使用标准软件如BLAST或FASTA确定
术语“嵌合抗原受体(CAR)”
嵌合抗原受体包括引导肽部分、细胞外靶标识别结构域、跨膜结构域和细胞内结构域。
特异性嵌合抗原受体(CAR)是可结合靶标抗原的人工设计的特异性抗体或配体，在本申请中是靶向结合人NKG2DL的特异性嵌合抗原受体（CAR）。CAR可以用于通过逆转录病毒载体促进其编码序列的转移将单克隆抗体的特异性移植到T细胞上。
CAR可以既结合抗原又转导T细胞激活的功能，其不依赖于MHC限制。因此，CAR是“通用”免疫抗原受体，其可以治疗具有抗原阳性肿瘤的患者群体，不论其HLA基因型如何。使用表达肿瘤特异性CAR的T淋巴细胞的过继性免疫疗法可以是用于治疗癌症的强大治疗策略。
术语“抗原”，是指肿瘤细胞细胞表面特异性高表达的可与配体特异性识别和结合的靶标分子。在本发明中指人NKG2DL。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。在具体的示例性实施方式中，细胞外抗原结合结构域是NKG2D。在具体的非限制性实施方式中，任何序列的NKG2D分子可以包含在具有异源序列的融合蛋白中以形成细胞外抗原结合结构域。
NKG2DL蛋白的受体为NKG2D，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。
术语“识别”是指选择性结合靶标。本文使用的术语“特异性结合”或“特异性结合到”或“特异性靶向”是指多肽或其片段识别并结合目的生物分子(例如多肽)，但其基本上不识别结合样品中的其他分子，例如天然地包括本发明多肽的生物样品中的其他分子。
术语“特异性结合”是指两个分子（例如配体和受体）之间的结合，其特征是甚至在存在许多其他不同分子时，一种分子（配体）与另一种特异分子（受体）结合的能力，即在分子的异质混合物中显示一种分子对另一分子的优先结合的能力。配体与受体的特异性结合也如下被证明：存在过量未标记的配体时，经可检测标记的配体与受体的结合降低（即 结合竞争实验）。
术语“受体”是可与靶标序列特异性结合的分子结构。特别地，受体结合另一个细胞上的配体，允许细胞到细胞的识别和/或相互作用。在本发明中，受体指靶向人NKG2DL靶标的人NKG2D。
术语“共刺激分子”是指淋巴细胞对抗原的有效应答所需的除抗原受体或其配体之外的细胞表面分子。
术语“载体”是指任何遗传元件，比如质粒、噬菌体、转座子、粘粒、染色体、病毒、病毒粒子等，其当关联有适当的控制元件时能够复制，并且其可以将基因序列转移到细胞中。因此，该术语包括克隆和表达载体，以及病毒载体和质粒载体。
术语“表达载体”是指一种重组核酸序列，即重组DNA分子，其含有期望编码序列和在特定宿主生物体中表达可操作连接的编码序列所必需的适当的核酸序列。用于在原核生物中表达所必需的核酸序列通常包括启动子、操纵子(可选的)和核糖体结合位点，通常伴随其它序列。已知真核细胞利用启动子、增强子和终止子及聚腺苷酸化信号。
本文使用的术语“免疫应答细胞”是指在免疫应答中起作用的细胞、或其祖细胞、或其子代。
术语“分离的细胞”是指与天然伴随细胞的分子和/或细胞组分分离的免疫细胞。
本文使用的术语“调节”是指正或负地改变。
本文使用的术语“病原体”是指能够引起疾病的病毒、细菌、真菌、寄生虫或原生动物。
术语“治疗(treating、treatment)”是指试图改变所治疗的个体或细胞的疾病过程的临床干预，并且可以用于预防，或在临床病理过程期间进行。治疗的治疗效果包括但不限于预防疾病的发生或复发、症状的缓解、疾病的任何直接或间接病理学后果的减少、预防转移、降低疾病进展的速度、减轻或缓和疾病状态、和缓解或改善的预后。通过预防疾病或病症的进展，治疗可以防止受侵袭或诊断的受试者或疑似患有病症的受试者中的病症引起的恶化，而且治疗可以预防具有病症风险或疑似患有病症的受试者中病症或病症症状的发作。
术语“杀伤效率”是一定比例的肿瘤拮抗剂和肿瘤细胞混合在体外测试体系中观察到的肿细胞失活比例。在本文是嵌合抗原受体修饰的免疫应答细胞造成肿瘤细胞死亡的比例。特别是效靶比为10:1时，的NKG2D嵌合抗原受体修饰的T细胞对表达NKG2DL的肿瘤细胞的杀伤比例。
本文使用的术语“疾病”是指破坏或干扰细胞、组织或器官的正常功能的任何病症或障碍。疾病的实例包括瘤形成或细胞的病原体感染。
本文使用的术语“有效量”是指足以具有治疗效果的量。在一个实施方式中，“有效量”是足以阻止、改善或抑制瘤形成的持续增殖、生长或转移(例如，侵袭或迁移)的量。
本文使用的术语“外源的”是指并非内源性存在于细胞中或不以足以达到过表达时获得的功能性效果的水平存在的核酸分子或多肽。因此，术语“外源的”将包括在细胞中表达的任何重组核酸分子或多肽，例如外源、异源和过表达的核酸分子和多肽。
本文使用的术语“外源核酸分子或多肽”是指并非正常存在于细胞中或由细胞获得的样品中的核酸分子(例如，cDNA、DNA或RNA分子)或多肽。该核酸可以来自另一生物体，或者其可以是例如细胞或样品中并非正常表达的mRNA分子。
术语“治疗(treating、treatment)”是指试图改变所治疗的个体或细胞的疾病过程的临床干预，并且可以用于预防，或在临床病理过程期间进行。治疗的治疗效果包括但不限于预防疾病的发生或复发、症状的缓解、疾病的任何直接或间接病理学后果的减少、预防转移、降低疾病进展的速度、减轻或缓和疾病状态、和缓解或改善的预后。通过预防疾病或病症的进展，治疗可以防止受侵袭或诊断的受试者或疑似患有病症的受试者中的病症引起的恶化，而且治疗可以预防具有病症风险或疑似患有病症的受试者中病症或病症症状的发作。
本文使用的术语“杀伤效率”是一定比例的肿瘤拮抗剂和肿瘤细胞混合在体外测试体系中观察到的肿细胞失活比例。在本文是嵌合抗原受体修饰的免疫应答细胞造成肿瘤细胞死亡的比例。特别是效靶比为10:1时，的NKG2D嵌合抗原受体修饰的T细胞对表达NKG2DL的肿瘤细胞的杀伤比例。
术语“有效量”是指足以具有治疗效果的量。在一个实施方式中，“有效量”是足以阻止、改善或抑制瘤形成的持续增殖、生长或转移(例如，侵袭或迁移)的量。
共刺激配体是在细胞表面上表达的蛋白质，其在结合其受体时产生共刺激应答，即实现当抗原结合其CAR分子时所提供的刺激的细胞内应。
术语“外源的”是指并非内源性存在于细胞中或不以足以达到过表达时获得的功能性效果的水平存在的核酸分子或多肽。因此，术语“外源的”将包括在细胞中表达的任何重组核酸分子或多肽，例如外源、异源和过表达的核酸分子和多肽。
第一方面，本申请提供了一种靶向结合人NKG2DL的人NKG2D蛋白及其功能性变体， 包含选自下组的序列：
（1）SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8 ，或SEQ ID No.9所示的氨基酸序列，或 (2) （1）在一处或多处氨基酸修饰产生的变体。
根据已有研究，靶向结合人NKG2DL的肽人NKG2D蛋白是基于配体人NKG2D蛋白的氨基酸序列。
根据已有的研究，如果以NKG2D的全长氨基酸序列来构建CAR，效果较差，因为NKG2D胞内区等功能域是自然杀伤细胞属性的，与T细胞并不一定兼容。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优化预测工具等手段来筛选分析了靶标人NKG2DL靶标和NKG2D片段，进一步设计构建包含改进的NKG2D的特异性靶向人NKG2DL的嵌合抗原受体（Chimeric Antigen Receptor，CAR）分子的氨基酸序列；其中如何选择适宜的、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，对于本领域普通技术人员来说也是一个难解的技术问题。
发明人经过创造性劳动，不断地进行氨基酸序列设计以及序列的排列组合和筛选，对10余条CAR分子的序列进行随机筛选试验和靶向性功能验证（例如构建病毒载体，以及进一步感染T细胞，获得修饰的T细胞，并检测所得修饰的T细胞的体外杀伤活性等试验），之后根据多个随机组合的结果比对，再进行序列调整，最终筛选出效果最好的序列，获得了本发明的高效价靶向人NKG2DL的人NKG2D氨基酸序列及其功能性变体。
在一些非限制性实施方式中，所述氨基酸修饰包括但不限于氨基酸的取代、缺失和添加，所述功能性变体包括但不限于通过氨基酸的取代、缺失和添加产生的靶向人NKG2DL的人NKG2D氨基酸序列的衍生多肽或肽类似物，所述衍生多肽或肽类似物与人NKG2D氨基酸序列具有同源性。
在一些非限制性实施方式中，所述同氨基酸修饰包括但不限于氨基酸侧链的化学修饰、天然或非天然氨基酸取代、突变、缺失、插入或翻译后修饰。
在一些非限制性实施方式中，所述经过靶向人NKG2DL的人NKG2D氨基酸序列的经过一处或多处氨基酸修饰的变体为与SEQ ID No.5所示氨基酸序列具有同源性的多肽。在一些非限制性实施方式中，所述靶向人NKG2DL的人NKG2D氨基酸序列经过一处或多处氨基酸修饰的变体为与SEQ ID No.6所示氨基酸序列具有同源性的多肽。在一些非限制性实施方式中，所述靶向人NKG2DL的人NKG2D氨基酸序列经过一处或多处氨基酸修饰的变体为与SEQ ID No.7所示氨基酸序列具有同源性的多肽。在一些非限制性实施方式中，所述靶向人NKG2DL的人NKG2D氨基酸序列经过一处或多处氨基酸修饰的变体为与SEQ ID No.8所示氨基酸序列具有同源性的多肽。在一些非限制性实施方式中，所述靶向人NKG2DL的人NKG2D氨基酸序列经过一处或多处氨基酸修饰的变体为与SEQ ID No.9所示氨基酸序列具有同源性的多肽。
所述同源性多肽为相对于参考多肽，在这里是指与靶向人NKG2DL靶标的NKG2D具有70~99％，优选80~99%，更优选90~99%，最优选95~99%同源性的多肽。
在一些非限制性实施方式中，所述与SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8 或SEQ ID No.9所示氨基酸序列具有同源性的多肽是将SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8 或SEQ ID No.9中任一所示氨基酸序列经过1~10个氨基酸残基，优选地经过1~5个氨基酸残基或更优选经过1~3个氨基酸残基的取代、缺失或添加而形成的衍生多肽。
除了全长多肽之外，本申请第一方面公开的主题还提供本申请公开的主题的多肽或肽结构域中任一段片段。在一些实施方案中，所述片段可以是至少5~15个氨基酸。在一些实施方案中，所述片段是至少20个连续氨基酸、至少30个连续氨基酸或至少50个连续氨基酸。在一些实施方案中，所述片段是至少60至80、100、200、300或更多个连续氨基酸。
本申请第一方面公开的主题的氨基酸片段可以通过本领域普通技术人员已知的方法产生，或者可以由正常的蛋白质加工策略产生非蛋白质类似物(例如从新生多肽中除去生物活性不需要的氨基酸或通过备选mRNA剪接或备选蛋白质加工事件除去氨基酸)。非蛋白质类似物可具有设计为模拟本发明蛋白质的功能活性的化学结构。此类类似物根据本发明公开的主题的方法施用。此类类似物可能超过原始多肽的生理活性。模拟设计的方法是本领域公知的，并且可以根据这样的方法通过修饰化学结构来进行类似物的合成，使得当在免疫应答细胞中表达时，所得的类似物增加原始多肽的抗肿瘤活性。这些化学修饰包括但不限于取代替代的R基团和改变参考多肽的特定碳原子的饱度。蛋白质类似物可以对体内降解相对耐受，导致施用时更长的治疗效果。
用于测量第一方面所述的肽或其功能性变体的功能活性的试验包括但不限于下文实施例中描述的试验方法。
第二方面，本申请提供了一种靶向结合人NKG2DL的特异性嵌合抗原受体，其包含从氨基端到羧基端顺次连接的引导序列氨基酸序列、靶向结合人NKG2DL的胞外识别结构域氨基酸序列、跨膜结构域氨基酸序列和胞内信号结构域氨基酸序列，所述靶向结合人NKG2DL的胞外识别结构域氨基酸序列包含本申请第一方面所述的靶向结合人NKG2DL的人NKG2D蛋白或其功能性变体，所述靶向NKG2DL的人NKG2D蛋白优选人NKG2DL的受体，所述靶向NKG2DL的人NKG2D蛋白修饰的免疫应答细胞在效靶比为10:1时对肿瘤细胞杀伤效率达40%~70%。
胞外识别结构域 (还称作胞外域或简单地由其含有的识别元件所指)包含特异性结合靶细胞的细胞表面上存在的分子的识别元件。跨膜域的功能是锚定结合细胞膜。胞内信号结构域包含免疫受体活化基序并任选地包含一个或多个共刺激信号传导域。
细胞外识别抗原的结构域还可以包含将新生蛋白质导入内质网的前导序列或信号肽。如果CAR将被糖基化并锚定在细胞膜中，信号肽或前导序列可能是必需的。信号序列或前导序列可以是存在于新合成的蛋白质的N-末端的肽序列(长度约5、约10、约15、约20、约25或约30个氨基酸)，其指导蛋白质进入分泌通路。
在一些非限制性实例中，前导序列共价连接到细胞外抗原结合结构域的5’末端。
在一些实施方式中，所述靶向结合人NKG2DL的特异性嵌合抗原受体包括铰链区。
在一些实施方式中，所述跨膜结构域包括跨膜区。
在一些非限制性实施方式中，所述胞内信号结构域包括免疫受体酪氨酸活化基序和共刺激信号域；
在某些非限制性实施方式中，CAR的跨膜结构域包含跨越膜的至少一部分的疏水性α螺旋。不同的跨膜结构域产生不同的受体稳定性。在抗原识别之后，受体簇和信号被传递到细胞。
在一些非限制性实施方式中，所述 CAR的跨膜结构域包含CD8跨膜结构区、CD28跨膜结构区、CD3ζ跨膜结构区、CD4跨膜结构区、4-1BB跨膜结构区、OX40跨膜结构区、ICOS跨膜结构区、CTLA-4跨膜结构区、PD-1跨膜结构区、LAG-3跨膜结构区、2B4跨膜结构区、BTLA跨膜结构区、合成肽(不基于与免疫应答相关的蛋白质)中的任一种。
在一些非限制性实施方式中，所述免疫受体酪氨酸活化基序包含CD3ζ链的胞内信号结构域或FcεRIγ胞内信号结构。
在一些非限制性实施方式中，所述共刺激信号域包含CD28胞内信号结构域、CD137/4-1BB胞内信号结构域、CD134/OX40胞内信号结构域、ICOS胞内信号结构域中的至少一种。
在一些非限制性实施方式中所述铰链区选自CD8α铰链区、IgG铰链区，或包含整个或部分的免疫球蛋白的铰链区或铰链区经过一个或多个氨基酸修饰的变体。
在一实施方式中，所述嵌合抗原受体的氨基酸序列从氨基端到羧基端由引导序列、本申请第一方面所述的靶向结合人NKG2DL的人NKG2D的氨基酸序列、人CD8铰链区的氨基酸序列、人CD8跨膜区的氨基酸序列、人CD28胞内结构域的氨基酸序列、人4-1BB胞内结构域的氨基酸序列和人CD3ζ胞内结构域的氨基酸序列依次串联而成；
在一实施方式中，所述嵌合抗原受体的氨基酸序列从氨基端到羧基端由引导肽的氨基酸序列、本申请第一方面所述靶向结合人NKG2DL的人NKG2D的氨基酸序列、人CD8跨膜区的氨基酸序列、人CD8铰链区序列的氨基酸序列、人CD28胞内结构域的氨基酸序列和CD3ζ胞内结构域的氨基酸序列依次串联而成。
在一实施方式中，所述嵌合抗原受体的氨基酸序列从氨基端到羧基端由引导序列、本申请第一方面所述靶向结合人NKG2DL的人NKG2D氨基酸序列、人CD8跨膜区的氨基酸序列、4-1BB胞内结构域的氨基酸序列和CD3ζ胞内结构域的氨基酸序列依次串联而成；
在一实施方式中，所述嵌合抗原受体的氨基酸序列从氨基端到羧基端由引导序列、本申请第一方面所述的靶向结合人NKG2DL的人NKG2D氨基酸序列、人CD8跨膜区氨基酸序列、人CD28胞内结构域的氨基酸序列和人CD3ζ序列胞内结构域的氨基酸序列依次串联而成。
在一实施方式中，所述嵌合抗原受体的氨基酸序列从氨基端到羧基端由引导肽序列的氨基酸谢、本申请第一方面所述的靶向结合人NKG2DL的人NKG2D的氨基酸序列、人CD8跨膜区的氨基酸序列、人CD8铰链区的氨基酸序列、人CD28胞内结构域的氨基酸序列和人CD3ζ的氨基酸序列依次串联而成。
在一优选实施例中，所述的嵌合抗原受体包括如下的从氨基端到羧基端顺序连接的引导肽的氨基酸序列、本申请第一方面所述的靶向结合人NKG2DL的人NKG2D的氨基酸序列、人CD8跨膜区的氨基酸序列、人4-1BB的氨基酸序列和人CD3ζ的氨基酸序列。
在一优选实施例中，所述的嵌合抗原受体包括如下的从氨基端到羧基端顺序连接的引导肽的氨基酸序列、本申请第一方面所述的靶向结合人NKG2DL的人NKG2D的氨基酸序列、人CD8跨膜区的氨基酸序列、人CD28的氨基酸序列和人CD3ζ的氨基酸序列。
在一优选实施例中，所述的嵌合抗原受体包括如下的从氨基端到羧基端顺序连接的引导肽的氨基酸序列、本申请第一方面所述的靶向结合人NKG2DL的人NKG2D的氨基酸序列、人CD8铰链区的氨基酸序列、人CD8跨膜区的氨基酸序列、人4-1BB的氨基酸序列和人CD3ζ的氨基酸序列。
在一优选实施例中，所述的嵌合抗原受体包括如下的从氨基端到羧基端顺序连接的引导肽的氨基酸序列、本申请第一方面所述的靶向结合人NKG2DL的人NKG2D的氨基酸序列、人CD8铰链区的氨基酸序列、人CD8跨膜区的氨基酸序列、人CD28的氨基酸序列和人CD3ζ的氨基酸序列。
在一些非限制性实施方式中，所述引导序列选自 (1)具有SEQ ID No.3 所示氨基酸序列的多肽，或 （2）（1）的经过氨基酸修饰的功能性变体；其中所述经过氨基酸修饰的功能性变体为与SEQ ID No.3所示氨基酸序列具有70~99％同源性，优选80~99%，更优选90~99%，最优选95~99%同源性的多肽。
在某些非限制性实施方式中，本申请第二方面公开的主题的CAR包含将抗原结合结构域连接至跨膜结构域的间隔物区(spacer)，又称铰链区。一间隔物区可以是足够柔性的，以允许抗原结合结构域在不同方向上定向，以利于抗原识别。间隔物区区可以是来自IgG1的铰链区、或免疫球蛋白的CH2CH3区和CD3的部分。一些间隔物区包含免疫球蛋白CH3域或CH3域和CH2域两者一些间隔物区包含整个或部分的免疫球蛋白(例如，IgG1、IgG2、IgG3、IgG4)铰链区，即落在免疫球蛋白的CH1和CH2域之间的序列，例如，IgG4Fc铰链或CD8铰链。免疫球蛋白来源的序列可以包含一个或多个氨基酸修饰，例如，1、2、3、4或5个取代。在一些非限制性实施方式中，所述间隔物区为铰链区。
在某些非限制性实施方式中，本申请第二方面公开的主题的CAR包括含有CD28多肽的跨膜结构域和含有CD28多肽的共刺激信号传导区。
在一些实施方式中，铰链区包含人CD8多肽。该铰链区CD8多肽选自 (1)具有SEQ ID No.10 所示氨基酸序列的多肽，或 （2）（1）的经过氨基酸修饰的功能性变体；其中所述经过氨基酸修饰的功能性变体为与SEQ ID No.310所示氨基酸序列具有70~99％同源性，优选80~99%，更优选90~99%，最优选95~99%同源性的氨基酸序列，或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。在非限制性的实施方式中，铰链区人CD8氨基酸序列具有SEQ ID NO:11的连续部分的氨基酸序列，其长度为至少20、或至少30、或至少40、或至少50，并且至多164个氨基酸。
在一些实施方式中，跨膜区包含人CD8多肽。该跨膜区CD8多肽选自 (1)具有SEQ ID No.11 所示氨基酸序列的多肽，或 （2）（1）的经过氨基酸修饰的功能性变体；其中所述经过氨基酸修饰的功能性变体为与SEQ ID No.11所示氨基酸序列具有70~99％同源性，优选80~99%，更优选90~99%，最优选95~99%同源性的氨基酸序列，或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。在非限制性的实施方式中，人跨膜区CD8氨基酸序列具有SEQ ID NO:11的连续部分的氨基酸序列，其长度为至少20、或至少30、或至少40、或至少50，并且至多164个氨基酸。
在一些实施方式中，所述人4-1BB胞内结构域的氨基酸序列选自：
（1）具有SEQ ID NO:12所示氨基酸序列的多肽；或 （2）（1）的经过氨基酸修饰的变体；其中所述经过氨基酸修饰的变体为与SEQ ID NO:12所示氨基酸序列具有70~99％同源性，优选80~99%，更优选90~99%，最优选95~99%同源性的多肽，或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代,。在非限制性的实施方式中，人4-1BB胞内结构域氨基酸序列具有SEQ ID NO:12的连续部分的氨基酸序列，其长度为至少20、或至少30、或至少40、或至少50，并且至多164个氨基酸。
在某些非限制性实施方式中，CAR的细胞内结构域可包含可激活或刺激细胞(例如，淋巴谱系的细胞，例如T细胞)的CD3ζ多肽。CD3ζ包含3个免疫受体酪氨酸激活基序（Immunoreceptor tyrosine-based activation motif，ITAM），并且在结合抗原后将激活信号传递到细胞(例如，淋巴谱系的细胞，例如T细胞)。
在一些实施方式中，所述CD3ζ胞内结构域选自(1)具有如SEQ ID No.14所示氨基酸序列的多肽；或 （2）（1）的经过氨基酸修饰的变体；其中所述经过氨基酸修饰的变体为与SEQ ID No.14所示氨基酸序列具有70~99％同源性，优选80~99%，更有选80~99%，最优选95~99%同源性的多肽，或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。在非限制性的实施方式中，人CD3ζ胞内结构域氨基酸序列具有SEQ ID NO:14的连续部分的氨基酸序列，其长度为至少20、或至少30、或至少40、或至少50，并且至多164个氨基酸。在一些非限制性实施方式中，所述人CD3ζ胞内结构域的氨基酸序列选自(1)具有如SEQ ID No.15所示氨基酸序列；或 （2）（1）的经过氨基酸修饰的变体；其中所述经过氨基酸修饰的变体为与SEQ ID No.15所示氨基酸序列具有70~99％同源性，优选80~99%，更有选80~99%，最优选95~99%同源性的多肽，或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。在非限制性的实施方式中，人CD3ζ胞内结构域氨基酸序列具有SEQ ID NO:13的连续部分的氨基酸序列，其长度为至少20、或至少30、或至少40、或至少50，并且至多164个氨基酸。
在一些实施方式中，所述人CD28ζ胞内结构域选自：
(1)具有如SEQ ID No.13或SEQ ID No.13所示氨基酸序列的多肽；或 2）（1）的经过氨基酸修饰的变体；其中所述经过氨基酸修饰的变体为与SEQ ID No.13或SEQ ID No.13所示氨基酸序列具有70~99％同源性，优选80~99%，更有选80~99%，最优选95~99%同源性的多肽，或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。在非限制性的实施方式中，人CD28ζ胞内结构域多肽具有SEQ ID NO:13的连续部分的氨基酸序列，其长度为至少20、或至少30、或至少40、或至少50，并且至多255个氨基酸。
在一示例性实施方式中，所述引导序列如SEQ ID No.3所示；。
在一示例性实施方式中，所述人NKG2D的氨基酸序列如SEQ ID No.4所示。在一示例性实施方式中，所述人NKG2D的氨基酸序列如SEQ ID No.5所示。在一示例性实施方式中，所述人NKG2D的氨基酸序列如SEQ ID No.6所示。在一示例性实施方式中，所述人NKG2D的氨基酸序列如SEQ ID No.7所示。在一示例性实施方式中，所述人NKG2D的氨基酸序列如SEQ ID No.8所示。在一示例性实施方式中，所述人NKG2D的氨基酸序列如SEQ ID No.9所示。
在一示例性实施方式实施例中，所述人CD8铰链区的氨基酸序列如SEQ ID No.10所示；
在一示例性实施方式实施例中，所述人CD8跨膜区的氨基酸序列如SEQ ID No.11所示；
在一示例性实施方式实施例中，所述人4-1BB胞内结构域的氨基酸序列如SEQ ID No.12所示；
在一示例性实施方式实施例中，所述CD28结构域的氨基酸序列如SEQ ID No.13所示；
在一示例性实施方式中，所述CD3ζ结构域的氨基酸序列如SEQ ID No.14所示。
在一示例性实施方式中，所述CD3ζ结构域的氨基酸序列如SEQ ID No.15所示。
在一非限制性实施方式中，靶向结合人NKG2DL的特异性嵌合抗原受体（CAR）是重组表达或由载体表达。
在某些非限制性的实施方式中，本申请的靶向结合人NKG2DL的CAR的细胞内结构域还包含至少一个共刺激信号传导区，其包含至少一个可提供最佳淋巴细胞活化的共刺激配体分子。其与CAR信号联合诱导CAR+T细胞的效应细胞功能。
其中，所述共刺激配体包括但不限于CD80、CD86、CD70、OX40L、4-1BBL、CD48、TNFRSF14和PD-L1。
在一些非限制性实施方式中，靶向结合人NKG2DL的CAR的细胞内结构域包含共刺激信号传导区，所述共刺激信号传导区，包含两种共刺激分子：CD28和4-1BB或CD28和OX40。
在一非限制性实施方式中，本申请的靶向结合人NKG2DL的的CAR细胞内结构域包含4-1BB多肽。4-1BB可以作为肿瘤坏死因子(TNF)配体并具有刺激活性。4-1BBL可以共价连接到的细胞外抗原结合结构域的5’末端。或者，4-1BBL可以共价连接到的细胞内结构域的3’末端。
本申请的非限制性的实施方式中中，4-1BB多肽选自（1）具有SEQ ID NO:12的氨基酸序列或（2）（1）的经过氨基酸修饰的功能性变体，过氨基酸修饰的变体为与SEQ ID No.12所示氨基酸序列具有70~99％同源性，优选80~99%，更有选80~99%，最优选95~99%同源性的多肽/或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。
在非限制性的实施方式中，4-1BB多肽具有SEQ ID NO:12的连续部分的氨基酸序列，其长度为至少20、或至少30、或至少40、或至少50，并且至多255个氨基酸。
在某些非限制性实施方式中，CAR还可包含将抗原结合结构域连接至跨膜结构域的间隔区(spacer)。间隔区可以是足够柔性的，以允许抗原结合结构域在不同方向上定向，以利于抗原识别。间隔区可以是来自IgG1的铰链区、或免疫球蛋白的CH2CH3区和CD3的部分。
在某些非限制性实施方式中，CAR的细胞内结构域可包含可激活或刺激细胞(例如，淋巴谱系的细胞，例如T细胞)的人CD3ζ多肽。CD3ζ包含3个ITAM，并且在结合抗原后将激活信号传递到细胞(例如，淋巴谱系的细胞，例如T细胞)。
在某些非限制性的实施方式中，CAR的细胞内结构域还包含至少一个共刺激信号传导区，其包含至少一个可提供最佳淋巴细胞活化的共刺激分子。本文使用的“共刺激分子”是指淋巴细胞对抗原的有效应答所需的除抗原受体或其配体之外的细胞表面分子。至少一个共刺激信号传导区可包含CD28多肽、4-1BB多肽、OX40多肽、ICOS多肽、、2B4多肽、BTLA多肽、合成肽(不基于与免疫应答相关的蛋白质)，或其组合。共刺激分子可以结合共刺激配体，共刺激配体是在细胞表面上表达的蛋白质，其在结合其受体时产生共刺激应答，即实现当抗原结合其CAR分子时所提供的刺激的细胞内应。共刺激配体包括但不限于CD80、CD86、CD70、OX40L、4-1BBL、CD48、TNFRSF14和PD-L1。作为一个实例，4-1BB配体(即4-1BBL)可结合4-1BB(也称为“CD137”)以提供细胞内信号，其与CAR信号联合诱导CAR+T细胞的效应细胞功能。
在一些实施方式中，CAR的细胞内结构域的共刺激信号传导区包含两种共刺激分子：CD28和4-1BB(参见Sadelain等人，Cancer Discovery，OF1-11，(2013))、或CD28和OX40。
4-1BB可以作为肿瘤坏死因子(TNF)配体并具有刺激活性。4-1BB可以作为肿瘤坏死因子(TNF)配体并具有刺激活性。4-1BB多肽具有SEQ ID NO:12的序列或其片段至少约85％、约90％、约5％、约96％、约97％、约98％、约99％或100％同源性的氨基酸序列，和/或可任选地包含至多一个或至多两个或至多三个保守氨基酸取代。在非限制性的实施方式中，4-1BB多肽具有SEQ ID NO:12的连续部分的氨基酸序列，其长度为至少20、或至少30、或至少40、或至少50，并且至多255个氨基酸。或者或另外，在非限制性的各种实施方式中，4-1BB多肽的氨基酸序列为SEQ ID NO:12的氨基酸。在一个实施方式中，本发明公开的主题的CAR细胞内结构域包含4-1BB多肽。
第三方面，本申请提供了一种编码第二方面所述的靶向结合人NKG2DL的特异性嵌合抗原受体的核酸分子，所述核酸分子包含从5‘到3‘依次串联连接的编码引导序列的核苷酸序列、编码靶向结合人NKG2DL的人NKG2D的核苷酸序列、编码跨膜结构域的核苷酸序列和编码胞内信号结构域的核苷酸序列。
在一些实施方式在中，所述核酸分子还包含编码铰链区的核苷酸序列。在一些实施方式中，所述胞内信号结构域包括免疫受体酪氨酸活化基序
和共刺激信号域；
在一些示例性实施方式中，所述核酸分子包含从5‘到3‘依次串联连接的分别编码引导序列、本申请第一方面所述的人NKG2D序列、人CD8铰链区序列、人CD8跨膜区序列、人CD28胞内结构域序列、人4-1BB胞内结构域序列和人CD3ζ胞内结构域的核苷酸序列。
在一些示例性实施方式中，所述核酸分子包含从5‘到3‘依次串联连接的分别编码引导序列、本申请第一方面所述的人NKG2D序列、人CD8跨膜区序列、人4-1BB胞内结构域序列和人CD3ζ胞内结构域序列的核苷酸序列。
在一些示例性实施方式中，所述核酸分子包含从5‘到3‘依次串联连接的分别编码引导序列、本申请第一方面所述的人NKG2D的核苷酸序列、人CD8铰链区的核苷酸、人CD8跨膜区的核苷酸、人4-1BB胞内结构域的核苷酸序列和人CD3ζ胞内结构域的核苷酸序列。
编码靶向结合人NKG2DL的细胞外识别结构域的多核苷酸可以通过密码子优化进行修饰。密码子优化可以改变天然存在的和重组的基因序列，以在任何给定的表达系统中实现最高可能水平的生产力。涉及蛋白质表达的不同阶段的因子包括密码子适应性、mRNA结构以及转录和翻译中的各种顺式元件。
在一些实施方式中，所述编码靶向结合人NKG2DL的细胞外识别结构域中人NKG2D的核苷酸序列如SEQ ID No.17所示或与SEQ ID No.17所示的核苷酸序列99%，95%，90%，85%或80%同源。
在一示例性实施例中，所述编码靶向结合人NKG2DL的细胞外识别结构域中人NKG2D的核苷酸序列如SEQ ID No.18所示或与SEQ ID No.18所示的核苷酸序列99%，95%，90%，85%或80%同源。
在一实施例中，所述编码靶向结合人NKG2DL的细胞外识别结构域中人NKG2D的核苷酸序列如SEQ ID No.19所示或与SEQ ID No.19所示的核苷酸序列99%，95%，90%，85%或80%同源。
在一实施例中，所述编码靶向结合人NKG2DL的细胞外识别结构域中人NKG2D的核苷酸序列如SEQ ID No.20所示或与SEQ ID No.20所示的核苷酸序列99%，95%，90%，85%或80%同源。
在一实施例中，所述编码靶向结合人NKG2DL的细胞外识别结构域中人NKG2D的核苷酸序列如SEQ ID No.21所示或与SEQ ID No.21所示的核苷酸序列99%，95%，90%，85%或80%同源。
在一实施例中，所述编码靶向结合人NKG2DL的细胞外识别结构域中人NKG2D的核苷酸序列如SEQ ID No22所示或与SEQ ID No.22所示的核苷酸序列99%，95%，90%，85%或80%同源。
在一些实施方式中，所述编码人CD8铰链区的核苷酸序列如SEQ ID No.23所示或与SEQ ID No.23所示核苷酸序列99%，95%，90%，85%或80%同源。
在一些实施方式中，所述编码人CD8跨膜区的核苷酸序列如SEQ ID No.24所示或与SEQ ID No.24所示核苷酸序列99%，95%，90%，85%或80%同源，
在一些实施方式中，所述编码人4-1BB胞内结构域的核苷酸序列如SEQ ID No.25所示或与SEQ ID No.25所示核苷酸序列99%，95%，90%，85%或80%同源。
在一些实施方式中，所述编码CD28胞内结构域的核苷酸序列如SEQ ID No.26所示或与SEQ ID No.26所示核苷酸序列99%，95%，90%，85%或80%同源。
在一些实施方式中，所述编码CD3ζ胞内结构域的核苷酸序列如SEQ ID No.27所示或与SEQ ID No.27所示核苷酸序列99%，95%，90%，85%或80%同源。
在一些实施方式中，所述编码CD3ζ胞内结构域的核苷酸序列如SEQ ID No.28所示或与SEQID No.28所示核苷酸序列99%，95%，90%，85%或80%同源。
第四方面，本申请提供了一种包含本申请第二方面的嵌合抗原受体或本申请第三方面的核酸的重组载体或表达质粒。
免疫应答细胞(例如，T细胞、CTL细胞、NK细胞)的基因修饰可以通过用重组DNA或RNA构建体转导基本上同源的细胞组合物来实现。在一个实施方案中，载体是逆转录病毒载体(例如，γ逆转录病毒或慢病毒)，其可将DNA或RNA构建体导入宿主细胞基因组。例如，编码NKG2D，靶向人NKG2DL特异性CAR的多核苷酸可以克隆到逆转录病毒载体中，并且可以从其内源启动子、逆转录病毒长末端重复序列或从替代的内部启动子驱动表达。
也可使用非病毒载体或RNA。可以使用随机染色体整合或靶向整合(例如使用核酸酶、转录激活子样效应物核酸酶(TALEN)、锌指核酸酶(ZFN)和/或规律成簇间隔短回文重复(CRISPR)或转基因表达(例如使用天然或化学修饰的RNA)。
在一些实施方式中，所述载体选自γ-逆转录病毒载体、慢病毒载体、腺病毒载体、腺联病毒载体。在一示例性实施方式中，所述载体是γ-逆转录病毒载体。
第五方面，本申请提供了一种启动子，用于构建本申请第四方面所述的重组载体和表达本申请第二方面所述的靶向结合人NKG2DL的特异性嵌合抗原受体，所述启动子包括但不限于核苷酸序列如SEQ No.29所示的EF1 alpha启动子和如SEQ No.30 所示的EFS启动子。
在一示例性优选实施例中，所述用于用于构建本申请第四方面所述的重组载体和表达本申请第二方面所述的靶向结合人NKG2DL的特异性嵌合抗原受体的启动子是如SEQ No.30 所示的EFS启动子。
在一示例性优选实施例中，所述用于用于构建本申请第四方面所述的重组载体和表达本申请第二方面所述的靶向结合人NKG2DL的特异性嵌合抗原受体的启动子是如如SEQ No.29所示的EF1 alpha启动子。
第六方面，本申请提供了一种重组病毒，其是能够表达本发明第二方面的靶向NKG2DL的特异性嵌合抗原受体、且能够侵染免疫应答细胞的病毒。
在一些实施方式中，所述免疫应答细胞为细胞毒性T淋巴细胞、NK细胞、NKT细胞或辅助性T细胞等。
在示例性实施方式中，所述免疫应答细胞为细胞毒性T淋巴细胞。
在一些实施方式中，所述病毒为慢病毒、腺病毒、腺联病毒或逆转录病毒等。
在一示例性实施方式中，所述病毒为慢病毒。
在一示例性实施方式中，所述病毒为逆转录病毒。
第七方面，本申请提供了一种分离的修饰的免疫应答细胞，其包含本申请第二方面所述的嵌合抗原受体，其由本申请第三方面所述的重组载体或表达质粒转化所得。
对于细胞的初始基因修饰以提供靶向人NKG2DL特异性免疫应答细胞，通常使用逆转录病毒载体进行转导，然而可以使用任何其它合适的病毒载体或非病毒递送系统。对于细胞的后续基因修饰以提供包含含有至少两种共刺激配体的抗原递呈复合物的细胞，逆转录病毒基因转移(转导)同样证明是有效的。逆转录病毒载体和合适的组装线的联合也是合适的，其中衣壳蛋白对于感染人细胞是有功能的。
在一些实施方式中，所述免疫应答细胞还包含至少一种外源的共刺激配体。
可能的转导方法还包括将细胞与生产细胞的直接共培养。转导病毒载体可用于在免疫应答细胞中表达共刺激配体(例如4-1BBL和IL-12)。优选地，选择的载体表现出高的感染效率和稳定的整合和表达。
在一些实施方式中，优选地，所述至少一种共刺激配体选自4-1BBL、CD80、CD86、CD70、OX40L、CD48、TNFRSF14及其组合，或更优选地，所述共刺激配体是4-1BBL。
在一些实施方式中，所述免疫应答细胞选自T细胞、自然杀伤(NK)细胞、细胞毒性T淋巴细胞(CTL)、调节性T细胞、人胚胎干细胞和可分化成淋巴样细胞的多能干细胞，优选T细胞和自然杀伤(NK)细胞，更优选为T细胞。
可以利用用于CAR表达的载体转导从患者分离的多个T细胞亚群。
第七方面公开的修饰免疫应答细胞可以表达特异性结合人NKG2DL的细胞外标识别结构域，用于治疗或预防瘤形成、免疫性疾病，或抗衰老。
在一示例性实施例中，其中所述修饰的免疫应答细胞为CAR-T细胞。
可以利用靶向人NKG2DL的人NKG2D嵌合抗原受体制备修饰的遗传修饰的中央记忆型T细胞，然后冷藏保存。
第八方面 本申请提供了本申请的第六方面的分离的嵌合抗原受体修饰的免疫应答细胞的制备方法，包括以下步骤：
首先，将第三方面所述的核酸分子，通过分子克隆的方式连接到表达载体中，获得靶向NKG2DL的特异性嵌合抗原受体的表达载体；
然后，将获得的所述靶向NKG2DL的特异性的CAR表达载体转染293T细胞，获得病毒液；
最后用所述病毒液感染免疫应答细胞，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修饰的免疫应答细胞。
在一些非限制性实施方式中，本发明修饰的的免疫应答细胞可以是淋巴谱系的细胞。所述淋巴谱系的细胞选自B、T和自然杀伤(NK)细胞，提供抗体的产生、细胞免疫系统的调节、血液中外来物质的检测、对宿主外源细胞的检测等功能。淋巴谱系的细胞的非限制性实例包括T细胞、自然杀伤(NK)细胞、细胞毒性T淋巴细胞(CTL)、调节性T细胞、胚胎干细胞和多能干细胞(例如，可分化成淋巴样细胞的多能干细胞)。
在一些实施方式中，所述免疫应答细胞选自T细胞、自然杀伤(NK)细胞、细胞毒性T淋巴细胞(CTL)、调节性T细胞、人胚胎干细胞和可分化成淋巴样细胞的多能干细胞，优选T细胞或自然杀伤(NK)细胞。在一些实例性实施方式中，T细胞是在胸腺中成熟的淋巴细胞，并且主要负责细胞介导的免疫。T细胞参与获得性免疫系统。
在一些非限制性实施方式中，T细胞包括但不限于T辅助细胞、细胞毒性T细胞、记忆T细胞(包括中心记忆T细胞、干细胞样记忆T细胞(或干样记忆T细胞)和两种类型的效应记忆T细胞(例如，TEM细胞和TEMRA细胞)、调节性T细胞(也称为抑制性T细胞)、自然杀伤T细胞、粘膜相关恒定T细胞、和γδT细胞。在一些实施方式中，表达CAR的T细胞表达Foxp3以实现和维持T调节表型。
本申请的修饰的免疫应答细胞可以进一步包含至少一种外源性共刺激性配体，使得免疫应答细胞外源性地共表达或被诱导为外源性地共表达基于NKG2D，靶向人NKG2DL的特异性嵌合抗体和至少一种外源性共刺激性配体。靶向人NKG2DL的特异性CAR和至少一种共刺激配体之间的相互作用提供了对于免疫应答细胞(例如，T细胞)的完全激活而言重要的非抗原特异性信号。在一些实施方式中，至少一种共刺激配体选自4-1BBL、CD80、CD86、CD70、OX40L、，及其组合。在一个实施方式中，共刺激性配体是4-1BBL。
在一优选实施例中，所述分离的修饰的免疫应答细胞为T细胞。
在一优选实施例中，所述分离的修饰的免疫应答细胞为自然杀伤(NK)细胞。
在一些非限制性实施方式中，所述分离的修饰的免疫应答细胞(例如T细胞)可以是自体的、非自体的(例如同种异体的)、或在体外衍生自工程化的祖细胞或干细胞。
第九方面，本申请提供了一种药物组合物，其包含有效量的如本发明第六方面所述分离的修饰的免疫应答细胞和药学上可接受的赋形剂。
本发申请公开的药物组合物，其包含表达靶向人NKG2DL的的特异性嵌合抗原受体的分离的修饰的免疫应答细胞和药学上可接受的载体。
所述药物组合物的施用可以是自体的或非自体的。例如，表达靶向人NKG2DL的特异性CAR的免疫应答细胞和包含它的组合物可以从一个受试者获得，并施用于相同受试者或不同的相容受试者。可以通过局部注射，包括导管给药、全身注射、局部注射、静脉内注射或胃肠外给药来施用本发明公开主题的外周血来源的T细胞或其子代(例如，体内、离体或体外衍生的)。当施用本发明公开主题的药物组合物(例如包含表达NKG2D特异性CAR的免疫应答细胞的药物组合物)时，通常将其配制成单位剂量可注射形式(溶液、悬浊液、乳浊液)。
本申请的组合物可以是制剂。本申请公开的表达靶向人NKG2DL的特异性嵌合抗原受体（CAR）的免疫应答细胞和包含其的组合物可以方便地作为无菌液体制剂提供，例如等渗水溶液、悬浊液、乳浊液、分散液或粘性组合物，其可以缓冲至选定的pH。液体制剂通常比凝胶、其它粘性组合物和固体组合物更容易制备。此外，液体组合物更方便施用，特别是通过注射。另一方面，粘性组合物可以在适当的粘度范围内配制，以提供与特定组织的更长的接触时间。液体或粘性组合物可以包含载体，其可以是包含例如水、生理盐水、磷酸盐缓冲盐水、多元醇(例如甘油、丙二醇、液体聚乙二醇等)和合适的其混合物的溶剂或分散介质。
可以加入增强组合物的稳定性和无菌性的各种添加剂，包括抗微生物防腐剂、抗氧化剂、螯合剂和缓冲剂。
根据本申请，使用的任何载体、稀释剂或添加剂必须与表达本发明公开主题的基于人NKG2D，靶向人NKG2DL的特异性嵌合抗原受体（CAR）的免疫应答细胞相容。
如果需要，组合物的粘度可以使用药学上可接受的增稠剂保持在选定的水平。合适的载体和其它添加剂的选择将取决于确切的给药途径和特定剂型的性质，例如液体剂型(例如，是否将组合物配制成溶液、悬浊液、凝胶或另一液体形式，比如时间释放形式或液体填充形式)。
第十方面，本申请提供了一种用于治疗或预防瘤形成、病原体感染、自身免疫性疾病、同种异体移植、或移植排斥或抗衰老的试剂盒，其包含本发明第六方面所述的免疫应答细胞或本发明第三方面所述的核酸。
如果需要，免疫应答细胞与将细胞施用于具有或有风险发生瘤形成、病原体感染、免疫病症或同种异体移植或衰老的受试者的说明书一起提供。说明书通常包括关于组合物用于治疗或预防瘤形成、病原体感染、免疫疾病或同种异体移植的用途的信息。在其它实施方案中，说明书包括以下中的至少一个：治疗剂的说明；用于治疗或预防瘤形成、病原体感染、免疫疾病或同种异体移植或其症状的剂量方案和施用方法；注意事项；禁忌；适应症；非适应症；过量信息；不良反应；动物药理学；临床研究；和/或参考文献。说明书可以直接印在容器上(当存在时)、或作为标签贴到容器上、或作为单独的页、小册子、卡片或折叠式印刷品，在容器中或与容器一起提供。
第十一方面，本申请提供了 本发明第一方面所述的靶向结合人 NKG2DL 的人 NKG2D 蛋白及其变体、第二方面所述的 靶向结合人NKG2DL 的特异性 嵌合抗原受体 第四方面所述重组载体或表达质粒，第五方面所述重组病毒、第七面所述制备方法获得的分离的CAR-修饰的免疫应答细胞，第八方面的组合物或第九方面的试剂盒在治 疗或预防 疾病的产品中的应用，所述治疗或预防 包括向患有与 NKG2DL 和 NKG2D 蛋白的特异性相互作用引起的疾病的患者施用治疗有销量的本申请第二方面所述的包含靶向 NKG2DL 的 NKG2D 的CAR-T细胞。
在一些实施方式中， 所述疾病包括 瘤形成、病原体感染、自身免疫性疾病、同种异体移植、移植排斥 、衰老。
在一些实施方式中，所述 瘤形成、病原体感染、自身免疫性疾病、炎性疾病、同种异体移植、或移植排斥 或衰老与 NKG2DL 和 NKG2D 蛋白的特异性相互作用有关。
在一些实施方式中， 所述治 疗或预防瘤形成包括减少受试者中肿瘤负荷 、 增加或延长患有瘤形成的受试者的存活或响应受试者的癌细胞或病原体而增加免疫激活细胞因子产生 。
在一些实施方式中， 所述肿瘤或 瘤形成选自 所述肿瘤或 瘤形成选自 肝癌 、胶质瘤 、 卵巢癌、胃癌、 随母 细胞瘤、胰腺癌、肺癌、前列腺癌 细胞 、乳腺癌 或神经母细胞瘤、膀胱癌、结肠癌、肾细胞癌、白血病、淋巴瘤、多发性骨髓瘤、黑色素瘤、 及其组合 。（和实施例的供试靶细胞对应）
在一示例性实施例中， 所述肿瘤或瘤形成为肝癌。
在一示例性实施例中， 所述肿瘤或瘤形成为胶质瘤。
在一示例性实施例中， 所述肿瘤或瘤形成为卵巢癌。
在一示例性实施例中， 所述肿瘤或瘤形成为胃癌。
在一示例性实施例中， 所述肿瘤或瘤形成为随母细胞瘤。
在一示例性实施例中， 所述肿瘤或瘤形成为胰腺癌。
在一示例性实施例中， 所述肿瘤或瘤形成为肺癌。
在一示例性实施例中， 所述肿瘤或瘤形成为前列腺癌。
在一示例性实施例中， 所述肿瘤或瘤形成为乳腺癌。
本发明基于人 NKG2D 分子构建靶向人 NKG2DL 的特异性嵌合抗原受体（CAR）以及该CAR修饰的T细胞（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 阳性的肿瘤的药物制剂。
本发明的发明人在研究中意外发现，采用本发明构建的包含特异性识别 人 NKG2DL 的分离的基于人 NKG2D 的 嵌合抗原受体修饰的 T细胞， 制备方法步骤简单， 在效靶比为10:1时， 具有高达 40 %~70% 的肿瘤细胞杀伤率 ，可 明显延长免疫细胞在患者体内的存活时间 ， 增强免疫细胞靶向识别肿瘤细胞特别是 表达 NKG2DL 的肿瘤 的能力，加强对肺癌细胞的特异杀伤活性 。 本发明的 基于 人 NKG2D 的 靶向 NKG2DL 的嵌合抗原受体 的 免疫应答 细胞为治疗 肿瘤 提供了一种新的 方案 选择，具有良好的产业应用前景 。
本申请提供了 使用此类细胞治疗例如需要增强的免疫应答的疾病的方法。
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）和全长核苷酸序列（如SEQ ID No.2所示）。
其次，构建靶向NKG2DL的特异性嵌合抗原受体。
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，由引导肽的氨基酸序列（如SEQ ID No.3所示）、人NKG2D氨基酸序列（如SEQ ID No.4、所示）、人CD8铰链区的氨基酸序列（如SEQ ID No.10所示）、人CD8跨膜区的氨基酸序列（如SEQ ID No.11所示）、人4-1BB胞内结构域的氨基酸序列（如SEQ ID No.12所示）、和人CD3ζ结构域的氨基酸序列（如SEQ ID No.14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，由编码引导序列的核苷酸序列（如SEQ ID No.16所示）、编码人NKG2D序列的核苷酸序列（如SEQ ID No.17、所示）、编码人CD8铰链区的核苷酸序列（如SEQ ID No.23所示）、编码人CD8跨膜区的核苷酸序列（如SEQ ID No.24所示）、编码人4-1BB胞内结构域的核苷酸序列（如SEQ ID No.25所示）、和编码CD3ζ结构域的核苷酸序列（如SEQ ID No.27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慢病毒载体lentiGuide-Puro（南京一道生物，图1），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.29所示）进行表达。具体操作步骤如下：
全基因合成含有启动子、引导序列、人NKG2D序列、CD8铰链区、CD8跨膜区、4-1BB胞内结构域、和CD3ζ结构域的靶向NKG2DL的特异性CAR分子的核苷酸序列（南京一道生物），利用 引物5’-cactttggcgccggctcgagggggcccgggtgcaaagatggataaagttttaaacagagagga-3’（序列表SEQ ID No.31）或5’-cactttggcgccggctcgagggggcccgggtaggtcttgaaaggagtgggaattggctcc-3’（序列表SEQ ID No.32）和
引物5'-tccagaggttgattgtcgacttaacgcgtttagcgagggggcagggcctgcatgtgaag-3' （序列表SEQ ID No.33）PCR扩增人工合成CAR分子序列，经Axygen胶回收试剂盒（杭州泽衡）回收，与经过限制性内切酶SmaI和MluI消化的载体lentiGuide-Puro(南京一道生物)进行同源重组连接。具体重组连接反应的体系和条件如下：
重组连接体系：
胶回收的PCR产物5μl，胶回收的SmaI和MluI酶切lentiGuide-Puro质粒(南京一道生物)3μl；4X 1402 QuickCloning Kit(南京基诺美) 5μl；去离子水7μl；连接反应体系体积20μl；
重组连接条件：将上述反应体系置于50℃水浴中，反应15min后置冰上1min.。
取10ul重组连接产物经感受态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（南京一道生物），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，EFS启动子）。
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Stbl3菌株在LB培养基中大量培养，采用Qiagen Plasmid Midi Kit（德国Qiagen公司）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。
 实施方式
施例2 制备靶向NKG2DL的特异性嵌合抗原受体的表达质粒
除了步骤1中，靶向NKG2DL特异性CAR分子的氨基酸序列中人NKG2D序列（如SEQ ID No.5所示），人CD3ζ结构域的氨基酸序列如SEQ ID No.15所示， 靶向NKG2DL特异性CAR分子的核苷酸序列中编码人NKG2D序列的核苷酸序列（如SEQ ID No.18所示），
和步骤2）表达靶向NKG2DL的特异性CAR分子的质粒的构建和鉴定部分
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慢病毒载体lentiGuide-Puro（南京一道生物，图1），构建单一编码框的全长CAR序列表达框阶段， 利用EFS启动子（序列表SEQ ID No.,30），其余与实施例1相同。
实施例3制备靶向NKG2DL的特异性嵌合抗原受体的表达质粒
除了步骤1中，靶向NKG2DL特异性CAR分子的氨基酸序列中人NKG2D序列（如SEQ ID No.6所示），靶向NKG2DL特异性CAR分子的核苷酸序列中编码人NKG2D序列的核苷酸序列（如SEQ ID No.19所示，其余与实施例1相同。
实施例4制备靶向NKG2DL的特异性嵌合抗原受体的表达质粒
除了步骤1中，靶向NKG2DL特异性CAR分子的氨基酸序列中人NKG2D序列（如SEQ ID No.7所示），人CD3ζ结构域的氨基酸序列如SEQ ID No.15所示，靶向NKG2DL特异性CAR分子的核苷酸序列中编码人NKG2D序列的核苷酸序列（如SEQ ID No.20所示，其余与实施例1相同。
实施例5制备靶向NKG2DL的特异性嵌合抗原受体的表达质粒
除了步骤1中，靶向NKG2DL特异性CAR分子的氨基酸序列中人NKG2D序列（如SEQ ID No.8所示），靶向NKG2DL特异性CAR分子的核苷酸序列中编码人NKG2D序列的核苷酸序列（如SEQ ID No.21所示，其余与实施例1相同。
实施例6制备靶向NKG2DL的特异性嵌合抗原受体的表达质粒
除了步骤1中，靶向NKG2DL特异性CAR分子的氨基酸序列中人NKG2D序列（如SEQ ID No.9所示），人CD3ζ结构域的氨基酸序列如SEQ ID No.15所示，靶向NKG2DL特异性CAR分子的核苷酸序列中编码人NKG2D序列的核苷酸序列（如SEQ ID No.22所示，，其余与实施例1相同。
实施例7 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OpTmizerT-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。T细胞纯度通过流式细胞术，利用抗PE anti-human CD3抗体（购买自美国BioLegend公司，货号：300408）进行检测，结果显示经过磁珠富集后的T细胞纯度超过97%（图3）。
实施例8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-3216）；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10~15分钟后经0.45μm滤膜过滤，最后于4℃，25000rpm 超离心2~3小时，浓缩病毒，将收集的病毒浓缩液转入-80℃保存。
实施例9
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30~60分钟, 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-025CAR-T细胞，即新型CAR-T细胞，CAR分子结构如图2，可以进行下一步的功能实验。
实施例10鉴定
之后利用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对制备好的CAR-T细胞进行细胞活性测试。流式细胞结果显示KD-025 CAR-T细胞活性均大于95% （图4）。
然后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。
空白对照组：没有感染病毒液的T细胞。
KD-019对照组：靶向CD19的特异性CAR-T细胞。
KD-025组：靶向NKG2DL的特异性CAR-T细胞。
细胞的制备过程中所收集的待检测细胞。
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APC标记的抗人NKG2D抗体（APC Mouse Anti-Human NKG2D, 558071, BD）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5所示（另注：图5中，横坐标PE-A表示APC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），对照组中几乎检测不到CAR分子的表达，KD-025组中CAR分子表达率达到46.7%。
从图5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。
应用例以其效果数据
供试肿瘤细胞系（也称靶细胞系）：肝癌细胞SMMC7721（购自南京科佰生物科技有限公司）、胶质瘤细胞U251（购自中国科学院上海生命科学研究院细胞资源中心 ）、卵巢癌细胞SKOV3 （购自中国科学院昆明动物研究所）、胃癌细胞NUGC4（购自南京科佰生物科技有限公司）、随母细胞瘤HTB186（购自广州吉妮欧）、胰腺癌细胞BXPC3（购自广州吉妮欧）、肺癌细胞A549（购自中国科学院昆明动物研究所）、前列腺癌细胞PC3（购自中国科学院昆明动物研究所）、乳腺癌细胞MCF7（购自中国科学院昆明动物研究所）。
首先检测NKG2DL主要亚型 MicA，MicB，ULBP1， ULBP2和ULBP3在各种供试肿瘤细胞系中的表达。胰酶消化收集供试肿瘤细胞，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，计数，调整细胞浓度至1x106/ml；分别加入MicA抗体（R&D Systems , FAB1300A-025），MicB抗体（R&D Systems ,FAB1599A-025）和同型对照（R&D Systems，IC0041A），以及ULBP1抗体(R&D Systems，FAB1380A), ULBP2抗体（R&D Systems , FAB1298A）， ULBP3抗体（R&D Systems，FAB1517A）和同型对照（R&D Systems，IC003A），4℃孵育60分钟；PBS清洗两次后重悬于FACS液，流式细胞仪（BD FacsCanto II）分析细胞荧光，并采用FlowJo软件分析结果。流式结果如图6所示（另注：图6中，横坐标表示APC荧光强度；纵坐标代表细胞数目），结果显示供试肿瘤细胞组都呈现出NKG2DL抗原的高表达, 对照组基本无表达。
接下来利用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；空白对照组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）；KD-019对照组添加的是靶向CD19的无关CAR-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，空白对照组和KD-019对照组，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7所示为以肝癌细胞SMMC7721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KD-025对肝癌细胞SMMC7721具有明显的杀伤作用（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超过60%。
图8所示为以胶质瘤细胞U251为靶细胞的肿瘤细胞杀伤率测试结果。从图8可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞KD-025对胶质瘤细胞U251具有明显的杀伤作用（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超过70%。
图9所示为以卵巢癌细胞SKOV3为靶细胞的肿瘤细胞杀伤率测试结果。从图9可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞KD-025对卵巢癌细胞SKOV3具有明显的杀伤作用（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超过50%。
图10所示为以胃癌细胞NUGC4为靶细胞的肿瘤细胞杀伤率测试结果。从图10可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞KD-025对胃癌细胞NUGC4具有明显的杀伤作用（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超过40%。
图11所示为以随母细胞瘤细胞HTB186为靶细胞的肿瘤细胞杀伤率测试结果。从图11可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞KD-025对随母细胞瘤细胞HTB186具有明显的杀伤作用（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超过70%。
图12所示为以胰腺癌细胞BXPC3为靶细胞的肿瘤细胞杀伤率测试结果。从图12可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞KD-025对胰腺癌细胞BXPC3具有明显的杀伤作用（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超过70%。
图13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13可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞KD-025对肺癌细胞A549具有明显的杀伤作用（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超过40%。
图14所示为以前列腺癌细胞PC3为靶细胞的肿瘤细胞杀伤率测试结果。从图14可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞KD-025对前列腺癌细胞PC3具有明显的杀伤作用（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超过70%。
图15所示为以乳腺癌细胞MCF7为靶细胞的肿瘤细胞杀伤率测试结果。从图15可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞KD-025对乳腺癌细胞MCF7具有明显的杀伤作用（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超过50%。
图16所示为CAR-T细胞对肝癌SMMC7721移植瘤裸鼠的肿瘤抑制测试结果。从图14可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞KD-025对肝癌SMMC7721移植瘤具有明显的抑制作用，在接种14天后，肿瘤基本完全消失（图14A），肿瘤荧光强度明显低于三个对照组（图14B）。
图17所示为CAR-T细胞对胶质瘤U251移植瘤裸鼠的肿瘤抑制测试结果。从图17可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞KD-025对胶质瘤U251移植瘤具有明显的抑制作用，在接种14天后，肿瘤基本完全消失（图17A），肿瘤荧光强度明显低于三个对照组（图17B）。
图18所示为CAR-T细胞对卵巢癌SKOV3移植瘤裸鼠的肿瘤抑制测试结果。从图18可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞KD-025对卵巢癌SKOV3移植瘤具有明显的抑制作用，在接种14天后，肿瘤生长明显被抑制（图18A），肿瘤荧光强度明显低于三个对照组（图18B）。
图19所示为CAR-T细胞对胃癌NUGC4移植瘤裸鼠的肿瘤抑制测试结果。从图19可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞KD-025对胃癌NUGC4移植瘤具有明显的抑制作用，在接种14天后，肿瘤生长被抑制（图19A），肿瘤荧光强度低于三个对照组（图19B）。
图20所示为不同剂量的KD-025 CAR-T细胞对小鼠主要脏器和生存周期的影响。从图17可以看出，实施例4的靶向NKG2DL的特异性CAR-T细胞KD-025不会引起小鼠心、肝、肺、肾等主要脏器发生炎症、水肿和坏死（图20A），并且对小鼠的生存周期没有任何负面影响（图20B）。
图21所示为KD-025 CAR-T在小鼠体内的存留时间。从图21可以看出，对照组和KD-025CAR-T细胞在注射小鼠10天后体内存留量降到大约50%，17天后下降到大约25%，31天后基本检测不到残留的CAR-T细胞（图21）。
图22所示为KD-025 CAR分子病毒转染T细胞后对原癌基因表达影响分析。从图22可以看出，对照组和KD-025CAR病毒转染的T细胞在对癌基因cMyc，kras BMl1和TP53表达进行实时定量PCR分析检测发现， KD-025CAR病毒转染T细胞并没有改变细胞内癌基因的表达水平（图22）。
从上述图7到图19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，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肝癌、卵巢癌、胃癌、肺癌、前列腺癌、乳腺癌、神经胶质瘤或神经母细胞瘤、胰腺癌、膀胱癌、结肠癌、肾细胞癌、白血病、淋巴瘤、多发性骨髓瘤、黑色素瘤、及其组合。
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。

Claims (32)

  1. 一种靶向结合人NKG2DL的人NKG2D蛋白或其功能性变体， 包含下组的：
    （1）SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8 或SEQ ID No.9所示的人NKG2D蛋白的氨基酸序列，或 （2） （1）经过氨基酸修饰产生的变体。
  2. 如权利要求1所述的靶向结合人NKG2DL的人NKG2D蛋白或其变体，其中所述与SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8 或SEQ ID No.9所示氨基酸序列具有70~99％同源性。
  3. 如权利要求1或2所述的靶向结合人NKG2DL的人NKG2D蛋白或其变体，其中所述与SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8 或SEQ ID No.9所示氨基酸序列具有70~99％同源性的多肽是将SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8 或SEQ ID No.9所示氨基酸序列经过1~10个氨基酸残基或优选地经过1~5个氨基酸残基或更优选经过1~3个氨基酸残基的取代、缺失或添加而形成的衍生多肽。
  4. 一种靶向结合人NKG2DL的特异性嵌合抗原受体，其特征是，所述嵌合抗原受体包含从氨基端到羧基端顺次连接的引导序列、靶向结合人NKG2DL的胞外识别结构域、跨膜结构域和胞内信号结构域，由所述嵌合抗原受体修饰的免疫应答细胞在效靶比为10:1时对肿瘤细胞杀伤效率达40%~70%，所述靶向人NKG2DL的胞外识别结构域包含权利要求1的任一项所述的靶向结合人NKG2DL的人NKG2D蛋白或其功能性变体，所述靶向结合人NKG2DL的人NKG2D蛋白优选人NKG2DL的受体。
  5. 如权利要求4所述的靶向结合人NKG2DL的特异性嵌合抗原受体，其中, 所述嵌合抗原受体的跨膜结构域和胞内信号结构域之间还包括铰链区。
  6. 如权利要求5所述的靶向结合人NKG2DL的特异性嵌合抗原受体，其中, 所述的跨膜结构域包括跨膜区，所述胞内信号结构域包括免疫受体酪氨酸活化基序和共刺激信号域。
  7. 如权利要求4，5，或6所述的靶向结合人NKG2DL的特异性嵌合抗原受体，其中，所述跨膜区包含CD8跨膜结构区、CD28跨膜结构区、CD3ζ跨膜结构区、CD4跨膜结构区、4-1BB跨膜结构区、OX40跨膜结构区、ICOS跨膜结构区、CTLA-4跨膜结构区、PD-1跨膜结构区、LAG-3跨膜结构区、2B4跨膜结构区、BTLA跨膜结构区、合成肽(不基于与免疫应答相关的蛋白质)中的任一种， 所述免疫受体酪氨酸活化基序包含CD3ζ链的胞内信号结构域或FcεRIγ胞内信号结构，所述共刺激信号域包含CD28胞内信号结构域、CD137/4-1BB胞内信号结构域、CD134/OX40胞内信号结构域、ICOS胞内信号结构域中的至少一种； 所述铰链区选自CD8α铰链区、IgG铰链区，或包含整个或部分的免疫球蛋白的铰链区或铰链区经过一个或多个氨基酸修饰的变体。
  8. 如权利要求7所述的靶向结合人NKG2DL的特异性嵌合抗原受体，
    其中所述引导序列选自下组：
    (1)具有SEQ ID No.3 所示氨基酸序列的多肽，或 （2） （1）的经过氨基酸修饰的功能性变体，其中所述氨基酸修饰的功能性变体与SEQ ID No.3所示氨基酸序列具有70~99％同源性，优选80~99%，更有选80~99%，最优选95~99%同源性的多肽；和/或
    所述铰链区为人CD8多肽或其功能性变体， 选自： (1) SEQ ID No.10所示氨基酸序列的多肽；或 （2）（1）的经过氨基酸修饰的功能性变体，其中所述氨基酸修饰的功能性变体与SEQ ID No.10所示氨基酸序列具有70~99％同源性，优选80~99%，更有选80~99%，最优选95~99%同源性的多肽；和/或所述跨膜区为人CD8多肽或其功能性变体， 选自：(1) SEQ ID No.11所示氨基酸序列的多肽；或 （2）（1）的经过氨基酸修饰的功能性变体，其中所述氨基酸修饰的功能性变体与SEQ ID No.11所示氨基酸序列具有70~99％同源性，优选80~99%，更有选80~99%，最优选95~99%同源性的多肽；和/或
    所述人4-1BB胞内结构域选自：
    （1）具有SEQ ID NO.12所示氨基酸序列的多肽；或 （2） （1）的经过氨基酸修饰的功能性变体，其中所述经过氨基酸修饰的变体为与SEQ ID NO.12所示氨基酸序列具有70~99％同源性的多肽，优选80~99%，更有选80~99%，最优选95~99%同源性的多肽；和/或
    所述人CD28ζ胞内结构域选自：
    (1)具有如SEQ ID No.13所示氨基酸序列的多肽；或 （2）（1）的经过氨基酸修饰的功能性变体；其中所述经过氨基酸修饰的变体为与SEQ ID No.13所示氨基酸序列具有70~99％同源性，优选80~99%，更有选80~99%，最优选95~99%同源性的，和/或
    所述人CD3ζ胞内结构域选自：
    (1)具有如SEQ ID No.14或SEQ ID No.15所示氨基酸序列的多肽；或 （2）（1）的经过氨基酸修饰的功能性变体；其中所述经过氨基酸修饰的变体为与SEQ ID No.14或SEQ ID No.15所示氨基酸序列具有70~99％同源性，优选80~99%，更有选80~99%，最优选95~99%同源性。
  9. 权利要求4, 5，6,7或8所述的靶向结合人NKG2DL的特异性嵌合抗原受体(CAR), 其是重组表达或由载体表达。
  10. 如权利要求4,5，6,7, 或8所述的靶向结合人NKG2DL的特异性嵌合抗原受体，其中，所述氨基酸修饰包括但不限于氨基酸的取代、缺失和添加，所述同源性的多肽包括但不限于通过氨基酸的取代、缺失和添加产生的衍生多肽。
  11. 如权利要求4所述的靶向结合人NKG2DL的特异性嵌合抗原受体，其中所述嵌合抗原受体为氨基酸序列从氨基端到羧基端由引导序列、权利要求1~3的任一项所述的人NKG2D序列、人CD8跨膜区序列、人CD8铰链区序列、人CD28胞内结构域序列、人4-1BB胞内结构域序列和CD3ζ胞内结构域序列依次串联而成；或为氨基酸序列从氨基端到羧基端由引导序列、权利要求1~3的任一项所述的人NKG2D序列、CD8跨膜区、CD8铰链区、CD28胞内结构域和CD3ζ胞内结构域序列依次串联而成，或为氨基酸序列从氨基端到羧基端由引导序列、权利要求1~3的任一项所述的人NKG2D序列、CD8跨膜区、4-1BB胞内结构域序列和CD3ζ胞内结构域序列依次串联而成；或为氨基酸序列从氨基端到羧基端由引导序列、权利要求1~3的任一项所述的人NKG2D序列、CD8跨膜区序列、CD28胞内结构域序列和CD3ζ胞内结构域序列依次串联而成。
  12. 如权利要求 8或11所述的 靶向结合人 NKG2DL 的特异性嵌合抗原受体， 其中
    所述引导序列如SEQ ID No.3所示；和/或
    所述人 NKG2D 序列如SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8 或SEQ ID No.9中任意一个所示，和/或
    所述人CD8铰链区的氨基酸序列如SEQ ID No.10所示；和/或
    所述人CD8跨膜区的氨基酸序列如SEQ ID No.11所示；和/或
    或所述人4-1BB胞内结构域的氨基酸序列如SEQ ID No.12所示；和/或
    所述 CD 28 结构域 的氨基酸序列如SEQ ID No.13所示；和/或
    所述 CD3 ζ 结构域 的氨基酸序列如SEQ ID No.14或SEQ ID No.15所示。
  13. 权利要求4所述的靶向结合人NKG2DL的特异性嵌合抗原受体(CAR), 其是重组表达或由载体表达。
  14. 如权利要求13所述的靶向结合人NKG2DL的特异性嵌合抗原受体(CAR)，其中所述载体选自γ-逆转录病毒载体、慢病毒载、腺病毒、腺联病毒或优选γ-逆转录病毒载体。
  15. 编码权利要求4任一项所述的靶向结合人NKG2DL的特异性嵌合抗原受体的核酸分子，其特征是，包含从5‘到3‘依次串联连接的编码引导序列的核苷酸序列、编码靶向结合人NKG2DL的人NKG2D蛋白的核苷酸序列、编码跨膜结构域的核苷酸序列和编码胞内信号结构域的核苷酸序列，其中,所述跨膜结构域包括跨膜区, 所述胞内信号结构域包括免疫受体酪氨酸活化基序和共刺激信号域；任选地，所述核酸分子还包括编码铰链区的核苷酸序列。
  16. 权利要求15所述的核酸分子，其中，所述跨膜区包含CD8跨膜结构区、CD28跨膜结构区、CD3ζ跨膜结构区、CD4跨膜结构区、4-1BB跨膜结构区、OX40跨膜结构区、ICOS跨膜结构区、2B4跨膜结构区、BTLA跨膜结构区、或跨膜区合成肽中的任一种，所述免疫受体酪氨酸活化基序包含CD3ζ链的胞内信号结构域或FcεRIγ胞内信号结构，所述共刺激信号域包含CD28胞内信号结构域、CD137/4-1BB胞内信号结构域、CD134/OX40胞内信号结构域、ICOS胞内信号结构域中的至少一种。
  17. 权利要求15所述的核酸分子，其中所述核酸分子包含从5‘到3‘依次串联连接的分别编码引导序列、编码权利要求1~3的任一项所述靶向结合人NKG2DL的人NKG2D序列、人CD8铰链区序列、人CD8跨膜区序列、CD28胞内结构域序列、人4-1BB胞内结构域序列和CD3ζ胞内结构域的核苷酸序列；或包含从5‘到3‘依次串联连接的分别编码引导序列、权利要求1~3的任一项所述靶向结合人NKG2DL的人NKG2D序列、CD8跨膜区、4-1BB胞内结构域序列和CD3ζ胞内结构域序列的核苷酸序列；或包含从5‘到3‘依次串联连接的分别编码引导序列、权利要求1~3的任一项所述靶向结合人NKG2DL的人NKG2D序列、CD8铰链区序列、CD8跨膜区序列、4-1BB胞内结构域序列和CD3ζ序列胞内结构域的核苷酸序列，或包含从5‘到3‘依次串联连接的分别编码引导序列、权利要求1~3的任一项所述靶向结合人NKG2DL的人NKG2D序列、CD8铰链区、CD8跨膜区、CD28和CD3ζ序列的核苷酸序列。
  18. 权利要求17所述的核酸分子，其中，
    编码所述引导序列的核苷序列如SEQ ID No.16所示或与SEQ ID No.16所示核苷酸序列99%，95%，90%，85%或80%同源，
    或编码所述靶向结合人NKG2DL的人NKG2D的核苷酸序列如SEQ ID No.17、SEQ ID No.18、SEQ ID No.19、SEQ ID No.20、SEQ ID No.21或SEQ ID No.22所示或与SEQ ID No.17、SEQ ID No.18、SEQ ID No.19、SEQ ID No.20、SEQ ID No.21或SEQ ID No.22所示核苷酸序列99%，95%，90%，85%或80%同源，
    或编码所述人CD8铰链区序列如SEQ ID No.23所示或与SEQ ID No.23所示核苷酸序列99%，95%，90%，85%或80%同源，
    或编码所述人CD8跨膜区序列如SEQ ID No.24所示或与SEQ ID No.24所示核苷酸序列99%，95%，90%，85%或80%同源，
    或编码所述人4-1BB胞内结构域序列如SEQ ID No.25所示或与SEQ ID No.25所示核苷酸序列99%，95%，90%，85%或80%同源，
    或编码所述CD28胞内结构域序列如SEQ ID No.26所示或与SEQ ID No.26所示核苷酸序列99%，95%，90%，85%或80%同源，
    或编码所述CD3ζ胞内结构域序列如SEQ ID No.27或SEQ ID No.28所示，或与SEQ ID No.27或SEQ ID No.28所示核苷酸序列99%，95%，90%，85%或80%同源。
  19. 一种重组载体或表达质粒，其包含权利要求15~18中任一项所述的核酸。
  20. 一种启动子，用于构建权利要求19所述的重组载体和表达权利要求4~14任一项所述的靶向结合人NKG2DL的特异性嵌合抗原受体。
  21. 如权利要求20所述的启动子，其特征是，所述启动子选自SEQ No.29所示的EF1 alpha启动子和如SEQ No.30 所示的EFS启动子，或所述启动子为SEQ No.30所示的EF1 alpha启动子。
  22. 一种重组病毒，其中： 所述重组病毒为能够表达权利要求4~14所述的靶向结合人NKG2DL的特异性嵌合抗原受体，且能够侵染免疫应答细胞的病毒；所述病毒包括慢病毒、腺病毒、腺联病毒或逆转录病毒； 所述免疫效应细胞包括细胞毒性T淋巴细胞、NK细胞、NKT细胞或辅助性T细胞。
  23. 一种分离的修饰的免疫应答细胞，其包含前述权利要求4~14中任一项所述的靶向结合人NKG2DL的特异性嵌合抗原受体，其由权利要求19所述的重组载体或表达质粒转化所得。
  24. 如权利要求23所述的分离的修饰的免疫应答细胞，其中，所述免疫应答细胞还包含至少一种外源的共刺激配体，优选地，所述至少一种外源的共刺激配体选自4-1BB、CD80、CD86、CD70、OX40L、CD48、TNFRSF14及其组合，或更优选地，其中所述共刺激配体是4-1BB。
  25. 如权利要求23或24所述的分离的修饰的免疫应答细胞，其中所述免疫应答细胞选自T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、调节性T细胞、人胚胎干细胞和可分化成淋巴样细胞的多能干细胞，或优选自T细胞和自然杀伤细胞或优选为T细胞。
  26. 权利要求25所述的分离的修饰的免疫应答细胞的制备方法，其特征在于：
    包括以下步骤：
    首先，将权利要求15~18任一项所述的核酸分子，通过分子克隆的方式连接到初始表达载体中，获得表达所述靶向NKG2DL的特异性嵌合抗原受体的表达载体；
    然后，将获得的所述靶向NKG2DL的特异性嵌合抗原受体的表达载体转染293T细胞，获得病毒液；
    最后用所述病毒液感染免疫应答细胞，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免疫应答细胞；
    其中所述免疫应答细胞选自T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、调节性T细胞、人胚胎干细胞和可分化成淋巴样细胞的多能干细胞，或所述免疫应答细胞选自T细胞或自然杀伤细胞。
  27. 一种用于治疗或预防瘤疾病、不适或健康失调的药物组合物，其特征是包含有效量的如权利要求23～25中任一项所述的分离的修饰的免疫应答细胞和药学上可接受的赋形剂，所述疾病、不适或健康失调与人NKG2DL靶标及其配体人NKG2D蛋白的特异性相互作用有关，所述 疾病、不适或健康失调包括、病原体感染、自身免疫性疾病、炎性疾病、同种异体移植、移植排斥和衰老。
  28. 一种用于治疗或预防瘤疾病、不适或健康失调或衰老的试剂盒，其特征是包含如权利要求23～25中任一项所述的分离的修饰的免疫应答细胞或如权利要求15~18任一项所述的核酸，所述疾病、不适或健康失调与人NKG2DL靶标及其配体人NKG2D蛋白的特异性相互作用有关，所述 疾病、不适或健康失调包括、病原体感染、自身免疫性疾病、炎性疾病、同种异体移植、移植排斥和衰老， 其中所述试剂盒还包含使用免疫应答细胞治疗患有肿瘤形成、病原体感染、自身免疫性疾病、炎性疾病、同种异体移植、或移植排斥或衰老的受试者的书面说明书。
  29. 如权利要求19所述重组载体或表达质粒，如权利要求20或21所述的启动子，权利要求 22所述重组病毒，权利要求23~25任一项所述的分离的修饰的免疫应答细胞、权利要求15~18所述的核酸或权利要求 1~3任一项1所述的靶向NKG2DL的NKG2D蛋白或其功能性变体、权利要求4~14任一项所述的靶向NKG2DL的特异性嵌合抗体在治疗或预防疾病、健康失调或不适或延缓衰老的产品中的应用，所述疾病、健康失调、不适或衰老是与人NKG2DL靶标及其配体人NKG2D蛋白的特异性相互作用有关，所述疾病、健康失调、不适包括NKG2DL在病灶部位细胞表面高表达的肿瘤形成、病原体感染、自身免疫性疾病、炎性疾病、同种异体移植、移植排斥；其中，所述肿瘤表达人的NKG2DL抗原蛋白。
  30. 如权利要求29所述的应用，其中，所述治疗或预防瘤形成包括减少受试者中肿瘤负荷、增加或延长患有瘤形成的受试者的存活或响应受试者的癌细胞或病原体而增加免疫激活细胞因子。
  31. 如权利要求29或30所述的应用，其中，所述肿瘤表达人NKG2DL蛋白，所述肿瘤或瘤形成选自肝癌、胶质瘤、卵巢癌、胃癌、随母细胞瘤、胰腺癌、肺癌、前列腺癌细胞、乳腺癌 或神经母细胞瘤、膀胱癌、结肠癌、肾细胞癌、白血病、淋巴瘤、多发性骨髓瘤、黑色素瘤、及其组合。
  32. 如权利要求29或30所述的应用，其中所述分离的修饰的免疫应答细胞是通过静脉回输的方式治疗所述肿瘤或瘤形成，或所述分离的修饰的免疫应答细胞是通过肿瘤内部注射的方式治疗所述肿瘤或瘤形成。
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CN111363046A (zh) * 2020-03-11 2020-07-03 深圳宾德生物技术有限公司 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
CN114702596A (zh) * 2022-03-11 2022-07-05 南京凯地医疗技术有限公司 靶向人cd33和nkg2dl的嵌合抗原受体细胞及其制备方法和应用
CN114702596B (zh) * 2022-03-11 2023-02-14 南京凯地医疗技术有限公司 靶向人cd33和nkg2dl的嵌合抗原受体细胞及其制备方法和应用

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