WO2022117049A1 - Nkg2d car-免疫细胞在抗衰老及老年相关疾病治疗中的应用 - Google Patents

Nkg2d car-免疫细胞在抗衰老及老年相关疾病治疗中的应用 Download PDF

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WO2022117049A1
WO2022117049A1 PCT/CN2021/135151 CN2021135151W WO2022117049A1 WO 2022117049 A1 WO2022117049 A1 WO 2022117049A1 CN 2021135151 W CN2021135151 W CN 2021135151W WO 2022117049 A1 WO2022117049 A1 WO 2022117049A1
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cells
car
nkg2d
senescent
cell
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PCT/CN2021/135151
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French (fr)
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赵旭东
杨东
李仕容
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四川大学华西医院
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Priority to US18/255,808 priority Critical patent/US20240016843A1/en
Publication of WO2022117049A1 publication Critical patent/WO2022117049A1/zh

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Definitions

  • the invention belongs to the field of immune cell therapy or biomedicine, and in particular relates to the application of an NKG2D CAR-immune cell in the treatment of anti-aging and age-related diseases.
  • mice can delay the onset of aging-related phenotypes, increase the median lifespan of mice by 24%, reduce age-related degeneration of multiple organ functions, and improve age-related phenotypes. Lipodystrophy, hepatic steatosis, cardiac function and bone loss, and tau-mediated neurodegeneration. Therefore, removing senescent cells is an important means to treat and prevent various age-related diseases.
  • the methods of removing senescent cells mainly focus on the search for small molecule compounds that can selectively remove senescent cells, such as dasatinib, quercetin, ABT263, etc., but these compounds are not effective in removing senescent cells, or there are obvious
  • the toxic and side effects are not conducive to over-the-counter medicines.
  • ABT-263 can cause transient thrombocytopenia and neutropenia; treating mice with osteoarthritis with UBX0101 cannot restore the pathological features of osteoarthritis in mice, but only changes in the expression levels of some molecules This will create an external environment conducive to repairing damage.
  • Immune cells modified by Chimeric Antigen Receptor can specifically recognize cell-associated antigens to target and kill target cells, which has broad clinical application prospects.
  • CAR-T cells targeting CD19 have excellent therapeutic effects on B-cell malignancies and have been approved by the FDA for marketing.
  • a large number of CAR-modified NK cells are currently in phase II clinical trials (NCT02742727, NCT02892695, NCT02944162, NCT03056339).
  • studies have also shown that macrophages expressing HER2CAR can specifically phagocytose tumor cells.
  • CAR-immune cell therapy in the field of anti-aging research, there is no better CAR-immune cell therapy. .
  • the purpose of the present invention is to provide a method for removing senescent cells specifically and efficiently using CAR-immune cell technology.
  • a CAR-immune cell targeting NKG2D ligands for preparing a medicine for:
  • the CAR-immune cells targeting NKG2D ligands express chimeric antigen receptors targeting NKG2D ligands, and the antigen binding domains in the chimeric antigen receptors include amino acid sequences such as SEQ ID NO: The polypeptide shown in 1, or a polypeptide that has more than 80% similarity with the sequence shown in SEQ ID NO: 1 and can bind to NKG2D ligands.
  • the CAR-immune cells are selected from the following group: CAR-T cells, CAR-NK cells, CAR-macrophages, or a combination thereof.
  • the CAR-immune cells are CAR-T cells.
  • the chimeric antigen receptor has the structure shown in formula I,
  • L is none or a signal peptide sequence
  • NKG2D is the NKG2D ligand binding domain sequence of claim 1;
  • H is no or CD8 ⁇ hinge region
  • TM is the human CD8 ⁇ transmembrane domain
  • C is 4-1BB or CD28 costimulatory signaling molecule
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇
  • Each "-" independently represents a linking peptide or peptide bond connecting each of the above elements.
  • amino acid sequence of the signal peptide sequence is shown in SEQ ID NO:2.
  • amino acid sequence of the CD8 ⁇ hinge region is shown in SEQ ID NO:3.
  • amino acid sequence of the CD8 ⁇ transmembrane domain is shown in SEQ ID NO:4.
  • amino acid sequence of the 4-1BB costimulatory signal molecule is shown in SEQ ID NO:5.
  • amino acid sequence of the CD3 ⁇ -derived cytoplasmic signaling sequence is shown in SEQ ID NO:6.
  • the expression of the chimeric antigen receptor is driven by the strong promoter EF1 ⁇ .
  • the senescent cells are selected from the group consisting of lung cells, fat cells, kidney cells, muscle cells, bone cells, or a combination thereof.
  • the senescent cells are human embryonic lung cells IMR90.
  • the senescent cells are naturally or artificially induced to senesce.
  • the artificially induced senescence method comprises: DNA damage-induced senescence, overexpression of P16-induced senescence, oncogenic signal-induced senescence, telomere shortening-induced senescence, or a combination thereof.
  • the method for artificially inducing senescence is to induce senescence with an inducer DOX.
  • the individual suffering from the senile disease contains senescent cells, and the NKG2D ligand in the senescent cells is up-regulated by 2-20 times, preferably 4-15 times, more preferably than normal cells 10-20 times the ground.
  • the senile disease is selected from the group consisting of muscular dystrophy, fatty liver, heart failure, atherosclerosis, diabetes, cardiac hypertrophy, osteoporosis, tissue/organ fibrosis, Alzheimer's disease Organ degenerative diseases caused by cellular aging such as Haimer's disease, Parkinson's syndrome, arthritis, or a combination thereof.
  • the senile disease is selected from the group consisting of senile osteoporosis, senile muscle atrophy, senile liver fibrosis, senile fatty liver, or a combination thereof.
  • composition comprising:
  • the antigen-binding domain in comprises a polypeptide whose amino acid sequence is shown in SEQ ID NO: 1, or a polypeptide that has more than 80% similarity with the sequence shown in SEQ ID NO: 1 and can bind to NKG2D ligands;
  • the other anti-aging drugs include other drugs that can specifically remove senescent cells.
  • component (b) includes a small molecule compound capable of specifically removing senescent cells, preferably selected from the group consisting of dasatinib, quercetin, ABT263, ABT737, piperonamide, or its combination.
  • the pharmaceutical composition is a liquid pharmaceutical composition.
  • the pharmaceutical composition is an injection.
  • the dose of the CAR-immune cells targeting NKG2D is 1 ⁇ 10 5 -5 ⁇ 10 7 cells/kg, preferably 5 ⁇ 10 6 - 1 ⁇ 10 7 cells/kg.
  • a method for delaying aging of an individual, or for preventing and/or treating senile diseases comprising the steps of: administering a CAR-immune cell targeting an NKG2D ligand to an object in need, wherein , the CAR-immune cells targeting NKG2D express a chimeric antigen receptor targeting NKG2D ligands, and the antigen binding domain in the chimeric antigen receptor includes an amino acid sequence as shown in SEQ ID NO: 1
  • the administration mode is intravenous injection.
  • a CAR-immune cell wherein a chimeric antigen receptor is expressed in the CAR-immune cell, and the chimeric antigen receptor has the structure shown in formula I,
  • L is none or a signal peptide sequence
  • NKG2D is the antigen binding domain sequence of claim 1;
  • H is no or CD8 ⁇ hinge region
  • TM is the human CD8 ⁇ transmembrane domain
  • C is 4-1BB costimulatory signaling molecule
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇
  • Each "-" independently represents a linking peptide or peptide bond connecting each of the above elements.
  • the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 12.
  • Figure 1 shows the results of the construction of a senescent cell model.
  • IMR90-Et is a DNA damage-induced senescence cell model. IMR90 cells were treated with the DNA damage drug Etoposide (Et) for 24 h, and the cells were stained with fresh medium for 8 days; IMR90 -p16 is a senescent cell model that overexpresses p16 protein by tetracycline-inducible system, and stained after 1 ⁇ g/ml dox treatment for 8 days; IMR90-Kras is a senescent cell model that overexpresses oncogene Kras G12D , and stained after overexpressing Kras G12D for 10 days; IMR90-Rep It is a replicative senescent cell model, which was observed after 37 consecutive passages in vitro; SC refers to senescent cells, and CON refers to young control cells; (B) Statistics of the positive rate of SA- ⁇ gal sta
  • Figure 2 shows the detection results of NKG2DL expression in a senescent cell model.
  • IMR90-Et is a DNA damage-induced senescent cell model
  • IMR90-p16 is a tetracycline-induced system overexpressing p16 protein senescent cell model
  • IMR90-Kras is an overexpressed cell model.
  • IMR90-Rep is a replicative senescent cell model, the mean and standard deviation of three measurements were calculated, two-tailed T test *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001.
  • B The expression of NKG2D ligands on the surface of senescent cells was detected by flow cytometry, and the percentage shown in the figure is the positive rate of cells.
  • Figure 3 shows the results of the killing of senescent cells by NKG2D CAR-T cells.
  • FIG. 1 Schematic diagram of the structure of MOCK and NKG2D CAR vectors;
  • C (D)
  • E MOCK and NKG2D CAR-T cells were co-cultured with IMR90-P16, IMR90-Et and IMR90-Rep senescent cells for 10 h, cell phenotype observation and killing rate analysis. The effect-target ratio was 2:1, and the experiment was repeated three times.
  • Figure 4 shows the safety assessment results of NKG2D-BBz CAR-T.
  • A 90 normal human tissues in HOrgN09PT02 microarray were stained with MICA antibody. Samples with significantly elevated MICA expression are marked in red.
  • A1-A4 thyroid gland; A5-A7: tongue; A8-A11: esophageal epithelium; A12-B5: gastric mucosa; B6-B7: duodenal mucosa; B8-C1: jejunal mucosa; C2-C4: ileal mucosa; C5-C9: appendix; C10-D1: colonic mucosa; D2-D3: rectal mucosa; D4-D5: liver; D6-D7: pancreas; D8-D10: trachea; D11-E3: lung; E4-E6: myocardium; E7-E9: artery; E10-F4: skeletal muscle; F5-F7: skin; F8: seminal vesicle; F9-F
  • Figure 5 shows the preparation results of monkey NKG2D CAR-T.
  • A Monkey T cell culture, the spheres are activated T cells;
  • B After NKG2D CAR-T virus infected monkey T cells, the CAR expression efficiency was detected by flow cytometry.
  • C CD4 and CD8 expression by flow cytometry after infection of monkey T cells with NKG2D CAR-T virus.
  • Figure 6 shows the monitoring results of basal signs in monkeys after NKG2D CAR-T infusion.
  • Figure 7 shows the detection results of monkey biochemical indicators after NKG2D CAR-T infusion.
  • WBC leukocyte
  • Lymphocyte lymphocyte
  • Monocyte monocyte
  • Granulocyte granulocyte
  • RBC erythrocyte
  • PLT platelet
  • Figure 8 shows the results of in vivo clearance of senescent cells by NKG2D CAR-T.
  • Figure 9 shows the establishment of an aging mouse model.
  • Figure 10 shows the results of NKG2D CAR-T treatment of aging-induced muscular dystrophy.
  • Figure 11 shows the results of NKG2D CAR-T treatment of aging-induced osteoporosis.
  • Figure 12 shows the results of NKG2D CAR-T cell treatment of aging-induced liver fibrosis.
  • Figure 13 shows the results of NKG2D CAR-T treatment of fatty liver by eliminating senescent cells.
  • CAR-immune cells such as CAR-T cells
  • the present invention has been completed on this basis.
  • the present invention constructed a series of senescent cell models of different cell lines, and confirmed the high expression of NKG2D ligand (NKG2DL) using the senescence model, and then used the NKG2D extracellular sequence as the target recognition domain, 4-1BB and CD3 ⁇ constructs second generation CAR-T cells for costimulatory signals.
  • NKG2D ligand NKG2D ligand
  • 4-1BB and CD3 ⁇ constructs second generation CAR-T cells for costimulatory signals.
  • the experimental results show that NKG2D CAR-T cells can efficiently kill senescent cells in vitro, releasing high concentrations of cytokines, perforin and granzymes.
  • lentivirus-mediated CAR expression had no significant effect on T cell proliferation, apoptosis, and genome stability.
  • monkey NKG2D CAR-T cells were isolated and prepared in the present invention, and then autologously infused into monkeys at a certain dose (eg, 1 ⁇ 10 6 /kg).
  • the monkeys' body temperature, body weight and cytokines were measured before and after the infusion of cells; the results showed that none of the monkeys had fever, no diarrhea symptoms, no abnormal changes in body weight, and no significant changes in serum cytokines before and after the infusion of NKG2D CAR-T cells. unusual changes.
  • the blood routine and biochemical test results of monkeys before and after the reinfusion of cells showed that the blood routine and biochemical indexes were within the normal range.
  • NKG2D CAR-T cells did not produce toxic side effects on monkey organs including heart, kidney and liver.
  • the muscle, fat, liver and kidney tissues of monkeys before and after treatment were taken for analysis, and the results showed that the CAR-T of the present invention can significantly reduce the expression of NKG2D ligands and aging markers in these tissues.
  • the CAR immune cells of the present invention can effectively remove accumulated senescent cells in vivo, thereby significantly improving aging-related symptoms (such as senile osteoporosis, senile muscle atrophy, senile liver fibrosis, senile fat). liver, etc.).
  • the term "about” can refer to a value or composition within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • administration and “administration” are used interchangeably and refer to the physical introduction of a product of the invention into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including Intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, eg, by injection or infusion.
  • Chimeric immune antigen receptors are composed of extracellular antigen recognition domain, usually scFv (single-chain variable Fragment), transmembrane region and intracellular costimulatory signaling domain.
  • the design of CARs has gone through the following process: the first-generation CAR has only one intracellular signal component, CD3 ⁇ or Fc ⁇ RI molecule. Since there is only one activation domain in the cell, it can only cause transient T cell proliferation and less cytokine secretion. , and can not provide long-term T cell proliferation signal and sustained anti-tumor effect in vivo, so it has not achieved good clinical efficacy.
  • the second-generation CARs introduce a costimulatory molecule on the basis of the original structure, such as CD28, 4-1BB, OX40, and ICOS. Compared with the first-generation CARs, the function is greatly improved, which further strengthens the persistence of CAR-T cells and promotes tumor cells. destructive ability. On the basis of second-generation CARs, some new immune costimulatory molecules such as CD27 and CD134 are connected in series to develop into third- and fourth-generation CARs.
  • the extracellular segment of CARs can recognize one or more specific antigenic determinants (epitopes), and then transduce the signal through the intracellular domain, causing cell activation and proliferation, cytolytic toxicity and secretion of cytokines, thereby eliminating target cells.
  • the patient's autologous or allogeneic (healthy donor) PBMCs are isolated, activated and genetically modified to produce CAR-producing immune cells, and then injected into the patient to specifically kill them by directly recognizing tumor cell surface antigens in a non-MHC-restricted manner.
  • the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes target-specific binding elements (also referred to as antigen binding domains).
  • the intracellular domain includes a costimulatory signaling region and/or a zeta chain portion.
  • a costimulatory signaling region refers to a portion of an intracellular domain that includes a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that are required for an efficient lymphocyte response to an antigen.
  • a linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.
  • linker As used herein, the terms “linker”, “hinge region” are used interchangeably and generally refer to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain.
  • the linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.
  • the CAR of the present invention when expressed in immune cells, is capable of antigen recognition based on antigen binding specificity. When it binds to an associated antigen on tumor cells, it causes tumor cell death and a reduction or elimination of the patient's tumor burden.
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and/or the zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of a combination of CD28 costimulatory signaling molecule, 4-1BB costimulatory signaling molecule and CD3 ⁇ signaling domain.
  • the basic structure of the chimeric antigen receptor of the present invention includes: an NKG2D antigen binding domain, an extracellular hinge region, a transmembrane region, and an intracellular signaling region.
  • NK cells are the main immune cells for the body to clear senescent cells
  • the NKG2D-NKG2D ligand axis is the main activation pathway for NK cells to recognize target cells. Therefore, compared with other CAR-immune cells, the CAR-immune cells constructed with the NKG2D extracellular domain as the recognition site should have more reliable safety.
  • NKG2D can recognize a variety of ligands such as MICA, MICB, ULBP1, ULBP2 and ULBP3, and the CAR-T cells constructed based on NKG2D can clear senescent cells more broadly.
  • CAR-immune cell refers to an immune cell expressing the chimeric antigen receptor of the present invention. It is particularly worth noting that the CAR-immune cells of the present invention can be different immune cells that perform effector functions in the organism, such as T cells, NK cells, macrophages, and the like.
  • CAR-T cells are the most thoroughly researched and widely studied immunotherapy, and a number of products have been approved for tumor treatment. Compared with CAR-T cells, CAR-NK cells have lower cytokine release and can also eliminate tumor cells through the NK cell receptor itself.
  • CAR-macrophages can convert nearby M2 macrophages into M1 by expressing pro-inflammatory cytokines and chemokines, upregulating antigen presentation mechanisms and activating the autoimmune system, with higher safety.
  • the extracellular region of NKG2D is selected as the antigen-binding domain targeting NKG2D in the CAR of the present invention.
  • amino acid sequence of the antigen binding domain is shown in SEQ ID NO: 1, which can efficiently bind to NKG2D ligand molecules.
  • the antigen binding domains targeting NKG2D ligands also include conservative variants of the sequences, which means that compared with the amino acid sequences of the antigen binding domains of the present invention, there are at most 10, preferably at most 8 One, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.
  • the number of added, deleted, modified and/or substituted amino acids is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, more preferably 1-33%, More preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • the number of amino acids added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, 1 is optimally.
  • the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.
  • the transmembrane domain naturally associated with one of the domains in the CAR is used.
  • transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with receptor complexes interactions with other members.
  • the CAR construct of the present invention has the following structure: signal peptide-antigen binding domain targeting NKG2D ligand-CD8 ⁇ hinge region-CD8 ⁇ TM-41BB-4-1BB costimulatory signaling molecule-CD3 ⁇ cytoplasmic signaling sequence.
  • amino acid sequence of the signal peptide is shown in SEQ ID NO:2.
  • amino acid sequence of the CD8 ⁇ hinge region is shown in SEQ ID NO:3.
  • amino acid sequence of the CD8 ⁇ transmembrane domain is shown in SEQ ID NO:4.
  • amino acid sequence of the 4-1BB costimulatory signal molecule is shown in SEQ ID NO:5.
  • the amino acid sequence of the CD3 ⁇ -derived cytoplasmic signaling sequence is shown in SEQ ID NO:6.
  • amino acid sequence of the CAR of the present invention is shown in SEQ ID NO: 12.
  • the present invention provides a CAR-immune cell targeting NKG2D ligand described in the first aspect of the present invention, other anti-aging drugs, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition is a liquid formulation.
  • the formulation is an injection.
  • the dose of the CAR-immune cells in the formulation is 1 ⁇ 10 5 -5 ⁇ 10 7 cells/kg, more preferably 5 ⁇ 10 5 -1 ⁇ 10 7 cells/kg.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine ; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, etc.
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins such as glucose, mannose, sucrose or dextran, mannitol
  • proteins such as glucose, mannose, sucrose or dextran, mannitol
  • proteins such as glucose, mannose, sucrose or dextran, mannitol
  • proteins such as glucose, mannose, sucrose or dextran, mannitol
  • proteins such as
  • the formulations of the present invention are preferably formulated for intravenous administration.
  • the present invention provides the use of the NKG2D-targeting CAR-immune cells of the present invention and the pharmaceutical composition of the present invention for preventing and/or treating senile diseases.
  • the present invention also provides the use of the CAR-immune cells targeting NKG2D of the present invention and the pharmaceutical composition of the present invention, for preparing a medicine for (i) removing senescent cells, wherein NKG2D is formulated with The body is up-regulated by 2-20 times, preferably 4-15 times, more preferably 10-20 times compared with normal cells; (ii) delay the aging of the individual; and/or (iii) prevent and/or treat senile diseases.
  • senile diseases or aging-related diseases include (but are not limited to) the following organ degenerative diseases caused by cell aging: muscular dystrophy, fatty liver, heart failure, atherosclerosis, diabetes, cardiac hypertrophy, osteoporosis , tissue/organ fibrosis, Alzheimer's disease, Parkinson's syndrome, arthritis, or other organ degenerative diseases due to cellular aging, or a combination thereof.
  • the universal CAR-immune cells of the present invention can also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals.
  • the mammal is a human.
  • CAR-modified cells are isolated from mammals (preferably human) and genetically modified (ie, transduced or transfected in vitro) with a vector expressing a CAR disclosed herein.
  • CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefit.
  • the mammalian recipient can be human, and the CAR-modified cells can be autologous or allogeneic, syngeneic, relative to the recipient.
  • the present invention also provides compositions and methods for use in vivo to enhance immune responses to targeted antigens in patients.
  • the present invention provides methods of treating senile diseases comprising administering to a subject in need thereof an effective amount of the CAR-immune cells of the present invention.
  • the CAR-immune cells of the present invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may include target cells as described herein in association with one or more pharmaceutically or clinically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelates Adjuvants such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • the compositions of the present invention are preferably formulated for intravenous administration.
  • compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the characteristics of the patient's condition, the type and severity of the disease - although appropriate doses may be determined by clinical trials.
  • the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the patient Individual differences in age, weight, aging tissue size, degree of aging, and conditions of (subjects). It may generally be indicated:
  • the pharmaceutical compositions comprising the T cells described herein may be administered at a dose of 104 to 109 cells/kg body weight, preferably 105 to 107 cells/kg body weight (including all integers within those ranges) value) application.
  • the T cell composition can also be administered multiple times at these doses.
  • Cells can be administered by using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regimen for a particular patient can be determined by one skilled in the medical arts by monitoring the patient for signs of disease.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous (i.v.) injection or intraperitoneally, intrapleurally.
  • the T cell composition of the present invention is preferably administered by i.v. intravenous injection.
  • Compositions of T cells can be injected directly into senescent tissue or a pathological tissue or site of infection caused by senescence.
  • cells are activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels, in conjunction with any number of relevant therapeutic modalities (eg, before, at the same time, or after) administration to a patient, such treatment modalities include, but are not limited to, treatment with the following agents: dasatinib, quercetin, ABT263, ABT737, longinamide.
  • the subject receives an infusion of the expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • the dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be carried out in accordance with art-accepted practice. Typically, 1 ⁇ 10 6 to 1 ⁇ 10 10 CAR-immune cells of the present invention can be administered to a patient, eg, by intravenous infusion, per treatment or per course of treatment.
  • the present invention develops for the first time a CAR-immune cell that can specifically clear senescent cells with high expression of NKG2D ligands.
  • the present invention targets NKG2D ligands for the first time, and NKG2D CAR-T cells are used to remove senescent cells in vitro and in vivo, and can Treatment of age-related diseases.
  • Lentivirus-mediated expression of the CAR of the present invention has no significant effect on T cell proliferation, apoptosis and genome stability.
  • the CAR-immune cells of the present invention have high safety.
  • the in vivo test results in monkeys confirmed that it did not produce toxic side effects on the organs of monkeys including heart, kidney and liver.
  • virus infection was performed after the cells adhered on the second day, pTomo-Kras G12D -EGFP virus (Kras virus) was added at MOI 50-100 per 10 cm dish, and polybrene (final concentration 8 ⁇ g/mL) was added to improve the infection efficiency;
  • the present invention adopts the second-generation CAR structure.
  • the human NKG2D extracellular domain chain is used as the recognition fragment, and its expression is driven by the strong promoter CMV.
  • the signal peptide (SP) and the NKG2D sequence are sequentially connected to human CD8 ⁇ hinge region (CD8Hinge), human CD8 ⁇ transmembrane region (CD8TM), 4- 1BB costimulatory domain and CD3 ⁇ activation domain.
  • the Mock vector without NKG2D extracellular domain sequence is used as a negative control.
  • the NKG2D CAR vector structure is shown in Figure 3A.
  • CD8SP-NKG2D EC-CD8Hinge and TM-41BB-CD3 ⁇ were synthesized by Beijing Qingke Biotechnology Co., Ltd. and cloned into pTomo lentiviral vector through XbaI and SalI restriction sites.
  • CD8 hinge and transmembrane regions (SEQ ID NO: 9):
  • 4-1BB costimulatory signaling molecule (SEQ ID NO: 10):
  • CD3 ⁇ intracellular signaling domain SEQ ID NO: 11:
  • Plasmid preparation Lentivirus-packaged core plasmids pCMV ⁇ 8.9 and pMD2.G were pumped with QIAGEN EndoFree Plasmid Maxi Kit, and the lentiviral expression core plasmid pTomo-NKG2D-CAR-T2A-mKATE was used for pTomo-Mock CAR QIAGEN Plasmid Midi Kit is in the middle draw.
  • 293T cell preparation 24h before virus packaging, 293T cells with an abundance of more than 90% were passaged at a ratio of 1:2.5, and 25 mL of DMEM containing 10% serum and no double antibody was added to each 15cm culture dish. base culture. 4 to 6 hours before packaging, the cells in the dish were subjected to "half-change medium" treatment, that is, 15 mL of the medium supernatant was aspirated, and 15 mL of DMEM medium containing only 10% serum and no double antibody was added to it to remove the cells. The state is adjusted to the best. When the cell density in the dish reaches 80% to 90%, virus packaging can be carried out.
  • Virus collection 15 mL of virus-containing medium supernatant was collected after 48 hours of packaging, and then 15 mL of DMEM medium containing 5% serum and 1% double antibody was added, and the virus supernatant was collected again after 72 hours of packaging;
  • Steps 3 and 4 have a total of 40 cycles.
  • step (vi) repeat operation step (v), take a certain amount of T cell culture medium to resuspend the cells, and count;
  • step c Mix the pre-cooled 1 ⁇ PBS with the liquid mixed in step b 1:1, and incubate it on a silent suspender at 4°C for 2 hours before use.
  • CD3-positive T cells were detected by flow cytometry.
  • CAR-T cells were collected by centrifugation at 300 g for 5 min, resuspended CAR-T cells with 1 mL of T cell complete medium, 10 ⁇ L of cell suspension was extracted, and the viable cells were counted after staining with 0.4% trypan blue;
  • the content of perforin and granzyme is determined by ELISA, and the sample used in the experiment is the cell supernatant of T cells and DNA damage-induced senescent IMR90 cells co-cultured for 12h (perforin) or 24h (granzyme). After collecting the supernatant, centrifuge at 2000g for 10min and temporarily store at -80°C. According to the instructions of Abcam's Perforin (PRF1) Human ELISA kit (ab46068) and Human Granzyme B SimpleStep ELISA kit (ab235635), respectively prepare standard products, draw standard curves, and analyze the contents of perforin and granzyme in the samples respectively. to measure. Subsequent data analysis was performed using GraphPad Prism software.
  • NKG2D CAR-T cells were reinfused into macaque/cynomolgus monkeys
  • Collect monkey T cells ie, CAR-T cells 72 hours after infection with the virus, remove the magnetic beads in the cell culture medium (transfer the cell suspension to a sterile flow tube, and then place the flow tube on the magnet Place for 1min in the flow tube, quickly add the liquid in the flow tube to a 15ml centrifuge tube), centrifuge in a 500g centrifuge for 5min and collect the centrifuged cell pellet;
  • Mouse sequences homologous to human NKG2D CAR sequences including murine NKG2D extracellular domain (NKG2D-EC), CD8 ⁇ hinge region (CD8Hinge), CD8 ⁇ transmembrane region (CD8TM), 4-1BB costimulatory domain and CD3 ⁇ activation structure
  • the domain was synthesized by Beijing Qingke Biotechnology Co., Ltd. and cloned into the MSCV-IRES-GFP retroviral vector through EcoRI and SalI restriction sites.
  • the Mock vector without NKG2D ectodomain sequence was used as a negative control in the experiment.
  • NKG2D EC (SEQ ID NO: 13):
  • CD8 hinge and transmembrane regions (SEQ ID NO: 14):
  • 4-1BB costimulatory signaling molecule (SEQ ID NO: 15):
  • CD3 ⁇ intracellular signaling domain SEQ ID NO: 16:
  • Plasmid preparation The retroviral plasmids MSCV-NKG2D, MSCV-MOCK, and the helper plasmid pCL-ECO were extracted with QIAGEN EndoFree Plasmid Maxi Kit (QIAGEN, 12362).
  • 293T cell preparation 24h before virus packaging, 293T cells with an abundance of more than 90% were passaged at a ratio of 1:3, and 25mL of DMEM medium containing 10% serum was added to each 15cm dish for culture. Virus packaging was performed when the cell density in the dish reached 80% to 90%.
  • Virus packaging The retroviral plasmid and the helper plasmid pCL-ECO were mixed according to the same quality, and then transfected according to the instructions of Beyotime lipo6000 (Beyotime, C0526). Collect the supernatant of the medium 48 hours and 72 hours after transfection, centrifuge at 3000 rmp for 15 min at 4 °C, and filter the cell debris with a 0.45 ⁇ m filter membrane; take the filtered virus solution and centrifuge it at 25000 rpm and 4 °C for low temperature ultracentrifugation. 2.5h. The virus was lysed in PBS containing 0.1% BSA overnight at 4°C. Take 5 ⁇ L of the virus stock solution, dilute it in a gradient of 10 times, 100 times, 1000 times, and 10000 times, and infect 3T3 cells respectively.
  • mice T cell isolation kit (stem cell, 19851) to separate T cells;
  • T cells were activated and cultured with mouse CD3/CD28 magnetic beads (life technologies, 11452D).
  • mice 8-week-old wild-type C57/B6J mice were irradiated with 4 Gy rays and raised in SPF animal room. After 3 months, the main tissues of the mice were taken to detect the expression of aging markers and NKG2D ligands. After confirming the successful model establishment, the mice were treated with control MOCK and mouse NKG2D CAR-T cells via tail vein infusion. After 1 month of treatment, the main tissues of the mice were collected to detect the expression of aging markers and NKG2D ligands, and 6 months later, the morphological changes of the mice's tissue damage and the changes of the mice's physical fitness were detected.
  • mice Eight-week-old male C57BL/6 mice were selected and fed with high-fat diet D12492 from Research Diet to establish a mouse fatty liver model, and were fed continuously for three months.
  • Example 1 Establishment of senescent cell model
  • senescence induced by different factors represents different types of cellular senescence, and involves different molecular markers and signaling pathways.
  • the present invention uses different inducing factors to construct a series of senescent cell models, which lays the foundation for subsequent research: 1. Treated by DNA damage drug Etoposide (Et) Human embryonic lung cells IMR90 induced senescence; 2. IMR90 cells were induced by DOX to overexpress p16 protein to induce senescence; 3. Oncogene Kras G12D was overexpressed in IMR90 cells to induce senescence; 4. IMR90 cells were continuously cultured in vitro to establish a natural senescence cell model . The above models were verified by SA- ⁇ gal staining, and the positive rates were all above 90% (Fig. 1A, B). Elevated expression of p16 is one of the typical features of senescent cells.
  • Example 2 Up-regulation of NKG2D ligand expression in an in vitro senescent cell model
  • Example 3 Detection of killing of senescent cells by NKG2D CAR-T cells
  • NKG2D ligands is up-regulated in senescent cells, indicating that NKG2D CAR-T cells should have the effect of eliminating senescent cells.
  • a second-generation CAR-T cell with CMV promoter-driven expression was constructed, with NKG2D extracellular domain as the recognition sequence, and 4-1BB and CD3 ⁇ as costimulatory structural signals (Figure 3A,B).
  • control T cells and CAR-T cells were co-cultured with IMR90-p16 senescent cells in vitro at a 2:1 effector-target ratio.
  • tumor-associated antigens on normal cells often leads to severe off-target phenomena, preventing the clinical application of CAR-T therapy.
  • CAR-T therapy In most human normal tissues, including thyroid, tongue, esophageal epithelium, gastric mucosa, jejunal mucosa, ileal mucosa, appendix, rectal mucosa, liver, pancreas, trachea, lung, myocardium, myocardium, arteries, skeletal muscle, seminal vesicles, prostate , bladder, testis, medulla oblongata, telencephalon, forebrain, brainstem, and spleen, the expression of MICA, the major ligand of NKG2D, was not detected, with only weak positivity in the skin (Fig. 4A).
  • karyotype analysis was also performed to evaluate the safety of lentiviral infection CAR expression. Compared with untransduced T cells, no abnormal karyotype was observed in NKG2D-BBz CAR-T cells 14 post-infection with virus (Fig. 4B).
  • the fresh blood collected from the peripheral blood of cynomolgus monkeys/cynomolgus monkeys was separated from non-human primate T cells by gradient centrifugation, and inoculated to 24 In the well plate (1x106 cells per well), 25ul of non-human primate-specific T cell magnetic beads (containing CD2/CD3/CD28 antibodies) were added to each well, and the isolated cells began to form spheres after 1 day of culture, indicating that the T cells It was successfully isolated from monkey peripheral blood and could be activated (Fig. 5A).
  • T cells When T cells are activated by antigen, they differentiate into CD4 and CD8 positive cells. After CAR-T cells proliferate and enter the human body, the proliferation rate of CD8-positive T cell populations will increase, and CD8-positive cells also play a major role in targeting and eliminating tumor cells. However, flow cytometry results showed that there was no change in CD4-positive T cells and CD8-positive T cells in T cells 3 days after virus infection (Fig. 5C).
  • CAR-T cells After CAR-T cells are reinfused into patients, the proliferation of a large number of T cells may lead to the up-regulation of cytokine secretion, which is clinically manifested as elevated body temperature, anorexia, diarrhea and vomiting.
  • the body temperature of the monkeys was measured before and after the infusion into five monkeys, and the basic signs of the monkeys were closely observed after the cells were reinfused.
  • the secretion of IL-6 in the serum of the two monkeys numbered 00065 and 98106 was almost unchanged before and after the infusion of cells; while the monkey numbered 00085 started to increase on the 14th day; the IL-6 secretion of the two monkeys numbered 01102 and 00102 -6 started to increase on day 1 after reinfusion of cells and then decreased on day 7 (Fig. 6D).
  • the changes of various cells in the blood of monkeys after NKG2D CAR-T treatment were detected, and the number was found to be 00085
  • the number of monocytes in the rhesus monkeys exceeded the normal range (0.22-2.22x109/L) on the 7th day after CAR-T cell reinfusion, but returned to the normal range on the 14th day.
  • the cynomolgus monkey platelet numbered 98106 exceeded the normal range on day 0 (211.84-669.06 ⁇ 109/L), and was within the normal range on day 7 and later (Fig. 7A).
  • the blood routine indexes of the other monkeys were all within the normal range.
  • NKG2D CAR-T cells have no effect on the blood routine of monkeys after infusion.
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase
  • GTT glutamyl transpeptidase
  • CRE2 creatinine
  • BUN blood urea nitrogen
  • CK cardiac marker creatine kinase
  • Example 6 Establishment of an aging mouse model
  • an aging model was first constructed by irradiating 8-week-old C57/B6J mice with 4 Gy rays. The hair of the mice turned white after irradiation (Fig. 9A), showing certain signs of aging.
  • P16 is considered to be a universal marker of senescent cells, and the literature has shown that its expression is significantly up-regulated in various senescent tissues in humans and mice. Therefore, the expression of p16 in the major organs of irradiated mice was examined. The results showed that irradiation significantly increased P16 expression in mouse fat, heart, skeletal muscle, lung, kidney, and liver, and the fold was positively correlated with time (Fig. 9B). At the same time, the expression of NKG2D ligands MULT-1 and RAE-1 was significantly increased in these tissues (Fig. 9C,D).
  • Example 7 NKG2D CAR-T treatment of aging-induced muscular dystrophy
  • T cells were isolated from mouse spleen, and mouse NKG2D CAR-T cells were established by retroviral infection, with a positive rate of 41.9% (Fig. 10A).
  • the MOCK and NKG2D CAR-T cells were injected intravenously to treat aging mice 11 months after irradiation (Example 6), and 4 weeks later, the skeletal muscle of the mice was extracted to extract RNA to detect the expression of NKG2D ligands.
  • Example 8 NKG2D CAR-T treatment of aging-induced osteoporosis
  • Osteoporosis is a common disease caused by aging of the body.
  • the efficacy of NKG2D CAR-T on osteoporosis was verified.
  • the mouse femurs were scanned by micro CT and 3D reconstructions were analyzed by Calipers Analyze software.
  • Example 9 NKG2D CAR-T cells in the treatment of senescence-induced liver fibrosis
  • mice NKG2D CAR-T cells and control mock T cells were injected back into the tail vein at a dose of 1 ⁇ 10 6 per mouse. in a mouse model of carbon tetrachloride-induced liver fibrosis. After 20 days of NKG2D CAR-T treatment, mouse livers were collected for SA- ⁇ -gal and Masson staining analysis.
  • NKG2D CAR-T cells effectively eliminated senescent cells in mouse liver tissue and effectively reduced the degree of liver fibrosis (Figure 12A).
  • the RNA of mouse liver tissue was further extracted, and the expression changes of senescent cell markers P16 and NKG2D ligands were detected.
  • the results showed that after NKG2D CAR-T cells were treated, the expressions of P16 and MULT1 in mouse liver tissue were significantly down-regulated (Fig. 12B), indicating that NKG2D CAR -T efficiently clears senescent cells expressing NKG2D ligands in vivo.
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • NKG2D CAR-T cells can effectively treat liver fibrosis caused by accumulation of senescent cells.
  • Example 10 NKG2D CAR-T cells treat senescent cell accumulation leading to fatty liver
  • SASP Cellular senescence-associated secretory phenotype
  • NKG2D CAR-T cells can treat aging-related fatty liver by eliminating senescent cells.
  • NKG2D ligands were significantly upregulated in an in vitro model of cellular senescence induced by DNA damage, replication stress, and oncogene expression. Elevated expression of P16 Ink4a is closely related to naturally senescent cells in vivo. However, studies have shown that the expression of NKG2D ligands is not significantly upregulated in senescent cells induced by P16 Ink4a overexpression. Unlike in vitro, the extracellular portion of NKG2D ligands expressed by senescent cells in vivo is cleared from the cell surface by various mechanisms (such as metalloproteinases MMP, ERp5, and GRP78, etc.), thereby evading the immune surveillance that targets NKG2D ligands.
  • MMP metalloproteinases
  • ERp5 metalloproteinases
  • NKG2D CAR-T cells that recognize mouse NKG2D ligands were constructed using mouse sequences homologous to human NKG2D CAR sequences.
  • the mouse NKG2D CAR-T cells were intravenously infused into the X-ray radiation-induced mouse aging model, and the expression of senescent cell markers in the main organs of the mice was down-regulated, and the symptoms of muscle atrophy and osteoporosis were significantly relieved in the mice. , the athletic ability was significantly improved.
  • the constructed mouse NKG2D CAR-T cells were used to treat carbon tetrachloride-induced liver fibrosis and high-fat diet-induced fatty liver, and the mouse liver was collected 20 days later for analysis: Realtime PCR results showed that NKG2D ligands and The expression of aging markers was significantly down-regulated; pathological analysis showed that NKG2D CAR-T cells significantly reduced the degree of liver lesions.
  • the venous blood of mice was drawn to further evaluate the changes in the expression of liver markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The results showed that NKG2D CAR-T significantly improved liver function. The above results all prove that NKG2D CAR-T cells can effectively treat related diseases caused by the accumulation of senescent cells.
  • the experimental results of the present invention unexpectedly prove that although the extracellular part of NKG2D ligands of senescent cells in vivo can be removed by mechanisms such as enzymatic cleavage to form soluble NKG2D ligands, the therapeutic method targeting NKG2D ligands of the present invention, It can still effectively kill senescent cells in the body, and effectively remove accumulated senescent cells in the body, thereby significantly improving aging-related symptoms (such as senile osteoporosis, senile muscle atrophy, senile liver fibrosis, senile fatty liver, etc. ).
  • the NKG2D CAR-immune cells of the present invention are feasible and provide a new idea for the development of anti-aging and the treatment of age-related diseases.

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Abstract

提供了一种靶向NKG2D配体的CAR-免疫细胞用于制备药物的用途。该药物用于：(i)清除衰老细胞，其中，衰老细胞中的NKG2D配体较正常细胞上调2-20倍，较佳地4-15倍，更佳地10-20倍；(ii)延缓个体衰老；和/或(iii)预防和/或治疗老年性疾病。靶向NKG2D配体的CAR-免疫细胞能够特异性地清除NKG2D配体高表达的衰老细胞，并具有较高的体内安全性。

Description

NKG2D CAR-免疫细胞在抗衰老及老年相关疾病治疗中的应用 技术领域
本发明属于免疫细胞治疗或生物医药领域，具体涉及一种NKG2D CAR-免疫细胞在抗衰老及老年相关疾病治疗中的应用。
背景技术
目前世界各国均面临着严重的人口老龄化，数据显示到2050年约三分之一的中国人口年龄将超过60岁。人口老龄化会带来各种各样的老年相关疾病，现有的医疗水平、医治能力尚不足以面对如此大规模的医疗需求，因此如何让老年人健康生活成了一项巨大挑战。大多数老年疾病如老年痴呆、骨质疏松、糖尿病、动脉粥样硬化、肾功能障碍等与机体的衰老有关，而从病理学上看，促进年龄相关慢性疾病发生的一个重要过程就是细胞衰老。
实验数据表明，清除衰老小鼠体内p16 Ink4a阳性细胞可使衰老相关表型发生延迟，小鼠的寿命中位数提高24％，并且能够减轻年龄相关的多种器官功能退化，改善与年龄相关的脂肪营养不良、肝脏脂肪变性、心功能和骨质流失以及tau介导的神经退行性变。因此，清除衰老细胞是治疗和预防各种年龄相关疾病的一种重要手段。
目前，清除衰老细胞的方法主要集中在寻找能够选择性清除衰老细胞的小分子化合物方面，例如达沙替尼、槲皮素、ABT263等，但这些化合物或清除衰老细胞效果欠佳，或存在明显的毒副作用，不利于成药。例如ABT-263可引起短暂性血小板减少症和中性粒细胞减少症；用UBX0101处理患有骨关节炎的小鼠，不能够挽回小鼠骨关节炎的病理特征，只是一些分子表达水平的改变从而将创造有利于修复损伤的外环境。
经过嵌合抗原受体(Chimeric Antigen Receptor，CAR)修饰的免疫细胞，可特异性地识别细胞相关抗原，从而靶向杀伤目标细胞，具有广阔的临床应用前景。例如靶向CD19的CAR-T细胞对B细胞恶性肿瘤具有极佳的治疗效果，已被FDA批准上市。此外，大量经过CAR改造NK细胞目前正处于临床II期实验(NCT02742727，NCT02892695，NCT02944162，NCT03056339)。目前，也有研究表明表达HER2CAR的巨噬细胞具有特异性吞噬肿瘤细胞的作用。鉴于CAR免疫细胞杀伤靶细胞的特异性、高效性和持续性，理应对衰老细胞的清除也有极佳的效果，然而，在针对抗衰老的研究领域，还没有效果较佳的CAR-免疫细胞疗法。
因此，本领域迫切需要开发一种利用CAR-免疫细胞技术来特异性地、高效地清除衰老细胞的方法，从而实现抗衰老和治疗老年相关疾病(aging-related disease)。
发明内容
本发明的目的是提供一种利用CAR-免疫细胞技术特异性地、高效地清除衰老细胞的方法。
在本发明的第一方面，提供了一种靶向NKG2D配体的CAR-免疫细胞的用途，用于制备一药物，所述药物用于：
(i)清除衰老细胞，其中，所述衰老细胞中的NKG2D配体较正常细胞上调2-20倍，较佳地4-15倍，更佳地10-20倍；
(ii)延缓个体衰老；和/或
(iii)预防和/或治疗老年性疾病；
并且，所述靶向NKG2D配体的CAR-免疫细胞中表达靶向NKG2D配体的嵌合抗原受体，所述的嵌合抗原受体中的抗原结合结构域包括氨基酸序列如SEQ ID NO:1所示的多肽，或与SEQ ID NO:1所示序列具有80％以上相似度并且可结合NKG2D配体的多肽。
在另一优选例中，所述的CAR-免疫细胞选自下组：CAR-T细胞、CAR-NK细胞、CAR-巨噬细胞、或其组合。
在另一优选例中，所述的CAR-免疫细胞是CAR-T细胞。
在另一优选例中，所述的嵌合抗原受体具有如式I所示的结构，
L-NKG2D-H-TM-C-CD3ζ   (式I)
式中，
L为无或信号肽序列；
NKG2D为如权利要求1所述的NKG2D配体结合结构域序列；
H为无或CD8α铰链区；
TM为人CD8α跨膜结构域；
C为4-1BB或CD28共刺激信号分子；
CD3ζ为源于CD3ζ的胞浆信号传导序列；
各“-”独立地表示连接上述各元件的连接肽或肽键。
在另一优选例中，所述信号肽序列的氨基酸序列如SEQ ID NO:2所示。
在另一优选例中，所述CD8α铰链区的氨基酸序列如SEQ ID NO:3所示。
在另一优选例中，所述CD8α跨膜结构域的氨基酸序列如SEQ ID NO:4所示。
在另一优选例中，所述4-1BB共刺激信号分子的氨基酸序列如SEQ ID NO:5所示。
在另一优选例中，所述源于CD3ζ的胞浆信号传导序列的氨基酸序列如SEQ ID NO:6所示。
在另一优选例中，所述的嵌合抗原受体由强启动子EF1α驱动表达。
在另一优选例中，所述的衰老细胞的选自下组：肺细胞、脂肪细胞、肾细胞、肌肉细胞、骨细胞，或其组合。
在另一优选例中，所述的衰老细胞为人胚肺细胞IMR90。
在另一优选例中，所述的衰老细胞是自然的或人工诱导衰老的。
在另一优选例中，所述的人工诱导衰老方法包括：DNA损伤诱导衰老、过表达P16诱导衰老、致癌信号诱导衰老、端粒缩短诱导衰老，或其组合。
在另一优选例中，所述的人工诱导衰老的方法为用诱导剂DOX诱导衰老。
在另一优选例中，患有所述老年性疾病的个体中，含有衰老细胞，所述衰老细胞中的NKG2D配体较正常细胞上调2-20倍，较佳地4-15倍，更佳地10-20倍。
在另一优选例中，所述的老年性疾病选自下组：肌肉萎缩症、脂肪肝、心衰、动脉粥样硬化、糖尿病、心肌肥厚、骨质疏松、组织/器官纤维化、阿尔茨海默病、帕金森综合征、关节炎等细胞衰老导致的器官退行性疾病，或其组合。
优选地，所述的老年性疾病选自下组：老年性骨质疏松、老年性肌肉萎缩、老年性肝纤维化、老年性脂肪肝、或其组合。
在本发明的第二方面，提供了一种药物组合物，所述药物组合物包括：
(a)靶向NKG2D配体的CAR-免疫细胞，其中，所述靶向NKG2D配体的CAR-免疫细胞中表达靶向NKG2D配体的嵌合抗原受体，所述的嵌合抗原受体中的抗原结合结构域包括氨基酸序列如SEQ ID NO:1所示的多肽，或与SEQ ID NO:1所示的序列具有80％以上相似度并且可结合NKG2D配体的多肽；
(b)除(a)之外的其他抗衰老药物；和
(c)药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中，在组分(b)中，所述的其他抗衰老药物包括其他能够特异性地清除衰老细胞的药物。
在另一优选例中，组分(b)包括能够特异性清除衰老细胞的小分子化合物，优选地选自下组：达沙替尼、槲皮素、ABT263、ABT737、荜茇酰胺，或其组合。
在另一优选例中，所述药物组合物是液态的药物组合物。
在另一优选例中，所述药物组合物是注射剂。
在另一优选例中，所述药物组合物中，所述靶向NKG2D的CAR-免疫细胞的剂量为1×10 5-5×10 7个细胞/kg，较佳地为5×10 6-1×10 7个细胞/kg。
在本发明的第三方面，提供了一种延缓个体衰老，或预防和/或治疗老年性疾病的方法，包括步骤：向有所需要的对象施用靶向NKG2D配体的CAR-免疫细胞，其中，所述靶向NKG2D的CAR-免疫细胞中表达靶向NKG2D配体的嵌合抗原受体， 所述的嵌合抗原受体中的抗原结合结构域包括氨基酸序列如SEQ ID NO:1所示的多肽，或与SEQ ID NO:1所示的序列具有80％以上相似度并且可结合NKG2D配体的多肽。
在另一优选例中，所述的施用方式为静脉注射。
在本发明的第四方面，提供了一种CAR-免疫细胞，所述CAR-免疫细胞中表达一嵌合抗原受体，所述的嵌合抗原受体具有如式I所示的结构，
L-NKG2D-H-TM-C-CD3ζ   (式I)
式中，
L为无或信号肽序列；
NKG2D为如权利要求1所述的抗原结合结构域序列；
H为无或CD8α铰链区；
TM为人CD8α跨膜结构域；
C为4-1BB共刺激信号分子；
CD3ζ为源于CD3ζ的胞浆信号传导序列；
各“-”独立地表示连接上述各元件的连接肽或肽键。
在另一优选例中，所述的嵌合抗原受体具有如SEQ ID NO:12所示的氨基酸序列。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了衰老细胞模型的构建结果。
(A)IMR90细胞不同衰老模型SA-βgal染色表型观察：IMR90-Et为DNA损伤诱导衰老细胞模型，DNA损伤药物Etoposide(Et)处理IMR90细胞24h，更换新鲜培养基持续培养8天后染色；IMR90-p16为通过四环素诱导系统过表达p16蛋白的衰老细胞模型，1μg/ml dox处理8天后染色；IMR90-Kras为过表达癌基因Kras G12D衰老细胞模型，过表达Kras G12D 10天后染色；IMR90-Rep为复制型衰老细胞模型，体外连续传代37次后染色观察；SC指衰老细胞，CON为年轻对照组细胞；(B)A图所示衰老IMR90细胞SA-βgal染色阳性率统计；(C)A图所示衰老IMR90细胞p16相对表达量(n＝3)。统计计算三次测量的平均值和标准差，双尾T检验*P<0.05,**P<0.01,***P<0.001。
图2显示了衰老细胞模型NKG2DL表达检测结果。
(A)实时荧光定量PCR检测不同衰老模型中NKG2D配体RNA表达，IMR90-Et为DNA损伤诱导衰老细胞模型，IMR90-p16为四环素诱导系统过表达p16蛋白衰老细胞模型，IMR90-Kras为过表达癌基因Kras G12D衰老细胞模型，IMR90-Rep为复制型衰老细胞模型，计算三次测量的平均值和标准差，双尾T检验*P<0.05,**P<0.01,***P<0.001。(B)流式细胞术检测衰老细胞表面NKG2D配体表达，图中所示百分比为细胞阳性率。
图3显示了NKG2D CAR-T细胞对衰老细胞杀伤检测结果。
(A)MOCK和NKG2D CAR载体结构示意图；(B)MOCK和NKG2D CAR慢病毒感染T细胞72h后，流式检测CAR表达，MOI＝50，图中所示百分比为阳性率，NTD为未感染病毒T细胞；(C)(D)(E)MOCK和NKG2D CAR-T细胞分别与IMR90-P16、IMR90-Et和IMR90-Rep衰老细胞共培养10h后细胞表型观察和杀伤率分析。效靶比为2：1，实验重复3次，双尾T检验*P<0.05,**P<0.01,***P<0.001。(F)MOCK和NKG2D CAR-T细胞分别与IMR90-Et衰老细胞共培养后10h后，ELISA检测上清中穿孔素和颗粒酶含量，计算三次测量的平均值和标准差，双尾T检验*P<0.05,**P<0.01,***P<0.001。
图4显示了NKG2D-BBz CAR-T的安全性评估结果。
(A)用MICA抗体将HOrgN09PT02微阵列中的90个正常人组织染色。MICA表达明显升高的样品以红色标记。A1-A4：甲状腺；A5-A7：舌头；A8-A11：食管上皮；A12-B5：胃黏膜；B6-B7：十二指肠粘膜；B8-C1：空肠黏膜；C2-C4：回肠粘膜；C5-C9：附录；C10-D1：结肠黏膜；D2-D3：直肠粘膜；D4–D5：肝脏；D6-D7：胰腺；D8-D10：气管；D11-E3：肺；E4-E6：心肌；E7-E9：动脉；E10-F4：骨骼肌；F5-F7：皮肤；F8：精囊；F9-F11：前列腺；F12-G6：睾丸；G7-G10：膀胱；G11：延髓；G12-H1：端脑；H2-H3：脑中脑；H4：脑干；H5-H6：脾脏，比例尺：500μm。(B)未转导的T细胞和NKG2D-BBz CAR-T细胞的核型分析。
图5显示了猴NKG2D CAR-T的制备结果。
(A)猴T细胞培养，球状物为被激活的T细胞；(B)NKG2D CAR-T病毒感染猴子T细胞后，流式细胞仪检测CAR表达效率。(C)NKG2D CAR-T病毒感染猴子T细胞后，流式细胞仪CD4和CD8表达。
图6显示了NKG2D CAR-T回输后猴基础体征监测结果。
(A)NKG2D CAR-T回输后realtime PCR检测血液中CAR-T的拷贝数；(B)(C)NKG2D CAR-T回输后猴体温和体重监测。(D)NKG2D CAR-T回输后，Elisa检测血清中细胞因子浓度。
图7显示了NKG2D CAR-T回输后猴生化指标检测结果。
(A)回输细胞前后猕猴/食蟹猴血常规各项指标(WBC:白细胞；Lymphocyte:淋巴细胞；Monocyte:单核细胞；Granulocyte:粒细胞；RBC:红细胞；PLT:血小板) 变化情况。(B、C、D)回输细胞前后猕猴/食蟹猴血清中与肝脏(B)、肾脏(C)和心脏(D)有关的分子标志的变化情况。
图8显示了NKG2D CAR-T体内清除衰老细胞的结果。
NKG2D CAR-T回输90天后，实时荧光定量PCR检测猴脂肪、肌肉、肝脏和肾脏组织中衰老细胞标志物P16、P14、P21、LGFBP2、IL6和MMP3表达变化，18SRNA作为内参。
图9显示了衰老小鼠模型的建立。
(A)小鼠辐照后3个月和11个月毛发状态；(B)Realtime PCR检测辐照后3个月和11个月小鼠腹股沟脂肪(IAT)、骨骼肌(SKM)、肾、肺、肝、心脏和皮肤P16表达，18S RNA作为内参。(B)(C)Realtime PCR检测辐照后3个月和11个月小鼠腹股沟脂肪(IAT)、骨骼肌(SKM)、肾、肺、肝、心脏和皮肤NKG2D配体MULT-1和RAE-1表达，18S RNA作为内参。
图10显示了NKG2D CAR-T治疗衰老引起的肌肉萎缩症的结果。
(A)MOCK和小鼠NKG2D CAR逆转录病毒感染小鼠脾T细胞后，流式检测阳性率；(B)MOCK和小鼠NKG2D CAR-T静脉回输衰老小鼠4周后，提取骨骼肌RNA，realtime PCR检测NKG2D配体RAE-1，MULT-1以及H60a表达，以18S RNA作为内参；(C,D)MOCK和小鼠NKG2D CAR-T静脉回输衰老小鼠4周后，提取骨骼肌RNA，realtime PCR检测细胞周期相关分子P16、P21以及衰老相关分泌表型分子IL-6,PAI-1和MMP3表达以18S RNA作为内参；(E)MOCK和小鼠NKG2D CAR-T静脉回输衰老小鼠6个月后，苏木素伊红染色观察肌肉纤维表型，并测量面积。(F)MOCK和小鼠NKG2D CAR-T静脉回输衰老小鼠6个月后，利用握力计对小鼠四肢握力进行检测。(G)MOCK和小鼠NKG2D CAR-T静脉回输衰老小鼠6个月后，利用转棒对小鼠最大步行速度进行检测，n＝5，双尾T检验*P<0.05,**P<0.01,***P<0.001。
图11显示了NKG2D CAR-T治疗衰老引起的骨质疏松症的结果。
(A)MOCK和小鼠NKG2D CAR-T静脉回输衰老小鼠6个月后，Quantum GX microCT成像系统检测小鼠股骨远端骨小梁数量。(B)MOCK和小鼠NKG2D CAR-T静脉回输衰老小鼠6个月后，Quantum GX microCT成像系统分析小鼠股骨密度，n＝5，双尾T检验*P<0.05。
图12显示了NKG2D CAR-T细胞治疗衰老导致的肝纤维化的结果。
(A)NKG2D CAR-T治疗20天后，采集小鼠肝脏进行SA-β-gal及Masson染色分析小鼠肝脏中衰老细胞数量和肝纤维化程度；(B)NKG2D CAR-T治疗20天后，realtime PCR检测肝脏中衰老标记物p16及NKG2D配体MULT1表达；(C,D)CAR-T治疗20天后，取小鼠静脉血检测血清谷草转氨酶及谷丙转氨酶表达。双尾T检验*P<0.05,**P<0.01,***P<0.001。
图13显示了NKG2D CAR-T通过清除衰老细胞治疗脂肪肝的结果。
(A)NKG2D CAR-T治疗20天后，采集小鼠肝脏进行SA-β-gal及Masson染色分析小鼠肝脏中衰老细胞数量和肝纤维化程度；(B)NKG2D CAR-T治疗20天后，realtime PCR检测肝脏中衰老标记物p16及NKG2D配体MULT1表达；双尾T检验*P<0.05,**P<0.01,***P<0.001。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发了一种利用CAR-免疫细胞(如CAR-T细胞)技术高特异性和高效地清除受试者中衰老细胞和治疗老年相关疾病的方法。在此基础上完成了本发明。
具体地，本发明构建了一系列不同细胞系的衰老细胞模型，并在利用衰老模型确认了NKG2D配体(NKG2DL)的高表达，然后以NKG2D胞外序列为靶点识别域，4-1BB和CD3ζ为共刺激信号构建了第二代CAR-T细胞。实验结果表明，NKG2D CAR-T细胞在体外可以高效地杀死衰老细胞，释放高浓度的细胞因子、穿孔素和颗粒酶。此外，慢病毒介导的CAR表达对T细胞的增殖、凋亡和基因组稳定性无显著作用。
此外，为了进一步验证NKG2D CAR-T细胞的安全性，本发明分离并制备了猴NKG2D CAR-T细胞，然后以一定剂量(如1x10 6/kg)自体回输到猴体内。在回输细胞前后对猴体温、体重和细胞因子进行测量；结果表明猴均未出现发热现象，无腹泻症状产生、体重无异常变化、血清中细胞因子在回输NKG2D CAR-T细胞前后无显著异常变化。在回输细胞前后对猴的血常规和生化检测结果表明，血常规以及生化指标均在正常范围之内。这说明NKG2D CAR-T细胞未对猴的器官包括心脏、肾脏和肝脏产生毒副作用。取治疗前后猴肌肉、脂肪、肝和肾组织进行分析，结果表明，本发明的CAR-T可显著降低这些组织中NKG2D配体和衰老标志物表达。
进一步的体内实验表明，本发明的CAR免疫细胞可在体内有效清除累积的衰老细胞，进而显著改善衰老相关症状(如老年性骨质疏松、老年性肌肉萎缩、老年性肝纤维化、老年性脂肪肝等)。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。
如本文所用，术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
如本文所用，术语“给予”、“施用”可互换使用，是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者，包括静脉内，肌内，皮下，腹膜内，脊髓或其它肠胃外给药途径，例如通过注射或输注。
嵌合抗原受体(CAR)
嵌合免疫抗原受体(Chimeric Antigen Receptors，CARs)由胞外抗原识别结构域，通常是scFv(single-chain variable Fragment)、跨膜区以及胞内共刺激信号结构域组成。CARs的设计经历了以下过程：第一代CAR只有一个胞内信号组份CD3ζ或者FcγRI分子，由于胞内只有一个活化结构域，因此它只能引起短暂的T细胞增殖和较少的细胞因子分泌，而并不能提供长时间的T细胞增殖信号和持续的体内抗肿瘤效应，所以并没有取得很好地临床疗效。第二代CARs在原有结构基础上引入一个共刺激分子，如CD28、4-1BB、OX40、ICOS，与一代CARs相比功能有很大提高，进一步加强CAR-T细胞的持续性和对肿瘤细胞的杀伤能力。在二代CARs基础上串联一些新的免疫共刺激分子如CD27、CD134，发展成为三代和四代CARs。
CARs的胞外段可识别一个或多个特异性的抗原决定簇(epitopes)，随后通过胞内结构域转导该信号，引起细胞的活化增殖、细胞溶解毒性和分泌细胞因子，进而清除靶细胞。首先分离病人自体或异体(健康供者)的PBMC，激活并进行基因改造产生CAR的免疫细胞，随后注入病人体内，以非MHC限制的方式通过直接识别肿瘤细胞表面抗原对其进行特异性杀伤。
具体地，本发明的嵌合抗原受体(CAR)包括细胞外结构域、跨膜结构域、和细胞内结构域。胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激信号传导区和/或ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子，而不是抗原受体或它们的配体。
在CAR的胞外结构域和跨膜结构域之间，或在CAR的胞浆结构域和跨膜结构域之间，可并入接头。
如本文所用，术语“接头”、“铰链区”可互换使用，通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸，优选地2至100个氨基酸和最优选地3至50个氨基酸。
本发明的CAR当在免疫细胞中表达时，能够基于抗原结合特异性进行抗原识别。当其结合肿瘤细胞上关联抗原时，导致肿瘤细胞死亡，患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和/或ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与CD28共刺激信号分子、4-1BB共刺激信号分子和CD3ζ信号结构域组合的细胞内结构域融合。
如本文所用，本发明嵌合抗原受体的基础结构包括：NKG2D抗原结合结构域、胞外铰链区、跨膜区和胞内信号区。
NK细胞是机体清除衰老细胞的主要免疫细胞，NKG2D-NKG2D配体轴是其识别靶细胞的主要活化通路。因此与其他CAR-免疫细胞相比，以NKG2D胞外域为识别位点 构建的CAR-免疫细胞理应具有更可靠的安全性。此外，NKG2D可识别MICA、MICB、ULBP1、ULBP2和ULBP3等多种配体，基于其构建的CAR-T细胞清除衰老细胞更具有广谱性。
如本文所用，术语“CAR-免疫细胞”是指表达本发明的嵌合抗原受体的免疫细胞。特别值得说明的是，本发明的CAR-免疫细胞可以是在生物体内行使效应功能的不同的免疫细胞，例如T细胞、NK细胞、巨噬细胞等。CAR-T细胞是目前研究最为彻底和广泛的免疫疗法，多款产品已经被批准用于肿瘤治疗。相较于CAR-T细胞，CAR-NK细胞具有较低的细胞因子释放，并且还可以通过NK细胞受体本身来消除肿瘤细胞。CAR-巨噬细胞可通过表达促炎细胞因子和趋化因子，将附近M2巨噬细胞转化为M1，上调抗原呈递机制并激活自身免疫系统，具有更高的安全性。
在本发明的一个优选例中，选择NKG2D的胞外区作为本发明CAR中靶向NKG2D的抗原结合结构域。
在一个优选的实施方式中，所述抗原结合结构域的氨基酸序列如SEQ ID NO:1所示，可以高效的与NKG2D配体分子结合。
Figure PCTCN2021135151-appb-000001
在本发明中，靶向NKG2D配体的抗原结合结构域还包括所述序列的保守性变异体，指与本发明抗原结合结构域的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。在本发明中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。在本发明中，所述添加、缺失、修饰和/或取代的氨基酸数量通常是1、2、3、4或5个，较佳地为1-3个，更佳地为1-2个，最佳地为1个。
对于绞链区和跨膜区(跨膜结构域)，CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中，使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中，可选择跨膜结构域，或通过氨基酸置换进行修饰，以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域，从而最小化与受体复合物的其他成员的相互作用。
优选地，本发明的CAR构建物具有以下结构：信号肽-靶向NKG2D配体的抗原结合结构域-CD8α铰链区-CD8αTM-41BB-4-1BB共刺激信号分子-CD3ζ胞浆信号传导序列。
在一个实施方式中，所述信号肽的氨基酸序列如SEQ ID NO:2所示。
Figure PCTCN2021135151-appb-000002
在一个实施方式中，所述CD8α铰链区的氨基酸序列如SEQ ID NO:3所示。
Figure PCTCN2021135151-appb-000003
在一个实施方式中，所述CD8α跨膜结构域的氨基酸序列如SEQ ID NO:4所示。
Figure PCTCN2021135151-appb-000004
在一个实施方式中，所述4-1BB共刺激信号分子的氨基酸序列如SEQ ID NO:5所示。
Figure PCTCN2021135151-appb-000005
在在一个实施方式中，所述源于CD3ζ的胞浆信号传导序列的氨基酸序列如SEQ ID NO:6所示。
Figure PCTCN2021135151-appb-000006
在一个优选的实施方式中，本发明CAR的氨基酸序列如SEQ ID NO:12所示。
Figure PCTCN2021135151-appb-000007
药物组合物
本发明提供了一种含有本发明第一方面所述的靶向NKG2D配体的CAR-免疫细胞、其他抗衰老药物，以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方式中，所述药物组合物为液态制剂。优选地，所述制剂为注射剂。优选地，所述制剂中所述CAR-免疫细胞的剂量为1×10 5-5×10 7个细胞/kg，更优地5×10 5-1×10 7个细胞/kg。
在一个实施方式中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。
本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明提供了本发明的靶向NKG2D的CAR-免疫细胞和本发明的药物组合物的用途，用于预防和/或治疗老年性疾病。
并且，本发明还提供了本发明的靶向NKG2D的CAR-免疫细胞和本发明的药物 组合物的用途，用于制备一药物，所述药物用于(i)清除衰老细胞，其中，NKG2D配体较正常细胞上调2-20倍，较佳地4-15倍，更佳地10-20倍；(ii)延缓个体衰老；和/或(iii)预防和/或治疗老年性疾病。
其中，老年性疾病或衰老相关疾病包括(但并不限于)以下因细胞衰老导致的器官退行性疾病：肌肉萎缩症、脂肪肝、心衰、动脉粥样硬化、糖尿病、心肌肥厚、骨质疏松、组织/器官纤维化、阿尔茨海默病、帕金森综合征、关节炎、或其他因细胞衰老导致的器官退行性疾病，或其组合。
本发明的通用型CAR-免疫细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
对于离体免疫细胞制备，以下中的至少一项在将细胞施用进入哺乳动物前在体外发生：i)扩增细胞，ii)将编码CAR的核酸引入细胞，和/或iii)冷冻保存细胞。
离体细胞处理程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体对上述细胞进行基因修饰(即体外转导或转染)。CAR-修饰的细胞可被施用于哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，CAR-修饰的细胞相对于接受者可为自体，也可为同种异基因的、同基因的(syngeneic)。
除了就离体免疫细胞而言使用基于细胞的疫苗之外，本发明也提供了用于体内以增强针对患者中靶向抗原的免疫应答的组合物和方法。
本发明提供了治疗老年性疾病的方法，其包括施用给需要的对象以有效量的本发明的CAR-免疫细胞。
本发明的CAR-免疫细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞，与一种或多种药学或临床上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的组合物优选配制用于静脉内施用。
本发明的药物组合物可以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症的特征、疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗衰老有效量”、“衰老性疾病-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、衰老组织大小、衰老程度和病症的个体差异。可通常指出： 包括本文描述的T细胞的药物组合物可以以10 4至10 9个细胞/kg体重的剂量，优选10 5至10 7个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，New Eng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象，治疗方案由医学领域技术人员确定。
对象组合物的施用可以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内、胸膜内施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.静脉内注射施用。T细胞的组合物可被直接注入衰老组织或由衰老引起的病理组织或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩增至治疗性水平的方法活化和扩增细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：达沙替尼、槲皮素、ABT263、ABT737、荜茇酰胺。
在一些实施方式中，在移植后，对象接受本发明的扩增的免疫细胞的注入。在一个额外的实施方式中，扩增的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 6个至1×10 10个本发明的CAR-免疫细胞，通过例如静脉回输的方式，施用于患者。
本发明的主要优点包括：
1)本发明首次开发了一种能够特异性地清除NKG2D配体高表达的衰老细胞的CAR-免疫细胞。
2)打破了体内衰老细胞NKG2D配体被各种机制清除而不能作为靶点的顾虑，本发明首次靶向NKG2D配体、将NKG2D CAR-T细胞用于在体外和体内清除衰老细胞，并可治疗老年相关疾病。
3)慢病毒介导的本发明的CAR表达对T细胞的增殖、凋亡和基因组稳定性无显著作用。
4)本发明的CAR-免疫细胞具有较高的安全性。在猴的体内试验结果证实了其对猴的器官包括心脏、肾脏和肝脏均未产生毒副作用。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通 常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实验方法
1.衰老细胞模型构建
(1)Tet-on系统过表达p16蛋白的细胞衰老模型构建
(i)将3×10 5个细胞分别铺于10cm皿，第二天贴壁后细胞密度在20％左右；
(ii)待细胞贴壁后，将Tet-on系统过表达p16蛋白的慢病毒以50-100的感染复数(multiplicity of infection,MOI)分别感染细胞，并按照1:1000的比例加入原液浓度为8mg/mL的polybrene以提高感染效率；
(iii)24h后以相同的病毒量进行二次感染；
(iv)感染病毒4天后，加入终浓度为3μg/mL的嘌呤霉素筛选；
(v)将构建好的过表达p16蛋白的细胞传于孔板或培养皿中，24h贴壁后加入1μg/mL的dox诱导p16蛋白的表达；
(vi)诱导8天后，用SA-βgal染色试剂盒(CS0030,Sigma)对细胞进行衰老染色，结果显示90％以上细胞呈阳性，表明此时细胞已衰老。
(2)癌基因KRAS G12D诱导衰老细胞模型的构建
(i)将细胞铺于10cm皿，使贴壁后的密度在20％-30％左右；
(ii)第二天细胞贴壁后进行病毒感染，每10cm皿以MOI 50-100加入pTomo-Kras G12D-EGFP病毒(Kras病毒)，并加入polybrene(终浓度8μg/mL)以提高感染效率；
(iii)细胞感染病毒的大约5天后基本停止生长，继续培养5天后，用SA-βgal染色试剂盒(CS0030,Sigma)对细胞进行衰老染色，结果显示90％以上细胞呈阳性，表明此时细胞已衰老。
(3)DNA损伤药物诱导细胞衰老模型的构建
(i)铺细胞于10cm皿中，使贴壁后的密度在50％左右
(ii)24小时后加入Etoposide(sigma,E1383)，使其终浓度为50μM；
(iii)36h后更换为新鲜培养基；
(iv)继续培养细胞，其间每三天更换一次培养基，8天后细胞出现衰老表型。用SA-βgal染色试剂盒(CS0030,Sigma)对细胞进行衰老鉴定。
(4)自然复制衰老模型的构建
(i)复苏非永生化人胚肺成纤维细胞HEL1，WI38或IMR90培养于10cm的细胞培养皿中；
(ii)当培养皿中细胞密度达到95％时进行传代，细胞会随着传代次数的增加， 增殖速度减慢，形态变大；
(iii)HEL1细胞代数达到P44，IMR90细胞达到P37PDL52，WI38细胞达到P38PDL52时，细胞几乎完全停止增殖，SA-βgal衰老染色呈阳性。
2.NKG2D配体表达检测
(1)NKG2D配体转录水平表达检测
(i)按上述方法准备衰老细胞后，在10cm皿中依据细胞密度加入1～2mL Trizol，冰上放置5min，枪头吹打混匀；
(ii)吸取1mL各孔裂解液加入1.5mL EP管中，加入氯仿200μL，用力振摇15s；室温放置5min离心(4℃，12000g，15min)；
(iii)在新的EP管中加入450μL的异丙醇；
(iv)小心吸取离心后的上层无色液体，加入含异丙醇的EP管中，混匀，室温孵育10min，离心(4℃，12000g，10min)；
(v)弃上清，加入1mL RNase free水配置的75％乙醇洗RNA，离心(4℃，7500g，5min)；
(vi)小心去上清，倒扣5min晾干，用枪头吸去管壁的液体；
(vii)加入30μL RNase Free水溶解，溶解后立即放在冰上，测定浓度
(viii)以提取的RNA为模板，使用Thermo Scientific RevertAidTM First Strand cDNA Synthesis Kit试剂盒将2μg RNA反转录为cDNA。反应体系如下所示：
Figure PCTCN2021135151-appb-000008
(ix)按以上体系将反应物加入PCR管中，PCR仪内65℃，5min，立即放于冰上，再向管中加入以下成份：
Figure PCTCN2021135151-appb-000009
轻轻混匀并瞬时离心，置于PCR仪中进行如下反应：25℃，5min；42℃，1h； 70℃，5min；
(x)荧光实时定量PCR检测NKG2D配体表达，具体操作根据Thermo powerup TM SYBR Green Master Mix(A25742)试剂盒说明书进行，程序：50℃，2min；95℃，2min；95℃，15s(40个循环)；60℃，1min(40个循环)；12℃，forever；
(xi)导出Excel格式的数据，计算目的基因的相对表达量。
(2)NKG2D配体膜表面表达检测
(i)胰酶消化并收集10cm皿的衰老组和正常组细胞，PBS洗细胞一次；
(ii)700μL含1％FBS的PBS重悬细胞(轻轻吹打混匀，以防产生过多气泡)；
(iii)将细胞平均分配到7个1.5mL EP中，每管含100μL PBS；
(iv)按1:50的比例稀释抗体；
(v)每管细胞中分别加入100μL抗体；
(vi)冰上避光孵育至少30min，每隔十分钟轻轻涡旋振荡一次；
(vii)加入1mL含1％FBS的PBS洗细胞2次，800g 5min；
(viii)加入200μL含1％FBS的PBS重悬细胞，流式检测NKG2D配体表达。
3.慢病毒表达载体构建
本发明采用了第二代CAR结构。以人NKG2D胞外域链为识别片段，由强启动子CMV驱动其表达，信号肽(SP)和NKG2D序列后依次连入人CD8α铰链区(CD8Hinge)、人CD8α跨膜区(CD8TM)，4-1BB共刺激结构域以及CD3ζ激活结构域。本发明以不含NKG2D胞外域序列的Mock载体为阴性对照。NKG2D CAR载体结构如图3A所示。CD8SP-NKG2D EC-CD8Hinge and TM-41BB-CD3ζ由北京擎科生物科技有限公司合成后，通过XbaI和SalI酶切位点克隆到pTomo慢病毒载体中。
Figure PCTCN2021135151-appb-000010
CD8铰链区和跨膜区(SEQ ID NO:9)：
Figure PCTCN2021135151-appb-000011
Figure PCTCN2021135151-appb-000012
4-1BB共刺激信号分子(SEQ ID NO:10)：
Figure PCTCN2021135151-appb-000013
CD3ζ胞内信号传导结构域(SEQ ID NO:11)：
Figure PCTCN2021135151-appb-000014
4.病毒包装
(i)质粒准备：慢病毒包装的核心质粒pCMV△8.9和pMD2.G均用QIAGEN EndoFree Plasmid Maxi Kit进行大抽，慢病毒表达核心质粒pTomo-NKG2D-CAR-T2A-mKATE，pTomo-Mock CAR用QIAGEN Plasmid Midi Kit进行中抽。
(ii)293T细胞准备：病毒包装前24h，丰度90％以上的293T细胞，按1：2.5的比例进行细胞传代，每15cm培养皿中加入25mL含有10％血清，不含双抗的DMEM培养基培养。包装前4～6h，对皿中的细胞进行“半换液”处理，即吸掉15mL培养基上清，并向其中补加15mL仅含10％血清，无双抗的DMEM培养基，以将细胞状态调整至最佳。当皿中细胞密度达到80％～90％时，即可进行病毒包装。
(iii)质粒转染过程：准备两个15mL的离心管，向其中分别加入2mL无血清无双抗的DMEM基础培养基。其中一管加入20μg核心质粒、10μg pCMV△8.9、4μg pMD2.G(核心质粒：pCMV8.9：pMD2.G＝5:2.5:1)混匀，记为A液；另一管加入68μL的Lipo6000转染试剂，与培养基混匀，并静置5min，计为B液；A液、B液混匀，室温静置20min。
(a)移液枪吸去14mL 293T培养皿中的上清(余11mL培养基)，将上述4mL混合液，轻轻滴加入293T细胞中，于37℃细胞培养箱中培养。
(b)8h后将皿中293T的培养基更换为16mL含5％血清，1％双抗的DMEM培养基。
(iv)收病毒：包装48h后收集15mL含病毒的培养基上清，再补加15m含5％血清，1％双抗的DMEM培养基，包装72h后再次收集病毒上清；
(v)浓缩病毒：将两次收集到的培养基上清在4℃条件下以3000rmp转速离心15min；0.45μm滤膜过滤，取30mL过滤后的病毒液于超速离心管中，并移液枪将5mL 20％蔗糖溶液匀速、缓慢地加入离心管底部；25000rmp 4℃条件下低温超速离 心2.5h；
(vi)溶解病毒：倒掉上清，将离心管倒扣在喷有酒精的纸巾上，室温干燥5min，吸掉离心管管壁剩余的液滴(或用喷有酒精的纸巾擦干液滴)；每管中加入适当体积的0.1％BSA的PBS于4℃过夜溶解病毒。
(vii)分装及保存病毒：过夜溶解后的病毒悬液于冰上分装成10μL每份，置于1.5mL EP管中，-80℃条件下储存。
(viii)QPCR法测定病毒滴度
取3μL病毒原液，梯度稀释为10倍，100倍，1000倍，10000倍，作为qPCR模板，每一梯度设置3个重复；参照
Figure PCTCN2021135151-appb-000015
Select Master Mix产品说明书，病毒滴度测定的反应体系及反应程序如下：
反应体系：
Figure PCTCN2021135151-appb-000016
反应程序：
50℃，2min；
95℃，2min；
95℃，15sec；
60℃，1min；
第3和第4步共40个循环。
5.人体外周血T淋巴细胞分离
(i)将人体外周血转移至50mL离心管中，添加RosetteSep TM Cocktail至血液中(50μL/mL血液)；
(ii)充分混匀后，室温孵育20min；
(iii)配制稀释液：将1640培养基与1*PBS按体积比1:2混合；
(iv)准备梯度离心管，加入15mL梯度分离液Ficol Lymphoprep；
(v)将稀释液与孵育血样按1:1混合；
(vi)将稀释血样轻柔转移至分离液上，1200g离心20min；
(vii)将离心后的全部上清液快速转移至新的离心管；
(viii)用25mL的稀释液与上清混合均匀，300g离心10min；
(ix)重复操作步骤(viii)；
(x)加入2mL T细胞完全培养基重悬T细胞，计数、分装、冻存。
6.猕猴/食蟹猴T细胞分离
(i)配制稀释液：含2％FBS的1xPBS；
(ii)向15ml离心管中加入采集的5ml猴子全血，加入等量的稀释液并缓慢混匀；
(iii)准备梯度离心管，加入15ml Ficol Lymphoprep离心液；
(iv)将移液器调至0档，而后把稀释的血液样本轻柔转移至分离液上，离心10min，离心速度为1200g；
(v)将上层含有淋巴细胞层的液体迅速转移到干净的50ml离心管中并加入等量稀释的液体轻轻混匀，离心10min，离心速度为500g；
(vi)重复操作步骤(v)，取一定量的T细胞培养基重悬细胞，计数；
(vii)将细胞直接用非人灵长类T细胞培养基培养，25ul磁珠(非人灵长类)/1x10 6细胞；
(viii)非人灵长类用T细胞培养基配制:
向1640基础培养基中加入10％胎牛血清，1％P/S，1％谷氨酸盐添加剂，1％巯基乙醇以及0.02％IL2，混匀。
(ix)非人灵长类T细胞培养磁珠激活：
a.准备1xPBS：将1ml 1xBPS分装到1.5ml EP管中并存放于4℃冰箱预冷；
b.将CD2、CD3、CD28和磁珠以1：1：1：1混匀；
c.将预冷的1×PBS和步骤b中混匀的液体1：1混匀，放于4℃静音混悬仪上孵育2h后即可使用。
7.T细胞纯度分析
(i)用200ul 1xPBS(含2％FBS)重悬分离的T细胞；
(ii)向重悬的细胞中加入2ul CD3抗体，混匀，放置于冰上孵育30min，期间每隔10min涡旋一次细胞，以500g的速度离心5min，弃上清；
(iii)加入1ml 1xPBS(含2％FBS)重悬细胞，500g离心5min；
(iv)重复步骤(iii)；
(v)加入200ul 1xPBS(含2％FBS)重悬细胞；
(vi)流式仪检测CD3阳性的T细胞。
8.T细胞杀伤实验
(1)NKG2D CAR-T细胞构建
(i)复苏T细胞，加入CD3/CD28磁珠以及含IL-2的T细胞培养基培养T细胞3天以激活T细胞；
(ii)取10 ^6个T细胞，于96孔板中，以MOI＝100加入相应体积的NKG2D CAR病毒，同时加入polybrene(终浓度8μg/mL)；
(iii)收集感染病毒后的T细胞于1.5mL离心管中，500g离心3min，弃上清；
(iv)500μL培养基将T细胞重悬，并转移至24孔板中培养，孔中培养基的总体积为1.5mL；
(v)培养4天后，流式检测荧光表达情况。
(2)CAR-T细胞和衰老细胞共培养
(i)提前8～9天准备衰老细胞于96孔板中，共培养时衰老细胞密度约为5000个/孔，正常组细胞于24h前以5000/孔铺板于96孔板中；
(ii)300g离心5min收集CAR-T细胞，用1mL的T细胞完全培养基重悬CAR-T细胞，抽取10μL细胞悬液，用0.4％的台盼蓝染色后计数活细胞；
(iii)接种CAR-T细胞：计数后调整CAR-T细胞密度，按效靶比1：2，1：1和2：1的比例，将CAR-T细胞轻轻加入靶细胞所在的96孔板中，每孔含150μL T细胞培养基。
(iv)CAR-T细胞与靶细胞在5％CO 2，37℃条件下共培养6～12h，直至出现衰老组细胞杀伤表型。
(3)CAR-T细胞杀伤效率定量
(i)共培养6～12h出现杀伤表型后，此时移除靶细胞中的培养基，并用PBS洗细胞一次；
(ii)用8道移液器吸取100μL 95％乙醇加入靶细胞中进行固定；
(iii)3min后移去95％酒精，按照1:1000比例稀释DAPI原液(浓度：1mg/mL)，避光染色2min；
(iv)移除DAPI，每孔加入200μL PBS；
(v)将96孔板放入高内涵仪器中，进行拍照计数统计，每孔选取中间9个视野，同一视野中使用IL-10，DAPI通道各拍摄一张照片；
(vi)使用高内涵仪器分析软件中的overlay模块将IL-10，DAPI通道照片叠加并导出；
(vii)用ImageJ软件对导出的照片进行计数，统计存活的靶细胞的个数；
(viii)杀伤效率＝(Blank组靶细胞数量-共培养组靶细胞数量)/Blank组靶细胞数量*100％
9.穿孔素、颗粒酶含量测定
本发明中使用ELISA法测定穿孔素与颗粒酶含量，实验所用样品为T细胞与DNA损伤诱导衰老IMR90细胞共培养12h(穿孔素)或24h(颗粒酶)的细胞上清。收集上清后，以2000g的转速离心10min，暂存于-80℃。按照Abcam公司的Perforin(PRF1)Human ELISA试剂盒(ab46068)以及Human Granzyme B SimpleStep ELISA试剂盒(ab235635)的使用说明分别配制标准品，绘制标准曲线，并对样品中穿孔素与颗粒酶的含量分别进行测定。后续使用GraphPad Prism软件进行数据分析。
10.感染病毒前后CD4/CD8阳性T细胞检测
(i)病毒感染猕猴/食蟹猴T细胞3天以后，将细胞吹散并收集液体到离心管中，将收集的细胞悬液离心，收集沉淀下来的CAR-T细胞(500g/5min)；
(ii)用4℃左右的1×PBS(含2％FBS)将细胞洗涤2次；
(iii)用400ul 1×PBS(含2％FBS)重悬细胞，每组细胞分成等量的两份，按1：200分别向CAR-T细胞悬液中加入CD4和CD8的抗体，在冰上避光孵育1h；
(iv)再次用1×PBS(含2％FBS)清洗孵育过抗体的T细胞2次；
(v)而后用适量的1×PBS(含2％FBS)重悬CAR-T细胞，并将其转移至流式管中，流式检测CD4/CD8阳性细胞率；
11.NKG2D CAR-T细胞回输到猕猴/食蟹猴体内
(i)收集感染病毒72h后的猴子T细胞(即CAR-T细胞)，去掉细胞培养基中的磁珠(将细胞悬液转移到无菌流式管中，而后将流式管放到磁体中放置1min，将流式管中的液体迅速加入到15ml大小体积的离心管中)，在500g转速的离心机中离心5min并收集离心下来的细胞沉淀；
(ii)用4℃左右的1xPBS清洗细胞，500g离心5min；
(iii)重复步骤2)；
(iv)将离心下来的细胞用1ml的1640基础培养基重悬；
(v)将重悬的细胞用1ml注射器回输入猴子大腿内测静脉中。
12.猕猴/食蟹猴血常规和生化指标检测
(i)血常规检测
a)取200ul新鲜血液到抗凝管中，轻晃摇匀；
b)上机(HEMAVET 950动物五分类血球仪)检测。
(ii)生化指标检测
a)采集1ml血液到无抗凝剂的采血管中，常温静置2h后离心，取上层血清400ul上机(罗氏I400全自动生化仪)检测。
13.猕猴/食蟹猴血清细胞因子检测
收集猕猴/食蟹猴在回输细胞前后不同时间段的血清，送优宁维公司(上海)进行多细胞因子检测。
14.逆转录病毒表达载体构建
与人NKG2D CAR序列同源的小鼠序列，包括鼠源NKG2D胞外域(NKG2D-EC)、CD8α铰链区(CD8Hinge)、CD8α跨膜区(CD8TM)，4-1BB共刺激结构域以及CD3ζ激活结构域，交由北京擎科生物科技有限公司合成后，通过EcoRI和SalI酶切位点克隆到MSCV-IRES-GFP逆转录病毒载体中。实验以不含NKG2D胞外域序列的Mock载体为阴性对照。
NKG2D EC(SEQ ID NO:13)：
Figure PCTCN2021135151-appb-000017
CD8铰链区和跨膜区(SEQ ID NO:14)：
Figure PCTCN2021135151-appb-000018
4-1BB共刺激信号分子(SEQ ID NO:15)：
Figure PCTCN2021135151-appb-000019
CD3ζ胞内信号传导结构域(SEQ ID NO:16)：
Figure PCTCN2021135151-appb-000020
15.逆转录病毒包装
(1)质粒准备：逆转录病毒质粒MSCV-NKG2D、MSCV-MOCK，以及辅助质粒pCL-ECO均用QIAGEN EndoFree Plasmid Maxi Kit(QIAGEN,12362)进行抽提。
(2)293T细胞准备：病毒包装前24h，丰度90％以上的293T细胞，按1：3的比例进行细胞传代，每15cm培养皿中加入25mL含有10％血清的DMEM培养基培 养。当培养皿中细胞密度达到80％～90％时进行病毒包装。
(3)病毒包装：逆转录病毒质粒和辅助质粒pCL-ECO按照等质量混合后，根据碧云天lipo6000(Beyotime,C0526)说明书进行转染。收取转染后48小时和72小时的培养基上清，在4℃条件下以3000rmp转速离心15min后0.45μm滤膜过滤细胞碎片；取过滤后的病毒液于25000rpm、4℃条件下低温超速离心2.5h。用含0.1％BSA的PBS 4℃过夜溶解病毒。取5μL病毒原液，梯度稀释为10倍，100倍，1000倍，10000倍，分别感染3T3细胞，48小时后流式检测细胞阳性率，计算病毒滴度。
16.小鼠CAR-T细胞制备
(1)按照小鼠T细胞分离试剂盒说明书(stem cell,19851)操作,分离T细胞；(2)取1x10 6个分离的T细胞，CD3抗体避光4℃孵育1小时后流式检测阳性率；
(3)剩余T细胞用小鼠CD3/CD28磁珠(life technologies,11452D)激活培养。
(4)小鼠T细胞激活24h后，CAR逆转录病毒感染。感染48h后，流式检测阳性率。继续培养5天后，通过尾静脉回输小鼠。
17.NKG2D CAR-T对衰老小鼠体内效果检测
取8周龄野生C57/B6J小鼠，4Gy射线辐射后饲养于SPF动物房。3个月后取小鼠主要组织检测衰老标志物和NKG2D配体表达。在确认模型构建成功后，用对照MOCK和小鼠NKG2D CAR-T细胞通过尾静脉回输进行治疗。治疗1个月后取小鼠主要组织检测衰老标志物和NKG2D配体表达，6个月后检测小鼠组织损伤的形态学变化和小鼠体能变化。
18.Micro CT扫描
整个实验在Quantum GX microCT成像系统((Quantum GX,PerkinElmer)中进行。在分析之前，小鼠用3％异氟烷/氧气进行连续麻醉并自然俯卧在Micro CT床上。扫描条件如下：voltage,80kV；Current,100μA；Voxel size,50μm.Time,14min。扫描结果用Calipers Analyze软件进行3D重建。
19.苏木精-肌肉染色
(1)依次将石蜡切片进行以下操作：二甲苯中脱蜡5-10分钟；换用新鲜的二甲苯，再脱蜡5-10分钟；无水乙醇5分钟；90％乙醇2分钟；80％乙醇2分钟；70％乙醇2分钟；蒸馏水2分钟。
(2)苏木素染色液染色5分钟，自来水冲洗去多余的染色液，1％盐酸分化30秒后，伊红染色液染色2分钟。
20.小鼠肝纤维化模型构建
选取8周龄雌性C57BL/6小鼠，用橄榄油配制体积分数25％的CCl4油溶液，并每只小鼠腹腔注射100ul每只小，每周2次，持续给药6周。
21.小鼠脂肪肝模型构建
选取8周龄雄性C57BL/6小鼠，饲喂Research Diet公司高脂饲料D12492进行小鼠脂肪肝模型构建，持续饲喂三个月。
实施例1：衰老细胞模型的建立
不同因素诱导的细胞衰老，代表了细胞衰老的不同类型，涉及的分子标记以及信号通路等均有所不同。为展开抗衰老的相关研究，并使研究结果更具有广谱性，本发明利用不同诱导因素构建了一系列的衰老细胞模型，为后续研究奠定基础：1.通过DNA损伤药物Etoposide(Et)处理人胚肺细胞IMR90诱导衰老；2.使用DOX诱导IMR90细胞过表达p16蛋白诱导衰老；3.在IMR90细胞中过表达癌基因Kras G12D诱导衰老；4.体外连续培养IMR90细胞，建立自然衰老细胞模型。利用SA-βgal染色对以上模型进行验证，可见阳性率均为90％以上(图1A，B)。p16表达升高是衰老细胞的典型特征之一。
荧光实时定量PCR检测衰老细胞模型的p16的表达，实验结果显示DOX诱导p16表达诱导衰老组细胞p16上调25～30倍，其他三种模型中p16表达上调2～3倍(图1C)。
实施例2：体外衰老细胞模型NKG2D配体表达上调
已有文献研究表明，衰老细胞表面NKG2D配体表达显著上调，是NK细胞是机体清除衰老细胞的主要靶点。
在本实施例中，检测了以上四种细胞衰老模型中NKG2D主要配体MICA、MICB、ULBP2、ULBP2以及ULBP3的表达，结果显示RNA和蛋白水平NKG2D配体的表达均存在上调，其中MICA、ULBP1、ULBP2上调最为显著(图2A、B)。
实施例3：NKG2D CAR-T细胞对衰老细胞杀伤检测
NKG2D配体在衰老细胞中表达上调，预示NKG2D CAR-T细胞理应具有清除衰老细胞的作用。为了验证以上猜想，构建了CMV启动子驱动表达，以NKG2D胞外域为识别序列，4-1BB和CD3ζ为共刺激结构信号的二代CAR-T细胞(图3A,B)。为了检测所构建的NKG2D CAR-T细胞对衰老细胞是否具有杀伤作用，本实施例中，把对照T细胞和CAR-T细胞分别与IMR90-p16衰老细胞按2:1效靶比体外共培养。
10小时后观察可见，与Mock对照共培养体系相比，衰老组IMR90-p16细胞数量明显减少，说明NKG2D对衰老IMR90-p16细胞具有显著杀伤作用(图3C)。
为了进一步验证NKG2D CAR-T细胞是否能够杀伤多种类型的衰老细胞，在IMR90-Rep和IMR90-Et衰老细胞模型中重复以上实验，均得到类似的实验结果(图3C，D)，说明NKG2D CAR-T细胞对衰老细胞的清除作用具有广谱性。取对照T细胞和NKG2D CAR-T细胞分别与IMR90-Et衰老细胞共培养体系中的上清分析，可见后 者穿孔素和颗粒酶释放显著上调(图3E)。
以上结果均说明：NKG2D CAR-T细胞对衰老细胞具有显著杀伤作用。
实施例4：NKG2D CAR-T安全性
肿瘤相关抗原在正常细胞上的表达通常会导致严重的脱靶现象，从而阻止了CAR-T治疗的临床应用。在大多数人体正常组织中，包括甲状腺、舌头、食道上皮、胃粘膜、空肠粘膜、回肠粘膜、阑尾、直肠粘膜、肝、胰腺、气管、肺、心肌、心肌、动脉、骨骼肌、精囊、前列腺、膀胱、睾丸、延髓、端脑、前脑、脑干和脾脏，均未检测到NKG2D主要配体MICA的表达，仅皮肤呈弱阳性(图4A)。
考虑到慢病毒介导的基因转移可能引起基因组不稳定和细胞恶性转化，因此还通过染色体核型分析以评估慢病毒感染CAR表达的安全性。与未转导的T细胞相比，NKG2D-BBz CAR-T细胞在感染病毒后14后未观测到异常核型(图4B)。
实施例5：NKG2D CAR-T体内效果检测
为了检测NKG2D CAR-T的体内效果，在本实施例中，将从猕猴/食蟹猴外周血采集得到的新鲜血液分别用梯度离心的方法分离非人灵长类的T细胞，并接种至24孔板中(每孔1x106个细胞)，每孔加入非人灵长类专用的T细胞磁珠25ul(含CD2/CD3/CD28抗体)，分离的细胞培养1天后开始成球，表明该T细胞成功从猴子外周血中分离且能够被激活(图5A)。
接着，将NKG2D CAR病毒以MOI＝100感染猴子的T细胞。由于猕猴和食蟹猴T细胞上具有抑制HIV病毒感染的限制性因子TRIM5α，而亲环素A(cyclophilin A；CypA)蛋白的存在能够调节TRIM5α对HIV病毒的感染效率。因此在病毒感染的同时，加入亲环素A的抑制剂CsA(5ug/mL)来促进病毒感染。猴子T细胞感染病毒3天后，用NKG2D抗体对T细胞进行孵育并运用流式技术检测病毒感染效率。病毒感染猴子T细胞的效率约为24.5％(图5B)。这表明该病毒成功整合到了猴子T细胞中让该细胞带上了NKG2D的CAR。
当T细胞被抗原激活后，其会分化成CD4和CD8阳性的细胞。在CAR-T细胞增殖和进入人体内部后CD8阳性的T细胞群增殖率将会升高，且CD8阳性的细胞在靶向和清除肿瘤细胞的过程中也起着最主要作用。但流式结果表明感染病毒3天后的T细胞中CD4阳性的T细胞和CD8阳性T细胞均无变化(图5C)。
为了检测NKG2D CAR-T细胞对猴子衰老细胞的作用，向每只猴子大腿内侧静脉回输1x10 6/kg自体NKG2D CAR-T细胞。CAR-T细胞在猴体内缓慢扩增，到18天左右达到峰值，然后开始下降(图6A)。
由于CAR-T细胞回输到病人体内后，大量T细胞的增殖可能会导致细胞因子分泌上调，在临床上表现为体温升高、厌食、腹泻及呕吐等症状，因此在NKG2D CAR-T 细胞回输到五只猴体内前后对猴子进行了体温的测量，并在回输细胞后对猴子的基础体征进行了严密的观察。
结果发现，五只猴子体温在回输细胞后21天内体温虽然有稍微的上下波动，但处于正常范围内，无发热症状出现(图6B)。同时，猴子进食正常，无腹泻和呕吐等异常情况出现。
除此之外，还监测了五只猴子两个月内的体重变化。其中编号为00085的猕猴体重在回输细胞一个月之后相比于回输细胞之前有所下降，但在第二个月的时候体重回升至回输细胞前；编号为00065的猕猴体重在实验前后两个月无太大差别；编号为00102、01102和98106三只食蟹猴在回输细胞后的两个月内体重有轻微下降(图6C)。
在回输NKG2D CAR-T细胞后，还检测了血清中与细胞因子释放综合征相关的主要的细胞因子IL-2、TNF-α、IFN-γ和IL-6的表达水平。所有检测样本中，均未发现IL-2和TNF-α的表达。编号为00102和00085的两只猴子在回输细胞后的14天里IFN-γ的分泌有所增加，而后编号为00102猴子的IFN-γ的量下降；编号为01102猴子的IFN-γ水平在回输后的第1天下降，但第7-14天有所增加；编号为00065猴子IFN-γ水平则在第7-14天有所增加；98106猴子的IFN-γ表达从回输后第0-7天先增加而后降低(图6D)。编号为00065和98106的两只猴子血清中IL-6的分泌在回输细胞前后几乎无变化；而编号为00085的猴子在第14天开始有所增加；编号为01102和00102两只猴子的IL-6在回输细胞后的第1天开始增加，而后在第7天开始下降(图6D)。
检测NKG2D CAR-T治疗后猴血液中各类细胞(白细胞:WBC；淋巴细胞:Lymphocyte；单核细胞:Monocyte；粒细胞Granulocyte；红细胞:RBC；和血小板:PLT)的变化情况，发现编号为00085的猕猴在CAR-T细胞回输后的第7天单核细胞数量超过正常范围(0.22～2.22x109/L)，但第14天时已回到正常范围。编号为98106的食蟹猴血小板在第0天时超过正常范围(211.84～669.06x109/L)，第7天及以后均在正常范围内(图7A)。其余猴子血常规的各项指标均在正常范围内。
以上的结果表明：NKG2D CAR-T细胞输入猴子体内后对其血常规无影响。在回输细胞后第7天和第14天分析血液中肝脏标志物谷草转氨酶(AST)、谷丙转氨酶(ALT)、碱性磷酸酶(ALP)、谷氨酰转肽酶(GGT)，肾脏标志物肌酐(CRE2)、尿素氮(BUN)，心脏标志物肌酸激酶(CK)表达，均未发现显著异常。这说明NKG2D CAR-T对猴子的肝脏、肾脏和心脏均无明显毒性(图7B,C)。
NKG2D CAR-T治疗后，提取猴皮下脂肪、肌肉、肝脏和肾脏组织RNA，检测衰老细胞标志物P16、P14、P21、LGFBP2、IL6和MMP3表达变化。实验结果显示CAR-T处理后，所有检测组织中，衰老标志物均下调，说明NKG2D CAR-T可有效地清除体内衰老细胞(图8)。
实施例6：衰老小鼠模型的建立
为了检测NKG2D CAR-T在衰老和年龄相关疾病中的作用，首先通过4Gy射线辐射8周龄C57/B6J小鼠构建了衰老模型。辐照后小鼠毛发变白(图9A)，表现出一定衰老症状。
P16被认为是衰老细胞通用的标志物，已有文献表明在人体和小鼠多种衰老组织中的表达显著上调。因此，检测了p16在辐照后小鼠主要器官中的表达。结果表明，辐照使小鼠脂肪、心脏、骨骼肌、肺、肾和肝脏中的P16表达显著升高，并且升高倍数与时间成正相关(图9B)。同时，在这些组织中NKG2D的配体MULT-1和RAE-1表达均明显升高(图9C,D)。
实施例7：NKG2D CAR-T治疗衰老引起的肌肉萎缩症
在本实施例中，从小鼠脾脏中分离了T细胞，并通过逆转录病毒感染建立了小鼠NKG2D CAR-T细胞，阳性率41.9％(图10A)。将MOCK和NKG2D CAR-T细胞通过静脉回输治疗辐照后11个月的衰老小鼠(实施例6)，4周后取小鼠骨骼肌提取RNA检测NKG2D配体的表达。
结果表明，与mock组相比，CAR-T治疗后的小鼠骨骼肌中NKG2D配体RAE-1和MULT-1明显减少(图10B)。与之对应的是，细胞周期相关因子P16和P21，以及衰老相关分泌表型分子IL-6,PAI-1和MMP3表达均下调(图10C,D)。取骨骼肌进行H&E染色观察，可见CAR-T治疗后的老鼠肌肉纤维直径大于未治疗组小鼠(图10E)。进一步地，对NKG2D CAR-T治疗后衰老老鼠的运动能力进行了检测，发现治疗后小鼠具有更好的握力和更快的奔跑速度(图10F,G)。以上结果说明，NKG2D CAR-T可治疗衰老引起的肌肉萎缩症。
实施例8：NKG2D CAR-T治疗衰老引起骨质疏松症
骨质疏松是机体衰老引起常见疾病。在本实施例中验证了NKG2D CAR-T对骨质疏松症的疗效。在NKG2D CAR-T细胞治疗衰老小鼠6个月后，利用micro CT对小鼠股骨进行扫描，并利用Calipers Analyze软件进行3D重建分析。
结果表明与对照组相比，NKG2D CAR-T治疗后，对骨细胞的衰老有改善，表现为骨小梁减少速度明显减慢(图11A)，同时NKG2D CAR-T处理后，衰老小鼠股骨密度有所上升(图11B)。结果说明，NKG2D CAR-T可治疗衰老引起的骨质疏松症。
实施例9：NKG2D CAR-T细胞治疗衰老导致的肝纤维化
研究表明衰老细胞及其分泌的SASP对肝纤维化具有明显促进作用。为验证NKG2D CAR-T细胞对肝纤维化的治疗作用，在本实施例中将构建好的小鼠NKG2D  CAR-T细胞及对照mock T细胞，以每只小鼠1x10 6的剂量尾静脉回输至四氯化碳诱导的肝纤维化小鼠模型中。NKG2D CAR-T治疗20天后，采集小鼠肝脏进行SA-β-gal及Masson染色分析。
结果表明NKG2D CAR-T细胞有效清除了小鼠肝脏组织的衰老细胞，并有效减轻了肝脏纤维化程度(图12A)。进一步提取小鼠肝脏组织RNA，检测衰老细胞标志物P16及NKG2D配体表达变化，结果显示NKG2D CAR-T细胞处理后，小鼠肝脏组织P16及MULT1表达均显著下调(图12B)，表明NKG2D CAR-T有效地清除体内表达NKG2D配体的衰老细胞。
抽取小鼠静脉血进一步评估肝脏标志物谷草转氨酶(AST)和谷丙转氨酶(ALT)表达变化。实验结果表明NKG2D CAR-T治疗的肝纤维化小鼠AST(图12C)及ALT(图12D)水平得到一定恢复。
以上的结果均证明NKG2D CAR-T细胞能有效治疗衰老细胞累积导致的肝纤维化。
实施例10：NKG2D CAR-T细胞治疗衰老细胞累积导致脂肪肝
细胞衰老相关分泌表型(SASP)产生对脂肪肝的发展具有促进作用。为了检测NKG2D CAR-T细胞对脂肪肝的治疗效果，在本实施例中，将构建好的小鼠NKG2D CAR-T细胞及对照mcok T细胞，以每只小鼠1x10 6的剂量通过尾静脉回输至高脂饲料诱导的脂肪肝小鼠模型中。CAR-T治疗20天后，取小鼠肝脏进行SA-β-gal和Masson染色分析。
结果表明NKG2D CAR-T细胞有效清除了小鼠肝脏组织的衰老细胞，并有效减轻了肝脏病变程度(图13A)。提取小鼠肝脏组织RNA检测衰老细胞标志物P16及NKG2D配体MULT1表达变化；发现NKG2D CAR-T细胞治疗后，小鼠肝脏组织P16及MULT1表达均明显下调(图13B)，说明NKG2D CAR-T有效地清除肝脏中NKG2D配体阳性的衰老细胞。
以上结果表明，NKG2D CAR-T细胞可通过清除衰老细胞从而治疗衰老相关的脂肪肝。
讨论
NKG2D配体在DNA损伤、复制压力和癌基因表达诱导的体外细胞衰老模型中表达显著上调。P16 Ink4a表达升高与体内自然衰老的细胞紧密相关。然而，研究表明，P16 Ink4a过表达诱导的衰老细胞中NKG2D配体的表达没有明显上调。与体外不同，体内的衰老细胞表达的NKG2D配体胞外部分会被各种机制(如金属蛋白酶MMP、ERp5和GRP78等)从细胞表面清除，从而逃避以NKG2D配体为靶点的免疫监视作用，从而导致衰老细胞在体内积累并促进个体衰老和衰老相关疾病。文献还表明，不同的 衰老细胞在NKG2D配体表达上存在差异。此外，在体内衰老细胞NKG2D配体胞外部分可被酶切等机制清除并形成可溶性的NKG2D配体。因此，在本发明之前，尚不清楚靶向NKG2D配体是否真正有效地在体内清除衰老细胞，从而治疗老年相关疾病。
在本发明中，由于伦理因素，无法直接在人体内检测NKG2D CAR-T对老年相关疾病的治疗效果。因此利用与人NKG2D CAR序列同源的小鼠序列构建了识别小鼠NKG2D配体的NKG2D CAR-T细胞。将小鼠NKG2D CAR-T细胞通过静脉回输至X射线辐射诱导的小鼠衰老模型中，可见小鼠主要器官中衰老细胞标志物表达下调，同时小鼠肌肉萎缩和骨质疏松症状均明显缓解，运动能力显著提升。
将构建的小鼠NKG2D CAR-T细胞用于治疗四氯化碳诱导的肝纤维化以及高脂食物诱导的脂肪肝，20天后采集小鼠肝脏进行分析：Realtime PCR结果表明治疗后NKG2D配体和衰老标志物表达显著下调；病理分析显示NKG2D CAR-T细胞明显减轻肝脏病变程度。抽取小鼠静脉血进一步评估肝脏标志物谷草转氨酶(AST)和谷丙转氨酶(ALT)表达变化，结果证明NKG2D CAR-T显著提高肝脏功能。以上的结果均证明NKG2D CAR-T细胞能有效治疗衰老细胞累积导致的相关疾病。
本发明的实验结果出乎意料地证明，虽然体内衰老细胞的NKG2D配体胞外部分会可被酶切等机制清除并形成可溶性的NKG2D配体，但是本发明的靶向NKG2D配体的治疗手段，仍能在体内有效杀死衰老细胞，并在体内有效清除累积的衰老细胞，进而显著改善衰老相关症状(如老年性骨质疏松、老年性肌肉萎缩、老年性肝纤维化、老年性脂肪肝等)。本发明的NKG2D CAR-免疫细胞具有可行性，为开发抗衰老和治疗老年相关疾病提供了新的思路。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种靶向NKG2D配体的CAR-免疫细胞的用途，其特征在于，用于制备一药物，所述药物用于：
    (i)清除衰老细胞，其中，所述衰老细胞中的NKG2D配体较正常细胞上调2-20倍，较佳地4-15倍，更佳地10-20倍；
    (ii)延缓个体衰老；和/或
    (iii)预防和/或治疗老年性疾病；
    并且，所述靶向NKG2D配体的CAR-免疫细胞中表达靶向NKG2D配体的嵌合抗原受体，所述的嵌合抗原受体中的抗原结合结构域包括氨基酸序列如SEQ ID NO:1所示的多肽，或与SEQ ID NO:1所示序列具有80％以上相似度并且可结合NKG2D配体的多肽。
  2. 如权利要求1所述的用途，其特征在于，所述的嵌合抗原受体具有如式I所示的结构，
    L-NKG2D-H-TM-C-CD3ζ  (式I)
    式中，
    L为无或信号肽序列；
    NKG2D为如权利要求1所述的NKG2D配体结合结构域序列；
    H为无或CD8α铰链区；
    TM为人CD8α跨膜结构域；
    C为4-1BB或CD28共刺激信号分子；
    CD3ζ为源于CD3ζ的胞浆信号传导序列；
    各“-”独立地表示连接上述各元件的连接肽或肽键。
  3. 如权利要求1所述的用途，其特征在于，所述的嵌合抗原受体由强启动子EF1α驱动表达。
  4. 如权利要求1所述的用途，其特征在于，所述的衰老细胞为人肺细胞脂肪细胞、肾细胞和肌肉细胞以及其他组织衰老细胞。
  5. 如权利要求1所述的用途，其特征在于，所述的衰老细胞是自然的或人工诱导衰老的。
  6. 如权利要求1所述的用途，其特征在于，所述的老年性疾病选自下组：肌肉萎缩症、脂肪肝、心衰、动脉粥样硬化、糖尿病、心肌肥厚、骨质疏松、组织/器官纤维化、阿尔茨海默病、帕金森综合征、关节炎等细胞衰老导致的器官退行性疾病，或其组合。
  7. 如权利要求1所述的用途，其特征在于，所述的老年性疾病选自下组：老年性骨质疏松、老年性肌肉萎缩、老年性肝纤维化、老年性脂肪肝、或其组合。
  8. 如权利要求1所述的用途，其特征在于，患有所述老年性疾病的个体中，含有衰老细胞，所述衰老细胞中的NKG2D配体较正常细胞上调2-20倍，较佳地4-15倍，更佳地10-20倍。
  9. 一种药物组合物，其特征在于，所述药物组合物包括：
    (a)靶向NKG2D配体的CAR-免疫细胞，其中，所述靶向NKG2D配体的CAR-免疫细胞中表达靶向NKG2D配体的嵌合抗原受体，所述的嵌合抗原受体中的抗原结合结构域包括氨基酸序列如SEQ ID NO:1所示的多肽，或与SEQ ID NO:1所示的序列具有80％以上相似度并且可结合NKG2D配体的多肽；
    (b)除(a)之外的其他抗衰老药物；和
    (c)药学上可接受的载体、稀释剂或赋形剂。
  10. 如权利要求9所述的药物组合物，其特征在于，组分(b)包括能够特异性清除衰老细胞的小分子化合物，优选地选自下组：达沙替尼、槲皮素、ABT263、ABT737、荜茇酰胺，或其组合。
  11. 如权利要求9所述的药物组合物，其特征在于，所述药物组合物是注射剂。
  12. 如权利要求9所述的药物组合物，其特征在于，所述药物组合物中，所述靶向NKG2D的CAR-免疫细胞的剂量为1×10 5-5×10 7个细胞/kg，较佳地为5×10 6-1×10 7个细胞/kg。
  13. 一种CAR-免疫细胞，所述CAR-免疫细胞中表达一嵌合抗原受体，所述的嵌合抗原受体具有如式I所示的结构，
    L-NKG2D-H-TM-C-CD3ζ  (式I)
    式中，
    L为无或信号肽序列；
    NKG2D为如权利要求1所述的抗原结合结构域序列；
    H为无或CD8α铰链区；
    TM为人CD8α跨膜结构域；
    C为4-1BB共刺激信号分子；
    CD3ζ为源于CD3ζ的胞浆信号传导序列；
    各“-”独立地表示连接上述各元件的连接肽或肽键。
  14. 如权利要求13所述的CAR-免疫细胞，其特征在于，所述的嵌合抗原受体具有如SEQ ID NO:12所示的氨基酸序列。
  15. 如权利要求13所述的CAR-免疫细胞，其特征在于，所述CAR-免疫细胞选自下组：CAR-T细胞、CAR-NK细胞、CAR-巨噬细胞、或其组合。
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