WO2022262130A1 - 巨噬细胞专属嵌合抗原受体、表达该受体的可控极化单核/巨噬细胞及其制备方法和应用 - Google Patents

巨噬细胞专属嵌合抗原受体、表达该受体的可控极化单核/巨噬细胞及其制备方法和应用 Download PDF

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WO2022262130A1
WO2022262130A1 PCT/CN2021/117808 CN2021117808W WO2022262130A1 WO 2022262130 A1 WO2022262130 A1 WO 2022262130A1 CN 2021117808 W CN2021117808 W CN 2021117808W WO 2022262130 A1 WO2022262130 A1 WO 2022262130A1
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macrophage
chimeric antigen
antigen receptor
macrophages
car
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张进
雷安华
田琳
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浙江大学
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Definitions

  • the present disclosure relates to the field of biotechnology, in particular to a macrophage-specific chimeric antigen receptor, a controllable polarized monocyte/macrophage expressing the receptor, and a preparation method and application thereof.
  • GBM Glioblastoma
  • CAR-T chimeric antigen receptor T cells
  • studies have attempted to extend the survival of GBM-bearing mice by developing CAR-T targeting the GBM-specific antigen EGFRvIII.
  • CAR-T therapy has been extensively explored in the treatment of many solid tumors including GBM, the effect is not ideal.
  • the main reasons are: 1. The possible off-target effect of adoptively transplanted CAR-T cells during treatment; 2. T cells have poor infiltration into solid tumors; 3. It is difficult for T cells to penetrate the blood-brain barrier; 4. Factors such as high tumor complexity and anaerobic microenvironment lead to irreversible exhaustion of cytotoxic T cells. Therefore, it is urgent to find new alternatives for immune cell therapy.
  • Macrophages this kind of non-specific immune cells that also have the ability to kill tumor cells, play a central role in the interaction between the adoptive immune system and the innate immune system, and have strong plasticity, so it is easy to break through the blood brain Barriers, as well as more effective infiltration into tumor tissue sites and long-term residence characteristics, are becoming new options for immune cell engineering.
  • adoptive transplantation of macrophages has the following problems: 1. M1 macrophages with cellular immune function are easily polarized to the M2 state under the influence of the tumor microenvironment, and then lose their immune function and promote tumor growth. 2.
  • the present disclosure provides a chimeric antigen receptor, comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular activation domain connected in sequence;
  • the extracellular antigen binding domain includes a signal peptide and/or scFv targeting EGFRvIII;
  • the transmembrane domain includes CD8 ⁇ ;
  • the intracellular activation domain includes at least one of TIR, CD3ZETA or GM-CSFR ⁇ / ⁇ .
  • the signal peptide is expressed by the nucleotide sequence shown in SEQ ID NO.1;
  • the scFv is expressed by the nucleotide sequence shown in SEQ ID NO.2.
  • the CD8 ⁇ is expressed by the nucleotide sequence shown in SEQ ID NO.3.
  • the TIR includes an intracellular signal transduction domain derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR13 or TLR19;
  • the intracellular signal transduction domain of the TLR4 is expressed by the nucleotide sequence shown in SEQ ID NO.4;
  • the CD3ZETA is expressed by the nucleotide sequence shown in SEQ ID NO.5;
  • the GM-CSFR ⁇ / ⁇ is expressed by the nucleotide sequence shown in SEQ ID NO.6.
  • the present disclosure provides a method for polarizing macrophages by expressing chimeric antigen receptors in macrophages.
  • the present disclosure provides a macrophage comprising a chimeric antigen receptor.
  • the present disclosure provides a method for preparing macrophages, comprising the following steps: constructing a lentiviral expression system containing the gene expression sequence of the chimeric antigen receptor, and expressing the gene of the chimeric antigen receptor by using the lentiviral expression system
  • the sequence is integrated into pluripotent stem cells, and macrophages are prepared after induction and differentiation.
  • the pluripotent stem cells are first induced to differentiate into monocytes, and then differentiated into macrophages.
  • the present disclosure provides a pluripotent stem cell capable of being differentiated into the above-mentioned macrophage, the pluripotent stem cell containing the gene encoding the chimeric antigen receptor.
  • the present disclosure provides a monocyte that can be differentiated to obtain the above-mentioned macrophage, the monocyte contains the gene encoding the chimeric antigen receptor;
  • the mononuclear cells are differentiated from pluripotent stem cells.
  • the present disclosure provides an application of macrophages, pluripotent stem cells or monocytes in preparing a product for treating glioma.
  • the present disclosure provides a product for treating glioma, the product comprising the above-mentioned macrophages.
  • the present disclosure provides a pharmaceutical preparation comprising the macrophage, the pluripotent stem cell or the monocyte.
  • the pharmaceutical formulation includes one or more of a pharmaceutically acceptable adjuvant, diluent, or carrier.
  • the present disclosure provides the use of the chimeric antigen receptor, the macrophage, the pluripotent stem cell, the monocyte or the pharmaceutical preparation in the treatment of glioma.
  • the present disclosure provides the use of the chimeric antigen receptor, the macrophage, the pluripotent stem cell, the monocyte or the pharmaceutical preparation in the treatment of glioma.
  • the present disclosure provides a method of treating glioma, comprising:
  • the present disclosure provides a method of treating glioma, comprising:
  • the present disclosure provides the use of an intracellular activation domain for preparing a drug for treating tumors or cancers, wherein the intracellular activation domain includes at least one of TIR, CD3ZETA or GM-CSFR ⁇ / ⁇ .
  • the agent is a macrophage; the macrophage comprises a chimeric antigen receptor comprising the intracellular activation domain;
  • the cancer is selected from cancers associated with solid tumors, and the tumor is selected from brain tumors.
  • the cancer related to solid tumors is selected from one of brain cancer, esophageal cancer, liver cancer, gastric cancer, intestinal cancer, lung cancer, and nasopharyngeal cancer.
  • the brain tumor is selected from glioma, neurofibroma, astrocytoma, oligodendroglioma, medulloblastoma, ependymoma, pineal tumor A sort of.
  • Figure 1 shows the construction of U87MG EGFRvIII cell line
  • A Construction of EGFRvIII lentivirus expression system
  • B Western Blotting (Western Blotting) detection of the expression level of total EGFR in U87MG cells after lentivirus infection
  • C Western Blotting detection of lentivirus The expression level of total EGFRvIII in U87MG cells after virus infection
  • D Immunofluorescence detection experiments showed that EGFRvIII was localized on the cell membrane surface after overexpression in U87MG cells;
  • FIG. 2 shows the construction of CAR-iMAC (chimeric antigen receptor-macrophage) and the inspection of its non-specific basal phagocytic function;
  • A Construction of 3 types with different intracellular activation signal transduction domains and can target GBM CAR of specific membrane protein EGFRvIII;
  • B iPSCs (induced pluripotent stem cells) stably overexpressing CAR were induced to differentiate into CAR-iMAC;
  • C CAR-iMAC (EGFP) phagocytized U87MG (tdTomato) cells in vitro;
  • Figure 3 shows CAR-iMAC targeting and killing the U87MG EGFRvIII cell line in vitro; A: the result after 12 hours of co-culture in vitro; B: the result of confocal scanning;
  • Figure 4 shows the results of flow cytometric analysis and ELISA experiment of CAR-iMAC targeting and killing U87MG EGFRvIII cell line in vitro;
  • A flow cytometric analysis technology counts the number of surviving tumor cells after 24 hours of co-culture;
  • B statistical analysis of data for remaining tumors in A Cell ratio;
  • C survival rate of tumor cells;
  • D ELISA test to detect the release of immune activation factors;
  • Figure 5 is the test of the survival time of iMAC derived from iPSC in vivo and the infiltration of solid tumor tissue;
  • A The survival signal and time of iMAC detected by in vivo imaging;
  • B The survival curve of iMAC in data statistics A;
  • C Experimental flow chart;
  • D the infiltration of iMAC in tumor tissue;
  • E the infiltration of iMAC in tumor tissue by immunohistochemistry;
  • Figure 6 is the verification of the tumor killing effect of CAR-iMAC in the GBM mouse tumor model; A: the killing effect of three CAR-iMACs on GBM (Luciferin biotin luminescence); B: the change of tumor signal; C: different CARs - time of survival of GBM mice after iMAC treatment;
  • Figure 7 is a structural diagram of CD8-GM-CSFR ⁇ / ⁇ -CAR and a schematic diagram of its activation
  • Figure 8 is the in vitro verification of the effects of CD8-GM-CSFR ⁇ / ⁇ -CAR-iMAC targeted killing and CAR-dependent immune activation;
  • FIG. 9 is a schematic diagram of the structure of the three-generation CAR designed in the present disclosure, and a schematic diagram of the comparison of immune activation of different CAR-iMACs depending on CAR.
  • macrophage generally refers to a myeloid immune cell developed from monocytes passing out of blood vessels, and is widely distributed in various organs of body tissues. Its main physiological functions in normal tissues are: mediate specific immune response by processing and presenting antigen; phagocytize and degrade necrotic cells, debris and foreign matter in the form of fixed cells or free cells, and then participate in non-specific reactions in the body ; Activate lymphocytes or other immune cells by secreting inflammatory factors, and then coordinate the inflammatory process.
  • CAR Chimeric Antigen Receptor
  • the extramembrane region is a single-chain variable region domain (single chain Fv domain, scFv) with the function of targeting and binding to tumor-specific antigen TAA.
  • the transmembrane region is usually composed of immunoglobulin superfamily, such as CD8 or CD28.
  • the intracellular signal transduction region is mainly composed of the intracellular signal transduction domain of the activation receptor that can activate immune cells, such as the costimulatory factor (4-1BB or CD28) exclusive to T cells, and the signal activation region CD3 ⁇ . After the immune cells loaded with chimeric antigen receptors specifically bind to the surface antigens of tumor cells, the extramembrane antigen-binding region transmits the signal to the intracellular signal activation region, and then initiates the immune cell activation response.
  • polarization of macrophages generally refers to the transformation of macrophages in response to various environmental factors (e.g., microbial products, damaged cells, activated lymphocytes) or under different pathophysiological conditions.
  • environmental factors e.g., microbial products, damaged cells, activated lymphocytes
  • M1 and M2 different functional phenotypes
  • M2 classically activated macrophages
  • alternatively activated macrophages alternatively activated macrophages
  • Mature macrophages show phenotype and morphological differentiation under various factors, that is, the polarization of macrophages.
  • macrophages are mainly activated into two phenotypes, M1 and M2.
  • the M1 type is activated by signals such as IFN- ⁇ and LPS, which mainly have anti-tumor and immune-enhancing effects, and can secrete inflammatory factors, chemokines, effector molecules and TNF- ⁇ , among which membrane Molecule CD80, surface marker CD64, etc. are the representatives.
  • the M2 type is activated by factors such as IL-4 and IL-13, which mainly have the potential to inhibit immune response, promote angiogenesis, tissue repair and promote tumor growth, and secrete more factors such as IL-10, TGF- ⁇ , and VEGF. Relatively high expression of CD163 and CD206.
  • pluripotent stem cell is a type of pluripotent cell with self-renewal and self-replication capabilities. Under certain conditions, it can differentiate into a variety of APSC pluripotent cells. Pluripotent stem cells (HSC) have the potential to differentiate into a variety of cell tissues, but they have lost the ability to develop into a complete individual, and their developmental potential is limited.
  • iPSC induced pluripotent stem cell
  • iMAC generally refers to Macrophage induced to differentiate from iPSCs.
  • monocyte refers to a cell that is differentiated from a hematopoietic stem cell in the bone marrow and develops in the bone marrow. Optional differentiation into mature macrophages and dendritic cells. Monocytes are characterized by obvious deformation movements, and have the ability to phagocytose and remove injured, senescent cells and their debris. In addition, monocytes also participate in the immune response, transfer the antigenic determinant carried by the phagocytized antigen to lymphocytes, and then induce the specific immune response of lymphocytes. Monocytes also have the ability to recognize and kill tumor cells.
  • the term "subject” refers to a vertebrate, optionally a mammal, optionally a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.
  • therapeutically effective amount refers to an amount of an agent sufficient to produce a beneficial or desired result.
  • Therapeutically effective amount may vary according to one or more of the following: the subject being treated and the condition of the disease, the weight and age of the subject, the severity of the condition, the mode of administration, etc., which can be determined by Easily determined by one of ordinary skill in the art. Dosage may vary depending on one or more of: the specific agent chosen, the dosing regimen followed, whether it is co-administered with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system carrying the agent .
  • Some embodiments of the present disclosure provide a chimeric antigen receptor, comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular activation domain connected in sequence;
  • the extracellular antigen binding domain includes a signal peptide and/or scFv targeting EGFRvIII;
  • the transmembrane domain includes CD8 ⁇ ;
  • the intracellular activation domain includes at least one of TIR, CD3ZETA or GM-CSFR ⁇ / ⁇ .
  • the present disclosure provides a chimeric antigen receptor, in which the extracellular antigen binding domain includes a signal peptide and/or scFv, the signal peptide can help the CAR protein sequence to locate on the cell membrane surface, and the scFv can specifically recognize the cell membrane surface protein EGFRvIII specifically expressed by GBM
  • the transmembrane domain includes CD8 ⁇ , which connects the extracellular antigen-binding domain and the intracellular activation domain; the intracellular activation domain includes TIR, CD3ZETA or GM-CSFR ⁇ / ⁇ , which promotes the polarization of macrophages to the M1 type.
  • the provided chimeric antigen receptors are introduced into macrophages, which not only endows the macrophages with a targeted killing effect on GBM, but also enhances the immunosuppressive effect of the immune cells against the tumor microenvironment, effectively promoting and maintaining Its M1 polarization state.
  • the signal peptide is expressed by the nucleotide sequence shown in SEQ ID NO.1;
  • the scFv is expressed by the nucleotide sequence shown in SEQ ID NO.2.
  • the signal peptide has an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.1;
  • the scFv has an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.2.
  • the CD8 ⁇ is expressed by the nucleotide sequence shown in SEQ ID NO.3.
  • CD8 ⁇ has an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.3.
  • the TIR includes but is not limited to an intracellular signal transduction domain derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR13 or TLR19;
  • the intracellular signal transduction domain of the TLR4 is expressed by the nucleotide sequence shown in SEQ ID NO.4;
  • the CD3ZETA is expressed by the nucleotide sequence shown in SEQ ID NO.5;
  • the GM-CSFR ⁇ / ⁇ is expressed by the nucleotide sequence shown in SEQ ID NO.6.
  • the intracellular signal transduction domain of TLR4 has the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.4;
  • CD3ZETA has the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.5;
  • GM-CSFR ⁇ / ⁇ has the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.6.
  • Some embodiments of the present disclosure provide a method for polarizing macrophages by expressing chimeric antigen receptors in macrophages.
  • the M1 polarization of macrophages is achieved by expressing the above chimeric antigen receptors in macrophages, which is simple and effective.
  • Some embodiments of the present disclosure provide a macrophage comprising a chimeric antigen receptor.
  • the macrophage can express the above-mentioned chimeric antigen receptor provided by the present disclosure, can maintain the M1 polarization state, and has strong targeting killing ability to GBM.
  • FIG. 9 The structural schematic diagram of the three-generation CAR designed in this disclosure, and the comparative schematic diagram of different CAR-iMAC dependent on CAR for immune activation are shown in FIG. 9 .
  • Truncate CAR is mainly composed of an extracellular scFv domain that specifically recognizes EGFRvIII protein and a CD8 ⁇ transmembrane domain. Lacks an intracellular signaling domain.
  • the first-generation CAR is mainly composed of an extracellular scFv domain that specifically recognizes EGFRvIII protein, a CD8 ⁇ transmembrane domain, and an intracellular CD3ZETA signal transduction domain.
  • the second-generation CAR mainly consists of an extracellular scFv domain that specifically recognizes EGFRvIII protein, a CD8 ⁇ transmembrane domain, and an intracellular signal transduction domain specific to macrophages.
  • the intracellular signaling domain is a single TIR or GM-CSFR ⁇ / ⁇ activation domain, respectively.
  • the third-generation CAR is an upgrade on the basis of the second-generation CAR. The difference is that the intracellular signal transduction domain of the third-generation CAR is composed of CD3ZETA, TIR, and GM-CSFR ⁇ / ⁇ through Linker connection to achieve a stronger and longer-lasting immune activation effect.
  • B-E in Figure 9 shows a schematic diagram of immune activation of different CAR-iMACs dependent on CAR. Due to the lack of intracellular signal transduction domain, Truncate CAR-iMAC does not have the function of immune activation dependent on CAR. After the first-generation CAR extracellular scFv targets and recognizes EGFRvIII, its intracellular CD3ZETA signal transduction domain is activated, thereby initiating the immune activation of the first-generation CAR-iMAC and the release of a small amount of inflammatory factors. The activation of TIR can stimulate the immune activation of macrophages, release inflammatory factors such as IL1, IL6, TNF ⁇ , and polarize to the M1 type.
  • TIR can stimulate the immune activation of macrophages, release inflammatory factors such as IL1, IL6, TNF ⁇ , and polarize to the M1 type.
  • GM-CSFR ⁇ / ⁇ can promote the massive expansion of macrophages, immune activation, release of inflammatory factors such as IL12, IL23, TNF ⁇ , and polarization to the M1 type. Therefore, compared with the first-generation CAR-iMAC, the second-generation CAR-iMAC, after targeting EGFRvIII, significantly improved anti-tumor abilities such as survival expansion, M1 polarization, and immune activation in the tumor microenvironment.
  • the third-generation CAR extracellular scFv targets and recognizes EGFRvIII, there will be two signal transduction domains among TIR, CD3ZETA and GM-CSFR ⁇ / ⁇ in the cell to cooperate to play an immune activation role. The advantage of activating the signaling pathway, and then exert a stronger and longer-lasting immune activation effect.
  • Some embodiments of the present disclosure provide a method for preparing macrophages, comprising the steps of: constructing a lentiviral expression system containing the gene expression sequence of the chimeric antigen receptor, and using the lentiviral expression system to express the chimeric antigen receptor
  • the gene expression sequence of the body is integrated into the pluripotent stem cells, and the macrophages are prepared after induction and differentiation;
  • the pluripotent stem cells are first induced to differentiate into monocytes, and then differentiated into macrophages.
  • Macrophages can overexpress chimeric antigen receptors stably for a long time, which can solve the problems of low editing efficiency, long cycle, heavy workload and untimely treatment in current immune cell therapy.
  • Some embodiments of the present disclosure provide a pluripotent stem cell capable of being differentiated into the above-mentioned macrophage, the pluripotent stem cell containing the gene encoding the chimeric antigen receptor.
  • the pluripotent stem cells contain the gene encoding the above-mentioned chimeric antigen receptor, their state is more inclined to the M1 state of pro-inflammation and tumor suppression, and can optionally differentiate into macrophages, which is more conducive to tumor suppression.
  • Some embodiments of the present disclosure provide a monocyte capable of being differentiated into the above-mentioned macrophage, the monocyte containing the gene encoding the chimeric antigen receptor;
  • the mononuclear cells are differentiated from pluripotent stem cells.
  • Some embodiments of the present disclosure provide an application of macrophages, pluripotent stem cells or monocytes in preparing a product for treating glioma.
  • the macrophages provided by the present disclosure can maintain the M1 polarized state and have strong targeting and lethality to GBM, they can be used to prepare products for the treatment of glioma, and the pluripotent stem cells can be differentiated to obtain the above-mentioned macrophages, Therefore, it can also be used to prepare products for treating glioma.
  • Some embodiments of the present disclosure provide a product for treating glioma, the product comprising the above-mentioned macrophages.
  • the macrophages provided by the present disclosure can maintain the M1 polarization state, and have strong targeting killing ability to GBM, and products including the macrophages also have the effect of treating glioma.
  • Some embodiments of the present disclosure provide a pharmaceutical preparation comprising macrophages, pluripotent stem cells or monocytes.
  • the pharmaceutical formulation includes one or more of a pharmaceutically acceptable adjuvant, diluent, or carrier.
  • Some embodiments of the present disclosure provide the use of chimeric antigen receptors, macrophages, pluripotent stem cells, monocytes or pharmaceutical preparations in the treatment of glioma.
  • Some embodiments of the present disclosure provide chimeric antigen receptors, macrophages, pluripotent stem cells, monocytes or pharmaceutical preparations for use in the treatment of glioma.
  • Some embodiments of the present disclosure provide a method of treating glioma, comprising:
  • Some embodiments of the present disclosure provide the use of the intracellular activation domain for preparing a drug for treating tumor or cancer, wherein the intracellular activation domain includes at least one of TIR, CD3ZETA or GM-CSFR ⁇ / ⁇ .
  • the drug is a macrophage; the macrophage comprises a chimeric antigen receptor comprising the intracellular activation domain; the cancer is selected from cancers associated with solid tumors, and the tumor is selected from brain tumor.
  • the cancer related to solid tumors is selected from one of brain cancer, esophageal cancer, liver cancer, gastric cancer, colon cancer, lung cancer, nasopharyngeal cancer and the like.
  • the brain tumor is selected from glioma, neurofibroma, astrocytoma, oligodendroglioma, medulloblastoma, ependymoma, pineal tumor, etc. kind of.
  • the intracellular activation domain of the present disclosure can be widely applied to a variety of macrophages targeting other tumor antigens, has wide applicability, can effectively activate macrophages, and promote the polarization of macrophages to the direction of immune activation.
  • the present disclosure provides a macrophage-specific chimeric antigen receptor, which includes an extracellular antigen-binding domain, a transmembrane domain and an intracellular activation domain connected in sequence.
  • the extracellular antigen binding domain includes a signal peptide and/or scFv, the signal peptide can help the CAR protein sequence to locate on the cell membrane surface, and the scFv can specifically recognize the cell membrane surface protein EGFRvIII specifically expressed by GBM;
  • the transmembrane domain includes CD8 ⁇ , which connects to the extracellular antigen Binding domain and intracellular activation domain;
  • the intracellular activation domain includes at least one of TIR, CD3ZETA or GM-CSFR ⁇ / ⁇ , and the intracellular activation domain of the present disclosure is a macrophage-specific intracellular activation domain , can effectively activate macrophages, and promote the polarization of macrophages to the direction of immune activation, that is, to promote the polarization of macrophages to the M
  • the GM-CSFR ⁇ / ⁇ domain can effectively maintain the monocyte-like state of macrophages. This state is conducive to the infiltration of macrophages into the tumor tissue.
  • Introducing the chimeric antigen receptor provided by the present disclosure into macrophages not only endows the macrophages with a targeted killing effect on GBM, but also enhances the immunosuppressive effect of the immune cells against the tumor microenvironment, effectively Promotes and maintains its M1 polarized state.
  • the chimeric antigen receptor provided by the present disclosure can endow macrophages with the characteristic of targeting GBM, reduce off-target effects, promote and maintain the M1 polarization state of macrophages in the tumor microenvironment, and improve their killing efficiency.
  • the method for polarizing macrophages provided by the present disclosure is simple and can realize M1 polarization of macrophages.
  • the macrophages provided in the present disclosure can solve at least one of the above problems.
  • the method for polarizing macrophages provided by the present disclosure is simple and effective by expressing the above-mentioned chimeric antigen receptor in macrophages to achieve M1 polarization of macrophages.
  • the macrophage provided by the present disclosure can express the chimeric antigen receptor provided by the present disclosure, can maintain the M1 polarization state, and has strong targeting and killing ability to GBM.
  • the present disclosure provides a method for preparing the above-mentioned macrophages, comprising constructing A lentiviral expression system that integrates the gene expression sequence of the antigen receptor, uses the lentivirus expression system to integrate the gene expression sequence of the chimeric antigen receptor into pluripotent stem cells, and prepares macrophages after induction and differentiation.
  • the macrophages The long-term and stable overexpression of chimeric antigen receptors can solve the problems of low editing efficiency, long cycle, heavy workload and untimely treatment in current immune cell therapy.
  • the pluripotent stem cells provided by the present disclosure contain the gene encoding the above-mentioned chimeric antigen receptor, and its state is more inclined to the M1 state of pro-inflammation and tumor suppression, and can be selectively differentiated into macrophages, which is more conducive to tumor suppression.
  • a GBM cell line expressing EGFRvIII antigen was constructed.
  • GBM-specific membrane protein EGFRvIII Obtain the expression sequence of GBM-specific membrane protein EGFRvIII, and clone it into the lentiviral expression plasmid Lenti-EF1A-PGK-Puro by means of molecular cloning, and construct the obtained EGFRvIII lentiviral expression system, as shown in A in Figure 1 .
  • the GBM-specific membrane protein EGFRvIII was expressed in the GBM cell lines U87MG and LN-229 (preserved by Liu Chong's research group of Zhejiang University School of Medicine) with the help of a lentiviral expression system, and the U87MG EGFRvIII cell line stably expressing the membrane protein was obtained.
  • Counterstain nuclei add DAPI dropwise and incubate in the dark for 5 minutes, stain the specimens for nuclei, wash off excess DAPI with PBST for 5 min ⁇ 4 times; blot the liquid on the slide with absorbent paper, and seal with an anti-fluorescence quencher. The slides were sealed, and images were collected under a fluorescence microscope.
  • PBMC peripheral blood mononuclear cells
  • PBMCs isolated from fresh blood have the advantages of large quantity and easy acquisition, and are excellent materials for inducing iPSCs.
  • the expression vectors expressing five transcription factors, OCT3/4, SOX2, KLF4, L-MYC, and LIN28A were transfected into PBMCs by electroporation to induce their reprogramming, and finally iPSCs were obtained.
  • EB formation day 0.
  • iPSCs When iPSCs have grown to cover 60-80% of the dish, they are digested with versene into single cells or smaller cell aggregates. After centrifugation at 600rpm for 3min, resuspend in mTeSR1 medium containing Y-27632, and inoculate into a low adsorption six-well plate at a ratio of 1:2 or 1:3. Incubate on a shaker in a 37°C/5% CO 2 incubator for about 24 hours. EB can be formed.
  • step 1 Primitive Streak and Mesoderm Induction (Day 1). Remove the medium in which the EBs were cultured in step 1. Add MI medium (APEL II medium (medium)+10ng/mL BMP4+5ng/mL bFGF). Continue culturing under the conditions of step 1 for 24 hours.
  • MI medium APEL II medium (medium)+10ng/mL BMP4+5ng/mL bFGF.
  • HSC Hematopoietic stem cell
  • step 4 Expansion of myeloid cells and mononuclear precursor cells (MPCs) (days 8-10). Remove the HS medium from step 3. Add ME-1 medium (APEL II medium+5ng/mL bFGF+50ng/ml VEGF+100ng/mL SCF+10ng/mL IGF1+25ng/mL IL-3+50ng/mL M-CSF+50ng/mL GM - CSF (granulocyte-macrophage colony stimulating factor)).
  • ME-1 medium APEL II medium+5ng/mL bFGF+50ng/ml VEGF+100ng/mL SCF+10ng/mL IGF1+25ng/mL IL-3+50ng/mL M-CSF+50ng/mL GM - CSF (granulocyte-macrophage colony stimulating factor)).
  • ME-1 medium APEL II medium+5ng/mL bFGF+50ng/ml VEGF+100ng
  • Maturity of iMAC (about 17th). Collect the iMONO collected in step 5 into a new Matrigel-coated six-well plate. Add MM medium (StemSpan-XF medium+5ng/mL bFGF+50ng/ml VEGF+100ng/mL SCF+10ng/mL IGF1+25ng/mL IL-3+100ng/mL M-CSF+100ng/mL GM- CSF) were cultured statically in a 37°C/5% CO 2 incubator. During this period of time, we will see that the smaller iMONO matures to the larger and vacuolated iMAC.
  • M1 polarization of iMAC Collect the mature iMAC in step 6, add MS medium (RPMI1640+100ng/mL M-CSF+100ng/mL GM-CSF) containing 100ng/ml LPS and 100ng/ml IFN- ⁇ , about 24 hours to get extremely Optimized iMAC.
  • iMONO and iMAC with high differentiation efficiency and good activity provide a guarantee for the subsequent demand for a large number of immune cells.
  • the marker antigens on the surface of iMAC were detected by flow cytometry, the gene expression profile of iMAC was analyzed by single-cell RNA-seq, and the secretion of cytokines was detected by ELISA to confirm that iPSCs were successfully induced to differentiate into M1 iMAC.
  • the signal peptide is a short peptide chain containing a hydrophobic amino acid sequence. It has the function of guiding the newly synthesized protein to transmembrane or secrete out of the cell.
  • the expression sequence of the signal peptide is expressed by the nucleic acid sequence shown in SEQ ID NO.1:
  • sc-Fv The expression sequence of sc-Fv is expressed by the nucleotide sequence shown in SEQ ID NO.2.
  • the sc-Fv has an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.2:
  • CD8 ⁇ is expressed by the nucleotide sequence shown in SEQ ID NO.3.
  • CD8 ⁇ has the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.3:
  • TIR is expressed by the nucleotide sequence shown in SEQ ID NO.4.
  • TIR has the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.4:
  • CD3ZETA is expressed by the nucleotide sequence shown in SEQ ID NO.5.
  • CD3ZETA has the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.5:
  • GM-CSFR ⁇ / ⁇ is expressed by the nucleotide sequence shown in SEQ ID NO.6.
  • GM-CSFR ⁇ / ⁇ has the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.6:
  • CAR-iPSC Cell lines stably expressing CAR (CAR-iPSC) were obtained by means of green fluorescence expression and single-cell RNA-seq. Then, the three CAR-iPSCs were induced to differentiate into CAR-iMONO or CAR-iMAC dependent on CAR activation.
  • CAR-iMAC expressing green fluorescent protein into a six-well plate, in a medium containing 100ng/ml LPS, 100ng/ml human INF- ⁇ ( ⁇ -interferon), 100ng/ml M-CSF (macro Phage colony-stimulating factor) and 100ng/ml GM-CSF in RPMI1640 medium for 24 hours. Place in a 37°C/5% CO2 cell culture incubator during the period.
  • the iMACs expressing the above three different CAR-iMACs and the control group were co-cultured in vitro with U87MG EGFRvIII cells expressing luciferase in a live cell workstation. Comparing CARs with different intracellular activation domains to enhance the effect of CAR-iMAC targeting GBM and promoting its immune activation. Luciferase assay and tumor cell flow cytometry assay were used to detect different CAR-iMAC targeted killing effects.
  • CAR-iMAC and WT-iMAC wild type-macrophage expressing green fluorescent protein into a six-well plate, in a medium containing 100ng/ml LPS (lipopolysaccharide), 100ng/ml human Polarized in RPMI1640 medium sourced from INF- ⁇ , 100ng/ml M-CSF and 100ng/ml GM-CSF for 24 hours. Place in a 37°C/5% CO2 cell culture incubator during the period.
  • LPS lipopolysaccharide
  • the setting parameters are: green fluorescent protein is FITC channel, tdTomato is PE channel.
  • the three CAR-iMACs expressing green fluorescent protein were polarized for 24 hours and co-cultured with U87MG EGFRvII cells expressing luciferase gene luciferase at a ratio of 10:1 in a dark 96-well plate 24 hours. And set only the culture well of U87MG EGFRvII cells as a negative control (NC) (the method is the same as that of Figure 3 in Figure 3).
  • NC negative control
  • FIG. 5 The results of the in vivo survival time of iMAC derived from iPSC and the infiltration of solid tumor tissues are shown in FIG. 5 .
  • NSG mice were intraperitoneally injected with fluorescent dye Dir-labeled iMAC, and the survival signal and time of iMAC were detected by intravital imaging, as shown in A in Figure 5.
  • Data Statistics The survival curve of iMAC in A in Fig. 5 (B in Fig. 5). It shows that iMAC derived from iPSC can exist in the tumor model for more than 30 days, which meets the requirement of survival time for immune cells in tumor immune cell therapy.
  • NSG mice were intraperitoneally injected with 1 ⁇ 10 6 U87MG cells overexpressing the luciferase gene, and 4 hours later, the same amount of iMAC was injected in situ. After 30 days, the tumor tissues were isolated and passed through Dir and luciferin ( Luciferin) imaging to observe the infiltration of iMAC in tumor tissue. The results are shown in D in Fig. 5, indicating the existence of iMAC in GBM. Immunohistochemical experiments were used to further detect the infiltration of iMAC in tumor tissues, and the experimental steps were as follows:
  • Embedding tissue first add some liquid paraffin to the iron mold, let it cool down slightly, then place the tumor tissue fixed in the previous step in paraffin, then cover the plastic mold box, and finally add a little liquid paraffin to carry out Freeze to solidify the paraffin.
  • Slicing Remove the embedded tumor tissue from the mold and place it on a paraffin slicer.
  • the slicer adjusts up, down, left, and right to make the tissue and the cutting direction consistent, and then adjusts the thickness of the slice, usually 5 ⁇ m, with a pen Pull the cut slide outward, and place the slide containing the intact tissue in warm water at 40°C with fine forceps.
  • Dewaxing dewax the slides in xylene-xylene, 100% alcohol, 100% alcohol, 95% alcohol, 90% alcohol, 80% alcohol and 70% alcohol in sequence. 10-15min.
  • Antigen restoration Rinse in clean water for a period of time after dewaxing, add citric acid buffer, put it in a microwave oven and cook for 3 minutes (medium heat), until it boils. Cool at room temperature, then cook again, and cool to room temperature to expose the antigenic sites.
  • Serum blocking After cooling to room temperature, pour off the citrate buffer, wash twice with water, place the slide in PBS for 5 minutes, wash twice, and dry the PBS around the tissue. Immediately add serum diluted ten times with PBS to block some non-specific sites, and then put it in a 37-degree incubator for half an hour.
  • Add secondary antibody take the slide out of the refrigerator, put it in PBS and wash it 3 times, 5 minutes each time, dry the PBS around the tissue, add the secondary antibody of the same species, and then place it in a 37-degree incubator half an hour.
  • Add SABC take the slides out of the incubator, put them in PBS and wash 3 times, 5 minutes each time, dry the PBS around the tissue, add SABC diluted 100 times with PBS, and then place in a 37°C incubator half an hour.
  • Add chromogen Take the slice out of the incubator, put it in PBS and wash 3 times, 5 minutes each time, dry the PBS around the tissue and add chromogen.
  • Color developer configuration add 1 drop of color developer A to 1ml of water, shake well, then add 1 drop of color developer B, shake well, add 1 drop of color developer C, shake well.
  • Counter-staining Rinse the color-developed slides with water for a period of time, then soak in hematoxylin for staining, usually for half a minute for animal tissues, and 3-5 minutes for plant tissues.
  • Dehydration Rinse the counterstained slides in water, then place the slides in 70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% alcohol-xylene-xylene. Place in each reagent for 2min, and finally soak in xylene and move to a fume hood.
  • Imaging Place the above-mentioned tumor histochemical samples in an OLYMPUS upright microscope, set up imaging and take pictures.
  • a chimeric antigen receptor CD8-GM-CSFR ⁇ / ⁇ -CAR, its structural schematic diagram and its activation diagram are shown in Figure 7, and A in Figure 7 shows that CD8-GM-CSFR ⁇ / ⁇ -CAR is recognized by extracellular
  • the scFv protein sequence of EGFRvIII, CD8 ⁇ transmembrane domain, and intracellular signal transduction domain constitute.
  • the intracellular signal transduction domain is composed of the intracellular signal activation domain of the receptor GM-CSFR ⁇ and the intracellular signal activation domain of the receptor GM-CSFR ⁇ through a linker (Linker) sequence. Linker sequences can flexibly change conformation.
  • B in Figure 7 shows that after the CD8-GM-CSFR ⁇ / ⁇ -CAR extracellular scFv contacts the EGFRvIII protein to form an immune synapse, the GM-CSFR ⁇ and GM-CSFR ⁇ activation domains in the package change the conformation of the linker Heterodimers are formed and then activated, ultimately promoting the proliferation, immune activation and phagocytosis of CAR-iMAC.
  • CD8-GM-CSFR ⁇ / ⁇ -CAR-iMAC targeted killing and CAR-dependent immune activation were verified in vitro.
  • CD8-GM-CSFR ⁇ / ⁇ -CAR-iMAC and CD8-DELTA-CAR-iMAC were co-combined with luciferase-expressing U87MG EGFRvIII cells in a live cell workstation. After culture and co-culture in vitro for 24 hours, confocal scanning showed that both CD8-GM-CSFR ⁇ / ⁇ -CAR-iMAC (EGFP) and CD8-DELTA-CAR-iMAC (EGFP) without the intracellular signal transduction domain had Ability to phagocytose U87MG EGFRvIII cells (A in FIG. 8 ).
  • WT-iMAC, CD8-DELTA-CAR-iMAC, and CD8-GM-CSFR ⁇ / ⁇ -CAR-iMAC were co-cultured with U87MG EGFRvIII cells for 24 hours in vitro at an effect-to-target ratio of 10/1, and macrophages were detected by ELISA assay.
  • Secretion of cellular immune activator IL-12 and TNF ⁇ The results are shown in B and C in Figure 8, indicating that CD8-GM-CSFR ⁇ / ⁇ -CAR-iMAC has a significant ability to secrete IL-12 and TNF ⁇ relative to WT-iMAC and CD8-DELTA-CAR-iMAC.
  • WT-iMAC, CD8-DELTA-CAR-iMAC and CD8-GM-CSFR ⁇ / ⁇ -CAR-iMAC were co-cultured with U87MG EGFRvIII cells expressing luciferase for 24 hours in vitro at an effect-to-target ratio of 10/1.
  • Fluorescence signals in U87MG EGFRvIII cells were detected by a multifunctional microplate reader. The results are shown in D in Figure 8, indicating that the fluorescent signal was significantly down-regulated after U87MG EGFRvIII cells were co-cultured with CD8-GM-CSFR ⁇ / ⁇ -CAR-iMAC. It was further confirmed that CD8-GM-CSFR ⁇ / ⁇ -CAR-iMAC had a more significant ability to kill U87MG EGFRvIII cells than WT-iMAC and CD8-DELTA-CAR-iMAC.
  • the chimeric antigen receptor provided by the present disclosure can endow macrophages with the characteristic of targeting GBM, reduce off-target effects, promote and maintain the M1 polarization state of macrophages in the tumor microenvironment, and improve their killing efficiency.
  • the method for polarizing macrophages provided by the present disclosure is simple and can realize M1 polarization of macrophages.
  • the macrophages provided by the present disclosure can maintain the M1 polarization state, and have strong targeting and lethality against GBM.
  • the macrophages can stably overexpress chimeric antigen receptors for a long time, and can solve the problems existing in current immune cell therapy. Problems such as low efficiency, long cycle, heavy workload and untimely treatment.
  • the pluripotent stem cells provided by the present disclosure contain the gene encoding the above-mentioned chimeric antigen receptor, and its state is more inclined to the M1 state of pro-inflammation and tumor suppression, and can be selectively differentiated into macrophages, which is more conducive to tumor suppression and has a wide range of Application prospect and high market value.

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Abstract

本公开提供了一种巨噬细胞专属嵌合抗原受体、表达该受体的可控极化单核/巨噬细胞及其制备方法和应用，涉及生物技术领域。本公开提供了一种嵌合抗原受体，包括依次连接的胞外抗原结合域、跨膜结构域和胞内激活结构域，其中胞外抗原结合域包括信号肽和/或scFv，能够异性识别GBM特异表达的细胞膜表面蛋白EGFRvIII；跨膜结构域包括CD8α，链接胞外抗原结合域和胞内激活结构域；胞内激活结构域包括TIR、CD3ZETA或GM-CSFRα/β，促进巨噬细胞向M1型极化，将本公开的嵌合抗原受体引入到巨噬细胞中，赋予了该巨噬细胞对GBM的靶向杀伤作用，有效的促进并维持其M1极化状态。

Description

巨噬细胞专属嵌合抗原受体、表达该受体的可控极化单核/巨噬细胞及其制备方法和应用
相关申请的交叉引用
本公开要求于2021年06月18日提交中国专利局的申请号为“202110682523.9”名称为“巨噬细胞专属嵌合抗原受体、表达该受体的可控极化单核/巨噬细胞及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及生物技术领域，尤其是涉及一种巨噬细胞专属嵌合抗原受体、表达该受体的可控极化单核/巨噬细胞及其制备方法和应用。
背景技术
胶质母细胞瘤(GBM)被认为起源于神经胶质祖细胞，是目前已发现的最普遍、最具侵袭性的原发性脑肿瘤。其发病率占据了颅内肿瘤的12％-15％和星型细胞肿瘤的50％-60％。罹患该肿瘤的患者中位数生存期约为15个月，5年生存率不到10％。因此，该肿瘤对人类生命健康构成了极大的威胁。虽然目前针对GBM的治疗方法有外科手术、放疗和化疗等手段，但由于该肿瘤向脑叶深部浸润生长，并具备生长迅速、易复发等特点，因此收效甚微，且容易对患者大脑造成难以恢复的二次伤害。
近年来兴起的以嵌合抗原受体T细胞(CAR-T)为代表的肿瘤免疫细胞疗法已逐步在肿瘤的临床治疗中发挥越来越重要的作用，尤其是在血液癌的治疗中展现出了较好的治疗效果。同时，已有研究试图通过开发靶向GBM特异抗原EGFRvIII的CAR-T来延长GBM携带小鼠的生存期。然而，尽管目前利用CAR-T疗法在针对GBM在内的诸多实体瘤的治疗中做了大量探索，但其效果并不理想。其中的主要原因在于：1.过继移植的CAR-T细胞在治疗中可能发生的脱靶效应；2.T细胞对实体肿瘤浸润性较差；3.T细胞难以穿透血脑屏障；4.实体肿瘤高度复杂性和厌氧的微环境等因素导致细胞毒性T细胞出现的难以恢复的耗竭。因此急需找到新的免疫细胞治疗替代方案。
巨噬细胞，这一类同样具有肿瘤细胞杀伤能力的非特异性免疫细胞，由于在过继性免疫系统和先天性免疫系统的相互作用中起着核心作用，且备较强的可塑性，易于突破血脑屏障，以及更有效的向肿瘤组织部位浸润并长时间驻存的特性，正在成为免疫细胞工程化改造的新选择。然而以巨噬细胞进行过继移植存在着如下问题：1.具有细胞免疫功能的M1型巨噬细胞在肿瘤微环境容易的影响下容易向M2状态极化，进而失去免疫功能并转而促进肿瘤的发生发展；2.传统的病人自体免疫细胞回输治疗的方法面临细胞来源少，分离效率低，改造难度大，耗时耗力等问题，并且原代巨噬细胞很难过慢病毒感染的途径进行基因工程化改造。
发明内容
本公开提供了一种嵌合抗原受体，包括依次连接的胞外抗原结合域、跨膜结构域和胞内激活结构域；
所述胞外抗原结合域包括信号肽和/或靶向EGFRvIII的scFv；
跨膜结构域包括CD8α；
所述胞内激活结构域包括TIR、CD3ZETA或GM-CSFRα/β中的至少一种。
作为可选的技术方案，所述信号肽由SEQ ID NO.1所示的核苷酸序列表达；
所述scFv由SEQ ID NO.2所示的核苷酸序列表达。
作为可选的技术方案，所述CD8α由SEQ ID NO.3所示的核苷酸序列表达。
作为可选的技术方案，所述TIR包括来源于TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、TLR13或TLR19的胞内信号转导结构域；
所述TLR4的胞内信号转导结构域由SEQ ID NO.4所示的核苷酸序列表达；
所述CD3ZETA由SEQ ID NO.5所示的核苷酸序列表达；
所述GM-CSFRα/β由SEQ ID NO.6所示的核苷酸序列表达。
本公开提供了一种巨噬细胞的极化方法，将嵌合抗原受体在巨噬细胞中表达。
本公开提供了一种巨噬细胞，包括嵌合抗原受体。
本公开提供了一种巨噬细胞的制备方法，包括如下步骤：构建含有所述嵌合抗原受体的基因表达序列的慢病毒表达体系，利用慢病毒表达体系将嵌合抗原受体的基因表达序列整合到多能干细胞中，经诱导分化后制备得到巨噬细胞。
作为可选的技术方案，所述多能干细胞先诱导分化为单核细胞，再分化得到巨噬细胞。
本公开提供了一种能够分化得到上述巨噬细胞的多能干细胞，所述多能干细胞含有编码所述嵌合抗原受体的基因。
本公开提供了一种能够分化得到上述巨噬细胞的单核细胞，所述单核细胞含有编码所述嵌合抗原受体的基因；
所述单核细胞由多能干细胞分化得到。
本公开提供了一种巨噬细胞、多能干细胞或单核细胞在制备用于治疗神经胶质瘤的产品中的应用。
本公开提供了一种用于治疗神经胶质瘤的产品，所述产品包括上述巨噬细胞。
本公开提供一种药物制剂，包括所述巨噬细胞、所述多能干细胞或所述单核细胞。
在一些实施方式中，所述药物制剂包括药学上可接受的佐剂、稀释剂或载体中的一种或多种。
本公开提供所述嵌合抗原受体、所述巨噬细胞、所述多能干细胞、所述单核细胞或所述药物制剂在神经胶质瘤治疗中的用途。
本公开提供所述嵌合抗原受体、所述巨噬细胞、所述多能干细胞、所述单核细胞或所述药物制剂，用于神经胶质瘤治疗中的用途。
本公开提供一种治疗神经胶质瘤的方法，包括：
向有此需要的受试者给药治疗有效量的所述巨噬细胞、所述治疗神经胶质瘤的产品或所述药物制剂。
本公开提供一种治疗神经胶质瘤的方法，包括：
向有此需要的受试者给药治疗有效量的权利要求5所述的巨噬细胞、权利要求10所述的治疗神经胶质瘤的产品或权利要求11-12中任一项所述的药物制剂。
本公开提供胞内激活结构域用于制备治疗肿瘤或癌症的药物的用途，其中，所述胞内激活结构域包括TIR、CD3ZETA或GM-CSFRα/β中的至少一种。
在一些实施方式中，所述药物为巨噬细胞；所述巨噬细胞包括含有所述胞内激活结构域的嵌合抗原受体；
所述癌症选自与实体肿瘤相关的癌症，所述肿瘤选自脑部肿瘤。
在一些实施方式中，所述与实体肿瘤相关的癌症选自脑癌、食管癌、肝癌、胃癌、肠癌、肺癌、鼻咽癌中的一种。
在一些实施方式中，所述脑部肿瘤选自神经胶质瘤、神经纤维瘤、星形细胞瘤、少突胶质细胞瘤、髓母细胞瘤、室管膜瘤、松果体瘤中的一种。
附图说明
为了更清楚地说明本公开实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本公开的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为构建U87MG EGFRvIII细胞系；A：构建EGFRvIII慢病毒表达体系；B：蛋白质印迹法(Western Blotting)检测慢病毒感染后总的EGFR在U87MG细胞中的表达水平；C：蛋白质印迹法检测慢病毒感染后总的EGFRvIII在U87MG细胞中的表达水平；D：免疫荧光检测实验表明EGFRvIII在U87MG细胞中过表达后定位在了细胞膜表面；
图2为CAR-iMAC(嵌合抗原受体-巨噬细胞)的构建及其非特异性基础吞噬功能的检验；A：构建3种带有不同胞内激活信号转导结构域并能靶向GBM特异的膜蛋白EGFRvIII的CAR；B：稳定过表达了CAR的iPSC(诱导性多能干细胞)诱导分化成CAR-iMAC；C：CAR-iMAC(EGFP)体外吞噬U87MG(tdTomato)细胞；
图3为CAR-iMAC体外靶向杀伤U87MG EGFRvIII细胞系；A：体外共培养12小时后结果；B：共聚焦扫描结果；
图4为CAR-iMAC体外靶向杀伤U87MG EGFRvIII细胞系的流式分析和ELISA实验结果；A：流式分析技术统计共培养24小时后肿瘤细胞存活的数量；B：数据统计分析A中剩余肿瘤细胞比例；C：肿瘤细胞的存活率；D：ELISA实验检测免疫激活因子的释放情况；
图5为iPSC来源的iMAC在体内生存时间和实体肿瘤组织浸润性的检验；A：活体成像检测iMAC的存活信号和时间；B：数据统计A中iMAC的存活曲线；C：实验流程图；D：肿瘤组织中iMAC的浸润情况；E：免疫组化实验检测肿瘤组织内部iMAC的浸润情况；
图6为GBM小鼠肿瘤模型中验证CAR-iMAC的肿瘤杀伤效果；A：三种CAR-iMAC分别对GBM(Luciferin生物素发光)的杀伤效果；B：肿瘤信号的变化情况；C：不同CAR-iMAC治疗后GBM小鼠生存的时间；
图7为CD8-GM-CSFRα/β-CAR的结构图及其激活示意图；
图8为体外验证CD8-GM-CSFRα/β-CAR-iMAC靶向杀伤和依赖CAR免疫激活的效果；
图9为本公开中设计的三代CAR的结构示意图，以及不同CAR-iMAC依赖CAR而免疫激活的对比示意图。
具体实施方式
下面将结合实施方式和实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施方式和实施例仅用于说明本公开，而不应视为限制本公开的范围。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
如本文所用，术语“巨噬细胞(Macrophage)”通常是指一种由单核细胞(Monocyte)穿出血管后发育而来的髓样免疫细胞，广泛分布于机体组织的各个脏器中。其在正常组织中的主要生理作用是：通过加工和呈递抗原的方式介导特异性免疫反应；以固定细胞或游离细胞的形式吞噬并降解坏死细胞、碎片和异物，继而参与机体内非特异性反应；通过分泌炎症因子激活淋巴球或其他免疫细胞，进而协调炎症过程。
如本文所用，术语“嵌合性抗原受体(Chimeric Antigen Receptor，CAR)”通常是指主要由胞膜外抗原结合区、跨膜区和胞内信号传导区三部分构成。膜外区是一段具有靶向并结合肿瘤特异性抗原TAA功能的单链可变区结构域(single chain Fv domain,scFv)。跨膜区通常是由免疫球蛋白超家族组成,如CD8或CD28。胞内信号传导区主要是由可活化免疫细胞的激活受体的胞内信号转导结构域构成，如T细胞专属的共刺激因子(4-1BB或CD28)、信号活化区CD3ζ。装载嵌合性抗原受体的免疫细胞与肿瘤细胞表面抗原特异性结合后，膜外抗原结合区将信号传递到胞内信号活化区，继而启动免疫细胞活化反应。
如本文所用，术语“巨噬细胞的极化”通常是指在响应各种环境因素(例如，微生物产物，受损细胞，活化的淋巴细胞)或在不同的病理生理条件下，巨噬细胞转化为不同的功能表型，即经典活化性巨噬细胞(classically activated macrophages，M1)和选择活化性巨噬细胞(alternatively activated macrophages，M2)。成熟的巨噬细胞在各种因素下出现表型及形态分化，即巨噬细胞的极化现象。根据对环境刺激反应的不同，巨噬细胞主要被激活为M1和M2两种表型。在长期肿瘤微环境下，经IFN-γ和LPS等信号活化得到M1型，主要具有抗肿瘤和增强免疫的作用，能分泌炎症因子、趋化因子、效应分子和TNF-α等，其中以膜分子CD80、表面标记CD64等为代表。经IL-4和IL-13等因子活化得到M2型，主要具有抑制免疫反应、促进血管发生、组织修复和促进肿瘤生长的潜能，更多地分泌IL-10、TGF-β、VEGF等因子，以CD163和CD206相对高表达。
如本文所用，术语“多能干细胞(Pluripotent Stem Cell)”是一类具有自我更新、自我复制能力的多潜能细胞。在一定条件下，它可以分化成多种APSC多能细胞，多能干细胞(HSC)具有分化出多种细胞组织的潜能，但失去了发育成完整个体的能力，发育潜能受到一定的限制。
如本文所用，术语“诱导多能干细胞(induced pluripotent stem cell，iPSC)”通常是指是通过在成体细胞中转入多能性因子继而起始基因组表达谱重新编程得到的具有分化为多种细胞潜能的多能性干细胞。
如本文所用，术语“iMAC”通常是指由iPSC诱导分化而来的Macrophage。
如本文所用，术语“单核细胞”是指由骨髓中的造血干细胞分化而来，并在骨髓中发育的细胞。可可选的分化为成熟的巨噬细胞和树突状细胞的。单核细胞具有明显的变形运动的特点，具有吞噬和清除受伤、衰老的细胞及其碎片的能力。此外，单核细胞还参与免疫反应，将吞噬抗原后所携带的抗原决定簇转交给淋巴细胞，继而诱导淋巴细胞的特异性免疫性反应。单核细胞还具有识别和杀伤肿瘤细胞的能力。
如本文所用，术语“受试者”是指脊椎动物，可选地是哺乳动物，可选地是人类。哺乳动物包括但不限于鼠类、猿类、人类、家畜、竞技动物和宠物。在体内获得的或在体外培养的生物实体的组织、细胞及其子代也包括在内。
如本文所用，术语“治疗有效量”是指足以产生有益的或期望的结果的药剂的量。治疗有效量可能会根据：接受治疗的受试者和疾病状况、受试者的体重和年龄、疾病状况的严重程度、给药的方式等中的一个或多个因素的不同而变化，其可由本领域普通技术人员容易地确定。剂量可能会根据：所选择的特定药剂、遵循的剂量方案、是否与其他化合物联合给药、给药时间、待成像的组织以及携带药剂的物理递送系统中的一个或多个因素的不同而变化。
本公开的一些实施方式提供了一种嵌合抗原受体，包括依次连接的胞外抗原结合域、跨膜结构域和胞内激活结构域；
所述胞外抗原结合域包括信号肽和/或靶向EGFRvIII的scFv；
跨膜结构域包括CD8α；
所述胞内激活结构域包括TIR、CD3ZETA或GM-CSFRα/β中的至少一种。
本公开提供了一种嵌合抗原受体中，胞外抗原结合域包括信号肽和/或scFv，信号肽可帮助CAR蛋白序列定位到细胞膜表面，scFv能够异性识别GBM特异表达的细胞膜表面蛋白EGFRvIII；跨膜结构域包括CD8α，连接胞外抗原结合域和胞内激活结构域；胞内激活结构域包括TIR、CD3ZETA或GM-CSFRα/β，促进巨噬细胞向M1型极化，将本公开提供的嵌合抗原受体引入到巨噬细胞中，在赋予该巨噬细胞对GBM的靶向杀伤作用的同时，也增强了该免疫细胞抵御肿瘤微环境的免疫抑制效应，有效的促进并维持其M1极化状态。
作为可选的技术方案，所述信号肽由SEQ ID NO.1所示的核苷酸序列表达；
所述scFv由SEQ ID NO.2所示的核苷酸序列表达。
在一些实施方式中，信号肽具有由SEQ ID NO.1所示的核苷酸序列编码的氨基酸序列；
在一些实施方式中，scFv具有由SEQ ID NO.2所示的核苷酸序列编码的氨基酸序列。
作为可选的技术方案，所述CD8α由SEQ ID NO.3所示的核苷酸序列表达。
在一些实施方式中，CD8α具有由SEQ ID NO.3所示的核苷酸序列编码的氨基酸序列。
作为可选的技术方案，所述TIR包括但不限于来源于TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、TLR13或TLR19的胞内信号转导结构域；
所述TLR4的胞内信号转导结构域由SEQ ID NO.4所示的核苷酸序列表达；
所述CD3ZETA由SEQ ID NO.5所示的核苷酸序列表达；
所述GM-CSFRα/β由SEQ ID NO.6所示的核苷酸序列表达。
在一些实施方式中，TLR4的胞内信号转导结构域具有由SEQ ID NO.4所示的核苷酸序列编码的氨基酸序列；
在一些实施方式中，CD3ZETA具有由SEQ ID NO.5所示的核苷酸序列编码的氨基酸序列；
在一些实施方式中，GM-CSFRα/β具有由SEQ ID NO.6所示的核苷酸序列编码的氨基酸序列。
本公开的一些实施方式提供了一种巨噬细胞的极化方法，将嵌合抗原受体在巨噬细胞中表达。
在本公开中，通过将上述嵌合抗原受体在巨噬细胞中表达，以实现巨噬细胞的M1极化，该方法简单有效。
本公开的一些实施方式提供了一种巨噬细胞，包括嵌合抗原受体。
该巨噬细胞能够表达上述本公开提供的嵌合抗原受体，能够维持M1极化状态，对GBM的靶向杀伤性强。
本公开中设计的三代CAR的结构示意图，以及不同CAR-iMAC依赖CAR而免疫激活的对比示意图如图9所示。
图9中的A展示Truncate CAR主要由胞外特异性识别EGFRvIII蛋白的scFv结构域和CD8α跨膜结构域组成。缺乏胞内信号转导结构域。第一代CAR主要由胞外特异性识别EGFRvIII蛋白的scFv结构域、CD8α跨膜结构域和胞内CD3ZETA信号转导结构域构成。第二代CAR主要由胞外特异性识别EGFRvIII蛋白的scFv结构域、CD8α跨膜结构域和专属于巨噬细胞的胞内信号转导结构域构成。该胞内信号转导结构域分别是单个的TIR或GM-CSFRα/β激活结构域。第三代CAR是在第二代CAR的基础上升级而来。区别在于第三代CAR的胞内信号转导结构域分别由CD3ZETA、TIR和GM-CSFRα/β两两之间通过Linker连接组合而成，以达到更强、更持久的免疫激活效果。
图9中的B-E展示了不同CAR-iMAC依赖CAR而免疫激活的示意图。由于缺乏胞内信号转导结构域，Truncate CAR-iMAC不具备依赖CAR而免疫激活的功能。第一代CAR胞外的scFv靶向识别EGFRvIII后，其胞内的CD3ZETA信号转导结构域被激活，进而起始第一代CAR-iMAC的免疫激活和少量的炎症因子释放。TIR的激活可刺激巨噬细胞免疫激活，释放IL1、IL6、TNFα等炎症因子，并向M1型极化。GM-CSFRα/β的激活可促进巨噬细胞的大量扩增，免疫激活，释放IL12、IL23、TNFα等炎症因子，并向M1型极化。因此，相对第一代CAR-iMAC，第二代CAR-iMAC在靶向EGFRvIII后，在肿瘤微环境中显著的提高了生存扩增，M1极化，以及免疫激活等抗肿瘤能力。当第三代CAR胞外的scFv靶向识别EGFRvIII后，其胞内将有TIR、CD3ZETA和GM-CSFRα/β当中的其中2个信号转导结构域协同发挥免疫激活的作用，可利用不同免疫激活信号通路的优势，进而发挥更强、更持久的免疫激活效果。
本公开的一些实施方式提供了一种巨噬细胞的制备方法，包括如下步骤：构建含有所述嵌合抗原受体的基因表达序列的慢病毒表达体系，利用慢病毒表达体系将嵌合抗原受体的基因表达序列整合到多能干细胞中，经诱导分化后制备得到巨噬细胞；
作为可选的技术方案，所述多能干细胞先诱导分化为单核细胞，再分化得到巨噬细胞。
公开人研究发现，原代巨噬细胞很难被慢病毒感染，很难被编辑和进行工程化改造，基于此，本公开提供了一种上述巨噬细胞的制备方法，采用本公开方法制备得到的巨噬细胞能够长期稳定的过表达嵌合抗原受体，能够解决目前免疫细胞治疗所存在的编辑效率低、周期长、工作量大和治疗不及时等方面的问题。
本公开的一些实施方式提供了一种能够分化得到上述巨噬细胞的多能干细胞，所述多能干细胞含有编码所述嵌合抗原受体的基因。
由于该多能干细胞含有编码上述嵌合抗原受体的基因，其状态更倾向于促炎抑肿瘤的M1状态，能够可选地分化得到巨噬细胞，更有利于抑制肿瘤。
本公开的一些实施方式提供了一种能够分化得到上述巨噬细胞的单核细胞，所述单核细胞含有编码所述嵌合抗原受体的基因；
所述单核细胞由多能干细胞分化得到。
本公开的一些实施方式提供了一种巨噬细胞、多能干细胞或单核细胞在制备用于治疗神经胶质瘤的产品中的应用。
由于本公开提供的巨噬细胞能够维持M1极化状态，对GBM的靶向杀伤性强，因此，能够用于制备治疗神经胶质瘤的产品，而多能干细胞能够分化得到上述巨噬细胞，因此同样能够用于制备治疗神经胶质瘤的产品。
本公开的一些实施方式提供了一种用于治疗神经胶质瘤的产品，所述产品包括上述巨噬细胞。
本公开提供的巨噬细胞能够维持M1极化状态，对GBM的靶向杀伤性强，包括该巨噬细胞在内的产品也同样具有治疗神经胶质瘤的作用。
本公开的一些实施方式提供了一种药物制剂，包括巨噬细胞、多能干细胞或单核细胞。
在一些实施方式中，药物制剂包括药学上可接受的佐剂、稀释剂或载体中的一种或多种。
本公开的一些实施方式提供了嵌合抗原受体、巨噬细胞、多能干细胞、单核细胞或药物制剂在神经胶质瘤治疗中的用途。
本公开的一些实施方式提供了嵌合抗原受体、巨噬细胞、多能干细胞、单核细胞或药物制剂，用于神经胶质瘤治疗中的用途。
本公开的一些实施方式提供了一种治疗神经胶质瘤的方法，包括：
向有此需要的受试者给药治疗有效量的巨噬细胞、治疗神经胶质瘤的产品或药物制剂。
本公开的一些实施方式提供胞内激活结构域用于制备治疗肿瘤或癌症的药物的用途，其中，胞内激活结构域包括TIR、CD3ZETA或GM-CSFRα/β中的至少一种。
在一些实施方式中，该药物为巨噬细胞；该巨噬细胞包括含有所述胞内激活结构域的嵌合抗原受体；所述癌症选自与实体肿瘤相关的癌症，所述肿瘤选自脑部肿瘤。
在一些实施方式中，所述与实体肿瘤相关的癌症选自脑癌、食管癌、肝癌、胃癌、肠癌、肺癌、鼻咽癌等中的一种。
在一些实施方式中，所述脑部肿瘤选自神经胶质瘤、神经纤维瘤、星形细胞瘤、少突胶质细胞瘤、髓母细胞瘤、室管膜瘤、松果体瘤等中的一种。
本公开的胞内激活结构域可广泛应用于多种靶向其他肿瘤抗原的巨噬细胞，具有广泛的适用性，均可以有效激活巨噬细胞，促进巨噬细胞向免疫激活方向的极化。
本公开提供了一种专属于巨噬细胞的嵌合抗原受体，包括依次连接的胞外抗原结合域、跨膜结构域和胞内激活结构域。其中胞外抗原结合域包括信号肽和/或scFv，信号肽可帮助CAR蛋白序列定位到细胞膜表面，scFv能够异性识别GBM特异表达的细胞膜表面蛋白EGFRvIII；跨膜结构域包括CD8α，连接胞外抗原结合域和胞内激活结构域；胞内激活结构域包括TIR、CD3ZETA或GM-CSFRα/β中的至少一种，本公开的胞内激活结构域为巨噬细胞特异性的胞内激活结构域，可以有效激活巨噬细胞，促进巨噬细胞向免疫激活方向的极化，即促进巨噬细胞向M1型极化；此外本公开的胞内激活结构域对于靶向其他肿瘤抗原的巨噬细胞具有广泛的适用性，均能实现上述的计划效果。同时，GM-CSFRα/β结构域可有效维持巨噬细胞的monocyte样状态。该状态有利于巨噬细胞向肿瘤组织内部浸润。将本公开提供的嵌合抗原受体引入到巨噬细胞中，在赋予该巨噬细胞对GBM的靶向杀伤作用的同时，也增强了该免疫细胞抵御肿瘤微环境的免疫抑制效应，有效的促进并维持其M1极化状态。
本公开提供的嵌合抗原受体，能够赋予巨噬细胞靶向GBM的特性，降低脱靶效应，促进和维持巨噬细胞在肿瘤微环境的M1极化状态，提高其杀伤效率。本公开提供的巨噬细胞的极化方法，该方法简单，能够实现巨噬细胞的M1极化。本公开提供的巨噬细胞，可以解决上述问题中的至少一种。
本公开提供的巨噬细胞的极化方法，通过将上述嵌合抗原受体在巨噬细胞中表达，以实现巨噬细胞的M1极化，该方法简单有效。
本公开提供的巨噬细胞，该巨噬细胞能够表达上述本公开提供的嵌合抗原受体，能够维持M1极化状态，对GBM的靶向杀伤性强。
发明人研究发现，原代巨噬细胞很难被慢病毒感染，很难被编辑和进行工程化改造，基于此，本公开提供了一种上述巨噬细胞的制备方法，包括构建含有所述嵌合抗原受体的基因表达序列的慢病毒表达体系，利用慢病毒表达体系将嵌合抗原受体的基因表达序列整合到多能干细胞中，经诱导分化后制备得到巨噬细胞，该巨噬细胞能够长期稳定的过表达嵌合抗原受体，能够解决目前免疫细胞治疗所存在的编辑效率低、周期长、工作量大和治疗不及时等方面的问题。
本公开提供了的多能干细胞含有编码上述嵌合抗原受体的基因，其状态更倾向于促炎抑肿瘤的M1状态，能够可选的分化得到巨噬细胞，更有利于抑制肿瘤。
下面通过实施例和对比例说明本公开，但是，应当理解为，这些实施例仅仅是用于更详细地说明之用，而不应理解为用于以任何形式限制本公开。
实施例
实施例1
构建表达EGFRvIII抗原的GBM细胞系。
获取GBM特异性膜蛋白EGFRvIII的表达序列，并借助分子克隆手段将其克隆到慢病毒表达质粒Lenti-EF1A-PGK-Puro中，构建得到的EGFRvIII慢病毒表达体系，如图1中的A所示。借助慢病毒表达体系将GBM特异性的膜蛋白EGFRvIII分别表达在GBM细胞系U87MG和LN-229(来源于浙大医学院刘冲课题组保存)中，得到稳定表达该膜蛋白的U87MG EGFRvIII细胞系。
采用Western Blotting分别检测慢病毒感染后总的EGFR和EGFRvIII在U87MG细胞中的表达水平，实验步骤如下：
1.细胞总蛋白样品的制备
(1)将野生型U87MG细胞和通过慢病毒过表达了EGFRvIII的U87MG细胞接种到6CM细胞培养皿中，于37℃/5％CO 2细胞培养箱中培养至长满培养皿。
(2)用移液器弃去细胞培养液。每个培养皿中加入3ml 4℃预冷的1×PBS(0.01M pH7.2～7.3)。平放轻轻摇动1min洗涤细胞，然后弃去洗液。重复以上操作两次，共洗细胞三次以洗去培养液。将PBS弃净后把培养皿置于冰上。
(3)每个细胞培养皿中加入500μl含蛋白酶抑制剂的RIPA裂解液，于冰上裂解30min，为使细胞充分裂解培养皿要置于摇床轻轻摇动。
(4)裂解完后，用干净的刮棒将细胞刮于培养瓶的一侧(动作要快)，然后用枪将细胞碎片和裂解液移至1.5ml离心管中。
(5)于4℃下12000rpm离心20min。
(6)将离心后的上清分装转移到新的1.5ml的离心管中。
(7)在上一步得到的细胞裂解液中加入使用终浓度为1×，并含有β-巯基乙醇的5×SDS上样缓冲液(loading buffer)。混匀后，与95℃金属浴中加热5分钟，使蛋白变性。-20℃或冰上保存备用。
2.SDS凝胶电泳及转膜
(8)制备好凝胶电泳体系，并将上一步得到的蛋白样品各10μl，以及5μl蛋白标志物(marker)加入到相应的上样孔中。
(9)设置电泳条件为100V恒压，电泳至溴酚兰刚跑出即可终止电泳，进行转膜。
(10)将上一步得到的凝胶置于湿式转膜体系中，于100V恒压条件下，电转约120分钟，使凝胶中的蛋白充分转移到PVDF膜上。
3.免疫反应
(11)将上一步转移有蛋白的PVDF膜快速取出，置于TBST溶液中洗净转膜液后，移至用TBST配制5％脱脂牛奶中，室温封闭1个小时。期间要在水平摇床上轻摇。
(12)通过PVDF上蛋白标志物的标识，预测目的蛋白(EGFRvIII、EFGR和内参β-ACTIN)的所在位置，用剪刀将含有目的蛋白的膜分别剪至适宜大小。
(13)将以上得到的含有目的蛋白的膜分别置于含有EGFRvIII、EFGR和β-ACTIN一抗的5％脱脂牛奶中，4℃过夜孵育(10-12小时)。
(14)从上一部中取出孵育了一抗的PVDF膜，在室温条件下，于水平摇床上用TBST清洗三次，每次10分钟。
(15)根据种属匹配原则，将上一步清洗后的PVDF膜置于稀释了相同种属来源且偶联了HRP的二抗的5％脱脂牛奶中，室温摇床孵育1小时。
(16)从上一部中取出孵育了一抗的PVDF膜，在室温条件下，于水平摇床上用TBST清洗三次，每次10分钟。
4.化学成像
(17)用吸水纸吸干上一步清洗过后的PVDF膜上的TBST，正面朝上，置于化学发光成像仪中。用移液器快速的向膜的正面适量预先配制好的ECL化学发光液。设置好成像条件后，成像保存。
结果如图1中的B和图1中的C所示，表明构建得到的U87MG EGFRvIII细胞系中，EGFR和EGFRvIII高表达。
免疫荧光实验步骤如下：
第一天：
1.将野生型U87MG细胞和通过慢病毒过表达了EGFRvIII的U87MG细胞接种到铺有细胞爬片的24孔细胞培养板中。
2.于37℃/5％CO 2细胞培养箱中培养12个小时。
第二天：
3.在培养板中将已爬好细胞的玻片用PBS浸洗3次，每次3min。
4.用4％的多聚甲醛固定爬片15min，PBS浸洗玻片3次，每次3min。
5. 0.5％Triton X-100(PBS配制)室温通透20min(细胞膜上表达的抗原省略此步骤)。
6.PBS浸洗玻片3次，每次3min，吸水纸吸干PBS，在玻片上滴加2.5％BSA，室温封闭30min(或37℃20min)。
7.吸水纸吸掉封闭液，不洗，每张玻片滴加足够量的稀释好的；EGFRvIII一抗并放入湿盒，4℃孵育过夜(一般要大于18小时)，或37℃，1-2小时。
第三天：
8.加荧光二抗：PBST浸洗爬片3次，每次3min，吸水纸吸干爬片上多余液体后滴加稀释好的荧光二抗，湿盒中20-37℃孵育1h，PBST浸洗切片3次，每次3min；注意：从加荧光二抗起，后面所有操作步骤都尽量在较暗处进行。
9.复染核：滴加DAPI避光孵育5min，对标本进行染核，PBST 5min×4次洗去多余的DAPI；用吸水纸吸干爬片上的液体，用含抗荧光淬灭剂的封片液封片，然后在荧光显微镜下观察采集图像。
免疫荧光检测实验表明EGFRvIII在U87MG细胞中过表达后定位在了细胞膜表面(图1中的D)。
实施例2
构建CAR-iMAC，并通过体外实验探究其对GBM细胞系的靶向杀伤功能。
1.构建iPSC诱导分化成iMAC的方法
1)将PBMC(外周血单个核细胞)重编程为iPSC
从新鲜血液中分离出来的PBMC具有数量多、容易获取的优点，是诱导iPSC的绝佳材料。分离得到PBMC后，利用电转法将表达OCT3/4、SOX2、KLF4、L-MYC和LIN28A五种转录因子的表达载体转染至PBMC中，诱发其重编程，最终得到iPSC。
2)基于拟配体(EB)形成诱导iPSC分化为单核细胞(iMONO)和M1型巨噬细胞(iMAC)，实验步骤如下：
1.EB形成(第0天)。当iPSC培养到覆盖培养皿的60-80％的面积的时候，利用versene将其消化成单个细胞或较小的细胞聚集块儿。600rpm，3min离心后，用含有Y-27632的mTeSR1培养基重悬，并以1:2或1：3的比例接种到低吸附六孔板中。于37℃/5％CO 2培养箱中，摇床培养约24小时。便可形成EB。
2.原条和中胚层诱导(第1天)。去除步骤1中培养EB的培养基。加入MI培养基(APEL II培养基(medium)+10ng/mL BMP4+5ng/mL bFGF)。继续以步骤1的条件培养24小时。
3.造血干细胞(HSC)诱导(第2-7天)。去除步骤2中的MI培养基。加入HS培养基(APEL II培养基+10ng/mL BMP4+5ng/mL bFGF+50ng/ml VEGF+100ng/mL SCF)。以步骤1的条件继续培养。
4.髓系细胞和单核前体细胞(MPC)扩增(第8-10天)。去除步骤3中的HS培养基。加入ME-1培养基(APEL II培养基+5ng/mL bFGF+50ng/ml VEGF+100ng/mL SCF+10ng/mL IGF1+25ng/mL IL-3+50ng/mL M-CSF+50ng/mL GM-CSF(粒细胞-巨噬细胞集落刺激因子))。以步骤1的条件继续培养。
5.iMONO的诱导(约第11天)。将步骤4中的EB转移到基质胶(Matrigel)包被的六孔板中。加入ME-2培养基(StemSpan-XF培养基+5ng/mL bFGF+50ng/ml VEGF+100ng/mL SCF+10ng/mL IGF1+25ng/mL IL-3 +50ng/mL M-CSF+50ng/mL GM-CSF),于37℃/5％CO 2培养箱中静置培养。在这个时间段里面会有呈单个细胞状态的iMONO不断的漂浮在培养基中。换液并离心收集iMONO。
6.iMAC的成熟(约第17)。将步骤5中收集到的iMONO收集到新的Matrigel包被的六孔板中。加入MM培养基(StemSpan-XF培养基+5ng/mL bFGF+50ng/ml VEGF+100ng/mL SCF+10ng/mL IGF1+25ng/mL IL-3+100ng/mL M-CSF+100ng/mL GM-CSF),于37℃/5％CO 2培养箱中静置培养。这个时间段里面会看到体积较小的iMONO向体积较大并呈空泡状的iMAC成熟。
7.iMAC的M1极化。收集步骤6中成熟的iMAC，加入含有100ng/ml LPS和100ng/ml IFN-γ的MS培养基(RPMI1640+100ng/mL M-CSF+100ng/mL GM-CSF)，大约24小时便可得到极化的iMAC。
该过程诱导分化只需要13-28天。这期间会有源源不断的iMONO和iMAC产生。因此分化效率高且活性好的iMONO和iMAC为后续大量的免疫细胞需求提供了保障。
3)检验iPSC来源的iMAC的巨噬细胞特性
通过流式分析技术检测iMAC表面标志性抗原，单细胞RNA-seq术分析iMAC基因表达谱，ELISA技术检测细胞因子分泌等方法确定iPSC成功诱导分化为了M1型iMAC。
2.构建具有靶向GBM功能的CAR-iMONO或CAR-iMAC
1)设计特异识别EGFRvIII的嵌合抗原受体
获取靶向EGFRvIII的sc-Fv的基因表达序列。利用分子克隆的方法将该序列与CD8α的跨膜结构域基因表达序列，以及TIR、CD3ZETA结构域表达序列组合克隆到慢病毒表达质粒Lenti-EF1A-T2A-EGFP-Puro中，形成融合表达的CAR序列(图2中的A)。同时，拟设计不含胞内激活结构域的CAR(scFv-CD8-DELTA-CAR)作为检验胞内信号转导结构域能否发挥免疫激活功能的阴性对照。
其中，信号肽是一段含有疏水性氨基酸序列的短肽链。具有引导新合成的蛋白质跨膜或者分泌到胞外的作用。信号肽的表达序列由SEQ ID NO.1所示的核酸序列表达：
Figure PCTCN2021117808-appb-000001
sc-Fv的表达序列为由SEQ ID NO.2所示的核苷酸序列表达。
在一种或多种实施方式中，sc-Fv具有由SEQ ID NO.2所示的核苷酸序列编码的氨基酸序列：
Figure PCTCN2021117808-appb-000002
CD8α由SEQ ID NO.3所示的核苷酸序列表达。
在一种或多种实施方式中，CD8α具有由SEQ ID NO.3所示的核苷酸序列编码的氨基酸序列：
Figure PCTCN2021117808-appb-000003
TIR由SEQ ID NO.4所示的核苷酸序列表达。
在一种或多种实施方式中，TIR具有由SEQ ID NO.4所示的核苷酸序列编码的氨基酸序列：
Figure PCTCN2021117808-appb-000004
CD3ZETA由SEQ ID NO.5所示的核苷酸序列表达。
在一种或多种实施方式中，CD3ZETA具有由SEQ ID NO.5所示的核苷酸序列编码的氨基酸序列：
Figure PCTCN2021117808-appb-000005
GM-CSFRα/β由SEQ ID NO.6所示的核苷酸序列表达。
在一种或多种实施方式中，GM-CSFRα/β具有由SEQ ID NO.6所示的核苷酸序列编码的氨基酸序列：
Figure PCTCN2021117808-appb-000006
2)构建CAR-iMONO或CAR-iMAC
利用慢病毒系统分别将三种不同CAR的表达序列整合到iPSC当中。借助绿色荧光表达和单细胞RNA-seq等技术手段获取稳定表达CAR的细胞系(CAR-iPSC)。而后，将该三种CAR-iPSC诱导分化成依赖CAR激活的CAR-iMONO或CAR-iMAC。
构建CAR-iMAC，并进行非特异性基础吞噬功能检验，实验步骤如下：
1.将一定数量的表达绿色荧光蛋白的CAR-iMAC接种到六孔板中，于含有100ng/ml LPS、100ng/ml人源INF-γ(γ干扰素)、100ng/ml M-CSF(巨噬细胞集落刺激因子)和100ng/ml GM-CSF的RPMI1640培养基中极化24小时。期间置于37℃/5％CO 2细胞培养箱中。
2.将以上得到的极化了的CAR-iMAC和表达樱桃红(tdTomato)红色荧光蛋白的野生型LN-229细胞以10/1的比例接种到玻璃皿中，用含有100ng/ml M-CSF和100ng/ml GM-CSF的RPMI1640培养基，于37℃/5％CO 2细胞培养箱中共培养24小时。
3.将上一步的玻璃皿置于共聚焦荧光扫描显微镜中，设置好参数后，扫描观察红色荧光和绿色荧光的共定位情况，并拍照保存。
CAR-iMAC的构建及其非特异性基础吞噬功能的检验如图2所示，结果表明，CAR-iMAC具有基础性吞噬功能。
实施例3
体外评估CAR-iMAC杀伤GBM细胞系的能力。
按3:1、5:1、10:1的效靶比分别将表达以上三种不同的CAR-iMAC和对照组细胞iMAC在体外与表达荧光素酶的U87MG EGFRvIII细胞在活细胞工作站中共培养，比对含有不同胞内激活结构域的CAR提升CAR-iMAC靶向GBM的效果和促进其免疫激活的效果。荧光素酶检测实验和肿瘤细胞流式计数实验检测不同的CAR-iMAC靶向杀伤作用。 依据以上指标对比scFv-CD8-TIR-CAR相比scFv-CD8-DELTA-CAR和scFv-CD8-CD3ZETA-CAR对CAR-iMAC极化和激活的效果。结果如图3所示。
其中，图3中的A的实验步骤如下：
1.将一定数量的表达绿色荧光蛋白的3种CAR-iMAC和WT-iMAC(野生型-巨噬细胞)接种到六孔板中，于含有100ng/ml LPS(脂多糖)、100ng/ml人源INF-γ、100ng/ml M-CSF和100ng/ml GM-CSF的RPMI1640培养基中极化24小时。期间置于37℃/5％CO 2细胞培养箱中。
2.将以上得到的极化了的3种CAR-iMAC和WT-iMAC分别与表达tdTomato红色荧光蛋白的U87MGEGFRvII细胞以10：1的比例接种到玻底皿中，用含有100ng/ml M-CSF和100ng/ml GM-CSF的RPMI1640培养基,于37℃/5％CO 2细胞培养箱中共培养12小时。
3.将上一步的玻底皿置于OLYPUS荧光显微镜中，设置好参数后，观察红色荧光和绿色荧光的分布情况，并拍照保存。
图3中的B的实验步骤如下：
1.将一定数量的表达绿色荧光蛋白的3种CAR-iMAC和WT-iMAC接种到六孔板中，于含有100ng/ml LPS、100ng/ml人源INF-γ、100ng/ml M-CSF和100ng/ml GM-CSF的RPMI1640培养基中极化24小时。期间置于37℃/5％CO 2细胞培养箱中。
2.将以上得到的极化了的3种CAR-iMAC和WT-iMAC分别与表达tdTomato红色荧光蛋白的U87MG EGFRvII细胞以10：1的比例接种到玻底皿中，用含有100ng/ml M-CSF和100ng/ml GM-CSF的RPMI1640培养基,于37℃/5％CO 2细胞培养箱中共培养24小时。
3.将上一步的玻底皿置于共聚焦荧光扫描显微镜中，设置好参数后，扫描观察红色荧光和绿色荧光的共定位情况，并拍照保存。
图4中的A和B图的实验步骤：
1.将表达绿色荧光蛋白的3种CAR-iMAC和WT-iMAC极化24小时后分别与表达tdTomato红色荧光蛋白的U87MG EGFRvII细胞分别以3：1,5:1,以及10:1的比例于六孔板中共培养24小时(方法同图3中的B图)。
2.胰蛋白酶消化并收集共培养的细胞，于流式分析仪中检测并统计分析3种CAR-iMAC、WT-iMAC和U87MG EGFRvII细胞的数量。设置参数为：绿色荧光蛋白为FITC通道，tdTomato为PE通道。
图4中的C图实验步骤：
1.将表达绿色荧光蛋白的3种CAR-iMAC极化24小时后分别与表达荧光素酶基因荧光素酶(luciferase)的U87MG EGFRvII细胞以10：1的比例于避光的96孔板中共培养24小时。并设置只有U87MG EGFRvII细胞的培养孔作为阴性对照(NC)(方法同图3中的B图)。
2.在共培养体系中每孔加入100μl浓度为10mg/ml的luciferin，并快速的置于多功能酶标仪中检测荧光信号。
图4中的D图实验步骤：
1.收集A图中共培养体系的培养基上清，1000rpm离心20分钟。
2.将上一步得到的培养基上清100μl加入到白介素12(IL-12)酶联免疫吸附测定试剂盒的相应检测孔中。通过ELISA实验检测3种CAR-iMAC和WT-iMAC分别与表达tdTomato红色荧光蛋白的U87MG EGFRvII细胞以10：1的比例于共培养后，IL-12的分泌情况，统计并分析检测数据。
结果显示，靶向EGFRvIII的三种CAR-iMAC(EGFP)与U87MG EGFRvIII(tdTomato)体外共培养12小时后，显示不同CAR-iMAC相对于WT-iMAC对U87MG EGFRvIII粘附性更强，同时U87MG EGFRvIII细胞胞体要明显大于CAR-iMAC(图3中的A)。共聚焦扫描显示三种CAR-iMAC和WT-iMAC都具有胞啃U87MG EGFRvIII的能力(图3中的B)。
采用流式分析技术统计三种CAR-iMAC和WT-iMAC分别以3:1、5:1以及10:1的效靶比与U87MG EGFRvIII共培养24小时后肿瘤细胞存活的数量。结果显示scFv-CD8-TIR-CAR-iMAC和scFv-CD8-CD3ZETA-CAR-iMAC均具备显著杀伤U87MG EGFRvIII的能力，并随着效靶比的增加杀伤效果越显著(图4中的A和B)。
三种CAR-iMAC按不同的效靶比分别与稳定表达荧光素酶U87MG EGFRvIII细胞体外共培养24小时后，通过检测检测荧光素酶活性来表征该肿瘤细胞的存活率。结果如图4中的C所示，表明在5/1和10/1的效靶比下，scFv-CD8-TIR-CAR-iMAC和scFv-CD8-CD3ZETA-CAR-iMAC相对于scFv-CD8-DELTA-CAR-iMAC具有显著杀伤U87MG EGFRvIII细胞的能力。
三种CAR-iMAC和WT-iMAC按10/1的效靶比分别与U87MG EGFRvIII共培养24小时后，ELISA实验检测免疫激活因子的释放情况。结果如3中的F所示，表明scFv-CD8-TIR-CAR-iMAC和scFv-CD8-CD3ZETA-CAR-iMAC相对WT-iMAC和scFv-CD8-DELTA-CAR-iMAC分泌的促炎因子IL12显著上调。
实施例4
iPSC来源的iMAC在体内生存时间和实体肿瘤组织浸润性的检验，结果如图5所示。NSG小鼠腹腔注射荧光染料Dir标记的iMAC，并通过活体成像检测iMAC的存活信号和时间，如图5中的A所示。数据统计图5中的A图中iMAC的存活曲线(图5中的B)。表明iPSC来源的iMAC可以在肿瘤模型中存在的时间超过30天，满足肿瘤免疫细胞治疗对免疫细胞所需生存时间的要求。
向NSG小鼠腹腔注射1×10 6个过表达荧光素酶(Luciferase)基因的U87MG细胞，4小时后原位注射相同数量的iMAC，30天后，分离肿瘤组织，并分别通过Dir和荧光素(Luciferin)成像观察肿瘤组织中iMAC的浸润情况。结果如图5中的D所示，表明在GBM中有iMAC的存在。采用免疫组化实验进一步检测肿瘤组织内部iMAC的浸润情况，实验步骤如下：
1.在腹腔GBM小鼠模型中注射iMAC 30天后，分离肿瘤组织。并于4％的PFA(多聚甲醛)中固定一周时间。
2.包埋组织：先在铁模具中加入一些液态石蜡，先稍微冷却，然后再将上一步固定的肿瘤组织置于石蜡之中，再将塑料模具盒盖上，最后加入少许液体石蜡，进行冷冻，使石蜡变成固态。
3.切片：将包埋好的肿瘤组织从模具上取下来，并置于石蜡切片机上，切片机通过调节上下左右来使组织和切割方向一致，然后调节切片的厚度，一般为5μm，用手笔将切割的载玻片向外拉，并用小镊子将包含有完整组织的载玻片置于40℃温水中。
4.捞组织：赶走水浴中的气泡，组织受热展开后，用载玻片下1/2以同一方向捞取5-6张组织，再将捞出来的载玻片置于架子上，放入37℃温箱中烘干。
5.脱蜡：依次将载玻片放入二甲苯-二甲苯、100％酒精、100％酒精、95％酒精、90％酒精、80％酒精和70％酒精中脱蜡，每个试剂中放10-15min。
6.抗原修复：脱蜡后在清水中冲洗一段时间，加入柠檬酸缓冲液，放入微波炉中蒸煮3min(中火)，刚到沸腾即可。室温冷却，然后再蒸煮一次，冷却至室温，以使抗原的位点暴露出来。
7.血清封闭：冷却至室温后，将柠檬酸缓冲液倒掉，水洗2次，并将载玻片置于PBS中5min，洗2次，擦干组织周围的PBS液。马上加上用PBS稀释十倍后的血清，使一些非特异性的位点封闭起来，然后放入37度温箱中半小时。
8.加一抗：将温箱中的载玻片取出，用吸水纸擦干载玻片的反面和正面组织周围的血清，分别滴加CD11b、CD86和CD206的一抗。于4度冰箱中保存过夜。
9.加二抗：将载玻片从冰箱中取出，放入PBS中洗3次，每次5min，擦干组织周围的PBS后加上相同属种的二抗，然后置于37度温箱中半小时。
10.加SABC：将片子从温箱中取出，放入PBS中洗3次，每次5min，擦干组织周围的PBS后加上用PBS稀释100倍的SABC，然后置于37℃温箱中半小时。
11.加显色剂：将片子从温箱中取出，放入PBS中洗3次，每次5min，擦干组织周围的PBS后加上显色剂。(显色剂的配置：在1ml水中加1滴显色剂A，摇匀，然后加1滴显色剂B，摇匀，再加1滴显色剂C,摇匀。A:DAB，B:H202，C:磷酸缓冲液。
12.复染：将显色后的片子用清水冲洗一段时间后，浸泡于苏木精中染色，一般动物组织为半分钟，植物组织3-5min。
13.脱水：将复染后的片子置于水中冲洗后，依次将载玻片放入70％酒精-80％酒精-90％酒精-95％酒精-100酒精-二甲苯-二甲苯。每个试剂中放置2min，最后浸泡在二甲苯中，搬到通风柜中。
14.封片：用中性树胶滴在组织旁边，再用盖玻片轻轻盖上(要先放平一侧，然后轻轻放下另一侧，以免产生气泡)，封好片子后置于通风柜中晾干。
15.成像：将以上的达到的肿瘤组化样品置于OLYMPUS正置显微镜中，设置成像拍照。
结果如图5中的E所示，表明iMAC具备向实体肿瘤内部浸润的能力，并以M2型巨噬细胞的形式存在。
实施例5
构建GBM小鼠腹腔肿瘤模型
分别将2×10 6个过表达了荧光素酶基因的U87MG EGFRvIII细胞通过腹腔注射的方式接种到NSG小鼠腹腔内，构建腹腔GBM小鼠模型。
GBM模型体内CAR-iMAC抗肿瘤能力的验证
将以上三种不同的CAR-iMAC，及其对照细胞iMAC过继移植到腹腔GBM小鼠模型中。通过小鼠活体成像技术适时观察并记录肿瘤的变化、小鼠体重和生存状态的变化情况。结果如图6所示。
在NSG小鼠GBM腹腔模型分别注射三种CAR-iMAC(scFv-CD8-DELTA-CAR-iMAC、scFv-CD8-TIR-CAR-iMAC、scFv-CD8-CD3ZETA-CAR-iMAC)(DiR)和PBS后，通过活体成像技术展示在效靶比为10/1的情况下，三种CAR-iMAC分别对GBM(Luciferin生物素发光)的杀伤效果如图6中的A所示，并分别GBM在三种CAR-iMAC存在的情况下肿瘤信号的变化情况以及治疗后GBM小鼠生存的时间(图6中的B和C)，结果表明scFv-CD8-TIR-CAR-iMAC相对scFv-CD8-CD3ZETA-CAR-iMAC具备更强的体内抗肿瘤功能。
实施例6
一种嵌合抗原受体CD8-GM-CSFRα/β-CAR，其结构示意图及其激活示意图如图7所示，图7中的A显示，CD8-GM-CSFRα/β-CAR由胞外识别EGFRvIII的scFv蛋白序列，CD8α跨膜结构域，以及胞内的信号转导结构域构成。胞内信号转导结构域由受体GM-CSFRα的胞内信号激活结构域和受体GM-CSFRβ的胞内信号激活结构域通过接头(Linker)序列链接构成。接头序列可以灵活改变构象。图7中的B显示，CD8-GM-CSFRα/β-CAR胞外的scFv在接触EGFRvIII蛋白从而形成免疫突触后，包内的GM-CSFRα和GM-CSFRβ激活结构域便通过接头构象的改变形成异源二聚体，继而得以激活，最终促进CAR-iMAC的增殖、免疫激活和吞噬作用。
实施例7
体外验证CD8-GM-CSFRα/β-CAR-iMAC靶向杀伤和依赖CAR免疫激活的效果。
按照实施例3图3中的B图的实验方法，将CD8-GM-CSFRα/β-CAR-iMAC和CD8-DELTA-CAR-iMAC在体外与表达荧光素酶的U87MG EGFRvIII细胞在活细胞工作站中共培养，体外共培养24h后，通过共聚焦扫描显示CD8-GM-CSFRα/β-CAR-iMAC(EGFP)和不含胞内信号转导结构域的CD8-DELTA-CAR-iMAC(EGFP)均具有吞噬U87MG EGFRvIII细胞的能力(图8中的A)。将WT-iMAC、CD8-DELTA-CAR-iMAC以及CD8-GM-CSFRα/β-CAR-iMAC以10/1的效靶比，分别与U87MG EGFRvIII细胞体外共培养24h后，通过ELISA实验检测巨噬细胞免疫激活因子IL-12和TNFα的分泌情况。结果如图8中的B和C所示，表明CD8-GM-CSFRα/β-CAR-iMAC相对于WT-iMAC和CD8-DELTA-CAR-iMAC有显著的分泌IL-12和TNFα的能力。将WT-iMAC、CD8-DELTA-CAR-iMAC以及CD8-GM-CSFRα/β-CAR-iMAC以10/1的效靶比，分别与表达荧光素酶luciferase的U87MG EGFRvIII细胞体外共培养24h后，通过多功能酶标仪检测U87MG EGFRvIII细胞中的荧光信号。结果如图8中的D所示，表明，U87MG EGFRvIII细胞与CD8-GM-CSFRα/β-CAR-iMAC共培养后，荧光信号显著下调。进一步证实CD8-GM-CSFRα/β-CAR-iMAC相对于WT-iMAC和CD8-DELTA-CAR-iMAC有更为显著的杀伤U87MG EGFRvIII细胞的能力。
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。
工业实用性
本公开提供的嵌合抗原受体，能够赋予巨噬细胞靶向GBM的特性，降低脱靶效应，促进和维持巨噬细胞在肿瘤微环境的M1极化状态，提高其杀伤效率。本公开提供的巨噬细胞的极化方法，该方法简单，能够实现巨噬细胞的M1极化。本公开提供的巨噬细胞，能够维持M1极化状态，对GBM的靶向杀伤性强，该巨噬细胞能够长期稳定的过表达嵌合抗原受体，能够解决目前免疫细胞治疗所存在的编辑效率低、周期长、工作量大和治疗不及时等方面的问题。本公开提供的多能干细胞含有编码上述嵌合抗原受体的基因，其状态更倾向于促炎抑肿瘤的M1状态，能够可选的分化得到巨噬细胞，更有利于抑制肿瘤，具有广泛的应用前景和较高的市场价值。

Claims (17)

  1. 一种嵌合抗原受体，其特征在于，包括依次连接的胞外抗原结合域、跨膜结构域和胞内激活结构域；
    所述胞外抗原结合域包括信号肽和/或靶向EGFRvIII的scFv；
    跨膜结构域包括CD8α；
    所述胞内激活结构域包括TIR、CD3ZETA或GM-CSFRα/β中的至少一种。
  2. 根据权利要求1所述的嵌合抗原受体，其特征在于，所述信号肽由SEQ ID NO.1所示的核苷酸序列表达；
    所述scFv由SEQ ID NO.2所示的核苷酸序列表达；
    优选地，所述CD8α由SEQ ID NO.3所示的核苷酸序列表达。
  3. 根据权利要求1或2所述的嵌合抗原受体，其特征在于，所述TIR包括来源于TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、TLR13或TLR19的胞内信号转导结构域；
    所述TLR4的胞内信号转导结构域由SEQ ID NO.4所示的核苷酸序列表达；
    所述CD3ZETA由SEQ ID NO.5所示的核苷酸序列表达；
    所述GM-CSFα/β由SEQ ID NO.6所示的核苷酸序列表达。
  4. 一种巨噬细胞的极化方法，其特征在于，将权利要求1-3任一项所述的嵌合抗原受体在巨噬细胞中表达。
  5. 一种巨噬细胞，其特征在于，包括权利要求1-3任一项所述的嵌合抗原受体。
  6. 权利要求5所述的巨噬细胞的制备方法，其特征在于，包括如下步骤：构建含有所述嵌合抗原受体的基因表达序列的慢病毒表达体系，利用慢病毒表达体系将嵌合抗原受体的基因表达序列整合到多能干细胞中，经诱导分化后制备得到巨噬细胞；
    优选地，所述多能干细胞先诱导分化为单核细胞，再分化得到巨噬细胞。
  7. 一种能够分化得到权利要求5所述巨噬细胞的多能干细胞，其特征在于，所述多能干细胞含有编码所述嵌合抗原受体的基因。
  8. 一种能够分化得到权利要求5所述巨噬细胞的单核细胞，其特征在于，所述单核细胞含有编码所述嵌合抗原受体的基因；
    所述单核细胞由多能干细胞分化得到。
  9. 权利要求5所述的巨噬细胞、权利要求7所述的多能干细胞或权利要求8所述的单核细胞在制备用于治疗神经胶质瘤的产品中的应用。
  10. 一种用于治疗神经胶质瘤的产品，其特征在于，所述产品包括权利要求5所述的巨噬细胞。
  11. 一种药物制剂，包括权利要求5所述的巨噬细胞、权利要求7所述的多能干细胞或权利要求8所述的单核细胞。
  12. 根据权利要求11所述的药物制剂，其特征在于，所述药物制剂包括药学上可接受的佐剂、稀释剂或载体中的一种或多种。
  13. 如权利要求1-3中任一项所述嵌合抗原受体、权利要求5所述的巨噬细胞、权利要求7所述的多能干细胞、权利要求8所述的单核细胞或权利要求11-12中任一项所述的药物制剂在神经胶质瘤治疗中的用途。
  14. 如权利要求1-3中任一项所述嵌合抗原受体、权利要求5所述的巨噬细胞、权利要求7所述的多能干细胞、权利要求8所述的单核细胞或权利要求11-12中任一项所述的药物制剂，用于神经胶质瘤治疗中的用途。
  15. 一种治疗神经胶质瘤的方法，包括：
    向有此需要的受试者给药治疗有效量的权利要求5所述的巨噬细胞、权利要求10所述的治疗神经胶质瘤的产品或权利要求11-12中任一项所述的药物制剂。
  16. 胞内激活结构域用于制备治疗肿瘤或癌症的药物的用途，其中，所述胞内激活结构域包括TIR、CD3ZETA或GM-CSFRα/β中的至少一种。
  17. 根据权利要求16所述的用途，其中，
    所述药物为巨噬细胞；所述巨噬细胞包括含有所述胞内激活结构域的嵌合抗原受体；
    所述癌症选自与实体肿瘤相关的癌症，所述肿瘤选自脑部肿瘤；
    优选地，所述与实体肿瘤相关的癌症选自脑癌、食管癌、肝癌、胃癌、肠癌、肺癌、鼻咽癌中的一种；
    优选地，所述脑部肿瘤选自神经胶质瘤、神经纤维瘤、星形细胞瘤、少突胶质细胞瘤、髓母细胞瘤、室管膜瘤、松果体瘤中的一种。
PCT/CN2021/117808 2021-06-18 2021-09-10 巨噬细胞专属嵌合抗原受体、表达该受体的可控极化单核/巨噬细胞及其制备方法和应用 WO2022262130A1 (zh)

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Publication number Priority date Publication date Assignee Title
CN114410588B (zh) * 2022-01-29 2022-11-04 西安电子科技大学 一种α1β1整合素依赖增强型CAR巨噬细胞及其制备方法和应用
CN114657143B (zh) * 2022-03-11 2022-10-25 西安电子科技大学 一种肿瘤微环境调控型car-单核/巨噬细胞及其制备方法和应用
CN117777312A (zh) * 2023-12-28 2024-03-29 北京磊顿生物科技有限公司 一种靶向EGFRvIII的嵌合抗原受体（CAR）及其抗癌的用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106636003A (zh) * 2017-01-24 2017-05-10 北京普瑞金科技有限公司 一种全人源化EGFRvIII嵌合抗原受体T细胞及其制备方法
CN106967685A (zh) * 2016-01-13 2017-07-21 北京马力喏生物科技有限公司 共表达抗EGFRvIII嵌合抗原受体和免疫检查点抑制分子的转基因淋巴细胞及其用途
CN111139256A (zh) * 2013-02-20 2020-05-12 诺华股份有限公司 使用人源化抗EGFRvIII嵌合抗原受体治疗癌症

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110564749B (zh) * 2018-06-05 2024-01-05 上海恒润达生生物科技股份有限公司 靶向egfr的嵌合抗原受体及其用途
CN109266618B (zh) * 2018-10-18 2021-04-23 赛元生物科技(杭州)有限公司 能够靶向肿瘤细胞的巨噬细胞及其制备方法
CN110183537B (zh) * 2019-05-28 2023-10-10 中国科学院苏州生物医学工程技术研究所 一种egfr特异性嵌合抗原受体、编码基因、car-t细胞及其制备方法、重组细胞和应用
CN110845623B (zh) * 2019-10-11 2021-07-09 厦门大学 一种egfr特异性嵌合抗原受体及其应用
CN111138548A (zh) * 2019-11-22 2020-05-12 中国科学院苏州生物医学工程技术研究所 一种egfr靶向性的嵌合抗原受体、car-nk细胞及其制备方法、应用
CN110904048A (zh) * 2019-12-04 2020-03-24 合肥中科干细胞再生医学有限公司 一种car-cd123t2嵌合抗原受体t细胞及其应用
CN111518219B (zh) * 2020-05-08 2021-06-22 浙江大学 嵌合性抗原受体及表达其的巨噬细胞、调节巨噬细胞极化的方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111139256A (zh) * 2013-02-20 2020-05-12 诺华股份有限公司 使用人源化抗EGFRvIII嵌合抗原受体治疗癌症
CN106967685A (zh) * 2016-01-13 2017-07-21 北京马力喏生物科技有限公司 共表达抗EGFRvIII嵌合抗原受体和免疫检查点抑制分子的转基因淋巴细胞及其用途
CN106636003A (zh) * 2017-01-24 2017-05-10 北京普瑞金科技有限公司 一种全人源化EGFRvIII嵌合抗原受体T细胞及其制备方法

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