WO2019014891A1 - 一种用于***的egfr单域抗体cart及其应用 - Google Patents

一种用于***的egfr单域抗体cart及其应用 Download PDF

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WO2019014891A1
WO2019014891A1 PCT/CN2017/093652 CN2017093652W WO2019014891A1 WO 2019014891 A1 WO2019014891 A1 WO 2019014891A1 CN 2017093652 W CN2017093652 W CN 2017093652W WO 2019014891 A1 WO2019014891 A1 WO 2019014891A1
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egfr
cart
domain antibody
gene
cells
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French (fr)
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张继帅
李胜华
栗红建
赵杨杨
余祥
郑孟韬
何昱
何鹏华
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深圳普瑞金生物药业有限公司
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Priority to PCT/CN2017/093652 priority patent/WO2019014891A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464404—Epidermal growth factor receptors [EGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated

Definitions

  • the invention belongs to the field of immunocyte therapy, in particular to an EGFR single domain antibody CART for treating tumors, and also relates to a lentiviral vector, an EGFR CAR gene, an EGFR CAR chimeric antigen receptor, an EGFR single domain antibody gene, a human
  • the EG2h amino acids are derived, as well as their preparation methods and uses.
  • Cancer treatment methods generally include surgical resection, chemotherapy, and radiation therapy.
  • the current research and development of cancer treatment methods is based on the idea of completely eliminating cancer cells.
  • the method of surgical resection is often limited by the spread of cancer cells to adjacent tissues or distant metastases.
  • Chemotherapy is limited by toxicity to other normal tissues in the body. Radiation therapy can also cause damage to normal tissues.
  • the treatment of cancer whether it is chemotherapy, surgery or radiotherapy, is extremely harmful to the body. In the advanced stage, it is difficult to completely cure the tumor in any way. Therefore, the treatment of cancer is still a huge challenge facing humanity.
  • Immune cell therapy technology is the latest technology for treating tumors in recent years. Compared with existing surgery, radiotherapy and chemotherapy, it has its own irreplaceable advantages and development potential. Therefore, it is called the latest breakthrough in cancer therapy. technology.
  • adoptive cellular immunotherapies ACIs
  • CAR chimeric antigen receptor
  • CART is not subject to major histocompatibility.
  • MHC major histocompatibility complex
  • CART cells have achieved great success in the treatment of lymphoma and leukemia, but CART cells have also exposed some problems as a means of immunotherapy, such as the effect of solid tumor treatment is not good, the main reason is the comparison of solid tumors. Dense, making it difficult for CART cells to enter the tumor tissue, even if it enters, it is difficult to continue to proliferate and exert a lasting effect.
  • the first generation of CART is mainly used
  • the signal of CD3 molecule has weaker proliferative ability in vivo
  • the second generation CART includes CD3 molecule signal and CD28 or 4-1BB signal, which has shown remarkable effect in clinical treatment of hematological tumors; compared with third generation CART
  • the second-generation CART which has all the signals of CD3 molecules and CD28 and 4-1BB molecules, suggests that the three-generation CART may be more potent and more proliferative than the second-generation CART cells, and the tumoricidal ability is stronger (Fig. 1).
  • VHH variable domain of heavy chain of heavy-chain antibody
  • Nb nanobody
  • Single-domain antibodies have some significant advantages over current single-chain antibodies for CART applications, and are more suitable for transformation into antibodies for CART.
  • single-domain antibodies are themselves a single strand. During the screening process, single-domain antibodies of one chain have been demonstrated. The binding affinity of the antigen is higher. Therefore, the single-domain antibody is directly transformed into CART with higher antibody success rate.
  • the single-domain antibody has a smaller antigen recognition site than the single-chain antibody, which may overcome steric hindrance and identify Many epitopes are easily accessible, enter the interior of complex antigen molecules or close to the antigenic epitope of the cell membrane, thereby increasing the killing ability of CART.
  • single domain antibody technology is a suitable choice for making CART ( Figure 2).
  • single-domain antibodies have been used in the manufacture of CART, and have been developed and reported at home and abroad. No single-domain antibody-modified three-generation CART cells have been studied and reported in the treatment of solid tumors.
  • EGFR is a member of the transmembrane protein tyrosine kinase of the erbB receptor family. When bound to a growth factor ligand, the receptor can homodimerize with an additional EGFR molecule or heterodimerize with another family member (eg, erbB2 (HER2), erbB3 (HER3)).
  • erbB2 HER2
  • HER3 erbB3
  • Deregulation of erbB family signaling promotes proliferation, invasion, metastasis, angiogenesis, and tumor cell survival, and has been described in many human cancers (such as human skin squamous cell carcinoma, lung cancer, colorectal cancer). Therefore, the erbB family represents a suitable target for the development of anticancer drugs.
  • cetuximab an antibody drug against EGFR targets, is used for the treatment of metastatic colorectal cancer, and TKI inhibitors against mutant EGFR are used for the treatment of lung cancer.
  • the patented technology aims at the solid tumors in which CART is difficult to play, and designs and manufactures a single-domain antibody-modified three-generation CART cell that recognizes the solid tumor target EGFR, combined with the advantages of the three-generation CART and the advantages of the single-domain antibody, the three generations of this patent Domain antibody CART has been proven through cell experiments and animal experiments. It has a strong ability to kill tumors and is an effective drug for the treatment of solid tumors.
  • the scFV used by CART if it is derived from traditional antibodies, needs to be genetically engineered, and the affinity of many antibodies after transformation is reduced, which cannot meet the needs.
  • the present invention provides the following technical solutions:
  • the invention provides a CART cell characterized by a single domain antibody engineered CART cell.
  • the single domain antibody is an EGFR single domain antibody.
  • the gene sequence of the EGFR single domain antibody is SEQ ID NO 2.
  • the CART is a three generation CART.
  • the structural sequence of the three generations of CART is SEQ ID NO 3.
  • the CART cell is an EGFR CAR gene-modified T cell, and the sequence of the EGFR CAR gene is SEQ ID NO 4.
  • the present invention provides a lentiviral vector, Pre-Lenti-EF1-EGFR CAR, which is characterized in that it expresses an EGFR CAR gene, and the sequence of the EGFR CAR gene is SEQ ID NO: 4.
  • the present invention provides an EGFR CAR gene, wherein the sequence of the EGFR CAR gene is SEQ ID NO 4.
  • the present invention provides an EGFR CAR chimeric antigen receptor, wherein the EGFR CAR chimeric antigen receptor is encoded by the EGFR CAR gene of claim 6.
  • the present invention provides an EGFR single domain antibody gene, wherein the sequence of the EGFR single domain antibody gene is SEQ ID NO 2.
  • the invention provides a humanized EG2h amino acid, characterized in that the sequence of the humanized EG2h amino acid is SEQ ID NO 1.
  • the present invention provides a lentiviral vector as described above, the EGFR CAR gene described above, the EGFR single domain antibody gene described above or the humanized EG2h amino acid described above for use in the preparation of CART cells the use of.
  • the present invention provides the use of a CART cell according to any of the above, in the manufacture of a medicament for treating a tumor.
  • the tumor is a solid tumor.
  • the solid tumor is a tumor with high or abnormal expression of EGFR.
  • the tumor is selected from the group consisting of glioma, lung cancer, esophageal cancer, gastric cancer, and colorectal cancer.
  • the single domain antibody is an EGFR single domain antibody and the gene sequence of the EGFR single domain antibody is SEQ ID NO 2.
  • the three generations of CART of the present invention incorporate more co-stimulatory signals, and the ability of CART cells to activate proliferation is stronger;
  • the present invention technically verifies that a single domain antibody can be engineered into a CART cell with strong killing ability
  • the present invention develops a CART cell drug that targets EGFR targets that can be used in the treatment of solid tumors.
  • CART cells of the present invention are not limited to the use of single domain antibodies of EGFR to engineer three generation CART cells, and those skilled in the art will appreciate that other single domain antibodies can also be used to engineer three generation CART cells and achieve the desired effect; Without being limited to solid tumors, those skilled in the art will foresee that the present invention is also expected to achieve good therapeutic effects for non-solid tumors.
  • the single domain antibody EG2 sequence that recognizes EGFR is from Andrea Bell et al., 2010, published in the journal Cancer Letters: Differential tumor-targeting abilities of three single-domain antibody formats. Based on the amino acid sequence of the EGFR-EG2 single domain antibody, according to the reference (Tomlinson, IM, Walter, G., Marks, JD, Llewelyn, MB, and Winter, G. (1992) J. Mol. Biol. 227, 776- The amino acid sequence of VH-DP-47 of the human antibody heavy chain of 798) was humanized.
  • FR1 (1-30) QVKLEESGGGLVQAGDSLRVSCAASGRDFS CDR1 (31-35) DYVMG FR2 (36-50) WFRQAPGKEREFVAA CDR2(51-66) ISRNGLTTRYADSVKG FR3 (67-97) RFTISRDNDKNMVYLQMNSLKPEDTAVYYCA CDR3 (98-114) VNSAGTYVSPRSREYDY FR4 (115-125) WGQGTQVTVSS
  • the amino acid sequence was codon-optimized to synthesize the gene.
  • One segment is a three-generation CAR structural gene (CD8 hinge region + CD28 transmembrane region, cytoplasmic region + 4-1BB cytoplasmic region + CD3 cytoplasmic region);
  • molecular cloning is performed. First, PCR was performed on the two gene fragments, and then overlapping PCR was performed to obtain an EGFR CAR gene containing three generations of CAR structures linked together, and the lentiviral vector Pre-Lenti-EF1-MCS and EGFR CAR gene were ligated. , transformation, extraction of plasmid, sequencing, to obtain the correct sequence of the lentiviral vector Pre-Lenti-EF1-EGFR CAR.
  • FIG 1 shows the evolution of the CART carrier design.
  • the first generation of the vector recognizes a tumor marker molecule, such as CD19, by a single-chain antibody (scFv), and transmits the signal intracellularly using the CD3 molecule ⁇ chain.
  • a tumor marker molecule such as CD19
  • scFv single-chain antibody
  • the second generation vectors are divided into two categories, one is the costimulatory molecule CD28+CD3 ⁇ , the other is the costimulatory molecule 4-1BB+CD3 ⁇ , the CD28 molecule promotes T cell expansion, and the 4-1BB molecule not only promotes T cell expansion, but also Prolong the survival time of CART cells in vivo.
  • the three-generation vector includes the co-stimulatory molecules CD28, 4-1BB plus CD3 ⁇ , which basically mimics the stimulation signals that T cells receive when they function normally. Compared with the second-generation vectors, the three-generation vectors will theoretically have better effects; A single domain antibody can be used as the antibody to the marker molecule.
  • Figure 2 shows that a single domain antibody is suitable for transformation into an antibody for CART.
  • the most commonly used single-chain antibody (scFV) of CART is derived from a traditional antibody, and the heavy and light chain variable regions are linked by a linker, and are transformed into an antibody recognition site of one strand, which is coupled to an element that activates T cells (see Figure 1). .
  • VHH Single-domain antibody is VHH when it is built and screened, and it is single-stranded. It has strong antigen binding ability. After coupling the elements of activated T cells, it is easy to be transformed into CART molecule. Compared with scFV, VHH has its own volume. Small, easy to bind to cell membrane surface molecules.
  • the scFV used by CART if derived from traditional antibodies, needs to be genetically engineered, and many of the antibodies have reduced affinity after transformation and cannot meet the needs.
  • the VHH of a single domain antibody has a high affinity for antigen binding, and can bind antigen alone, and has a high probability of obtaining antibodies for CART.
  • Figure 3 is a flow chart showing the construction of EGFR CAR lentiviral vector.
  • Figure 4 is an electrophoresis pattern of a single domain antibody gene and a three generation CAR structural gene PCR product.
  • the left panel shows the single domain antibody gene (438 bp) and the right panel shows the three generation CAR gene (939 bp).
  • Figure 5 is an electropherogram of overlapping PCR products.
  • the left panel shows the markers (1000, 2000, 3000, 4000, 5000, 6000, 8000, 10000 bp), and the right panel shows the EGFR CAR gene (1377 bp).
  • Figure 6 shows the positive rate of CART cells by flow cytometry.
  • the left panel shows the addition of a control antibody (ISO-FITC) and the right panel shows the CAR molecule positive rate of EGFR CART cells (goat anti-human Fab fragment FITC-labeled antibody).
  • Figure 7 is a Western blot to identify the expression of EGFR in different tumor cells.
  • Figure 8 shows the killing effect of EGFR-CART.
  • the target cells are A431 cells, and the effector cells are controls.
  • T cells and EGFR-CART cells from the kill rate, EGFR-CART cells can kill A431 cells, indicating that CART was successfully constructed.
  • Figure 9 is a EGFR-CART specific killer EGFR overexpressing cell.
  • the target cells are MC38 parental cells, MC38 overexpresses HER2 molecule cells (MC38-HER2), and MC38 overexpresses EGFR molecule cells (MC38-EGFR).
  • MC38-HER2 molecule cells MC38-HER2
  • MC38-EGFR MC38 overexpresses EGFR molecule cells
  • EGFR-CART specifically kills cells overexpressing EGFR, and does not kill structurally similar HER2-expressing MC38 cells, indicating that EGFR-CART has a stronger killing specificity.
  • Figure 10 shows that EGFR-CART effectively inhibits subcutaneous tumor growth in mice.
  • mice on the left side used the control CAR-T, and the 5 mice on the right side used the 3 generation EGFR CAR-T.
  • the EGFR single domain antibody gene (SEQ ID NO 2) and the third generation CAR structural gene (SEQ ID NO 3) were synthesized, and the two genes were subjected to overlapping PCR splicing to obtain an EGFR CAR gene (SEQ ID NO 4).
  • the EGFR CAR gene sequence was analyzed using the sequence analysis software pDRAW32.
  • the restriction enzyme sites EcoRI and BamHI were suitable for cloning genes and matched with the MCS (multiple cloning site) of Pre-Lenti-EF1-MCS vector.
  • the primers at both ends were designed as follows.
  • the EcoRI-upstream sequence is shown in SEQ ID NO: 5:
  • the BamHI-downstream sequence is set forth in SEQ ID NO: 6:
  • the primers for overlapping PCR are:
  • PCR amplification After receiving the synthesized gene and primer, PCR is performed, and the system is as follows (using the KOD Fx enzyme of Toyobo):
  • the PCR program is as follows:
  • Step 1 94 ° C 2 minutes
  • Step 2 98 ° C 10 seconds
  • Step 3 50 ° C 30 seconds
  • Step 4 68 ° C 1 minute, to step 2 5 cycles
  • Step 6 60 ° C 30 seconds
  • the PCR program is:
  • Step 1 94 ° C 2 minutes
  • Step 2 98 ° C 10 seconds
  • Step 3 50 ° C 30 seconds
  • Step 4 68 ° C, 2 minutes, to step 2 5 cycles
  • Step 6 60 ° C 30 seconds
  • the reaction system is as follows:
  • the tea bath was incubated at 37 ° C for 2 hours.
  • fragment nanomolar number 1:3, ie
  • Packaging cells 293T were cultured in a 37 ° C, 5% CO 2 incubator with DMEM/10% FBS (Hyclone).
  • Transfected cells in addition to Pre-Lenti-EF1-MCS-EGFRCAR plasmid, need to be co-transfected with the packaging plasmid psPAX2, pMD2.0G.
  • Pre-Lenti-EF1-MCS-EGFR CAR used 5 ⁇ g
  • psPAX2 used 3.75 ⁇ g
  • pMD2.0G used 1.25 ⁇ g.
  • a mixture of the three plasmids was added to 500 ⁇ l of opti-MEM medium, and 25 ⁇ l of Lipofectamine 2000 transfection reagent was added to 500 ⁇ L of opti-MEM medium in another microcentrifuge tube, and then the diluted transfection reagent was The mixture was added dropwise to the diluted plasmid, mixed, centrifuged, and allowed to stand at room temperature for 20 minutes. Finally, the mixture of the plasmid and the transfection reagent was added to a 10 cm 2 culture dish, shaken, mixed, and placed in an incubator.
  • lentivirus can be harvested, 10 ml of virus-containing medium is transferred to a 10 ml centrifuge tube, centrifuged at 1250 rpm for 5 minutes at 4 ° C, the dead 293T cells are removed, and then the virus-containing medium is concentrated. Sterile filtration, sub-package, frozen at -80 °C. A portion of the virus is retained to determine the titer.
  • lymphocytes are in the white layer between the upper plasma layer and the separation solution.
  • lymphocyte count take 1 ⁇ 10 7 lymphocytes in a microcentrifuge tube.
  • the T cells were fully activated and proliferated. At this time, the T cells were transferred to a 25 cm 2 culture flask.
  • the effect of killing target cells by CART cells is determined by measuring the LDH released by dead cells into the cell culture supernatant.
  • step 2.5 The killing experiment in step 2.5 was incubated overnight. After approximately 18 hours, the largest release well was added to 20 ⁇ L of 10 ⁇ cell lysate.
  • the “experiment” is the OD492 obtained by different target ratios
  • the "effect cell spontaneous” is the OD492 of the control T cell or the EGFR CART cell
  • the "target cell spontaneous” is the minimum release of the A431 cell
  • the "target cell maximum” is the maximum release of the A431 cell.
  • the calculated killing rate is shown in Figure 8, indicating that Pre-Lenti-EF1-MCS-EGFR CART has better killing function.
  • the single domain antibody that recognizes the EGFR molecule is from the llama. After humanization, it is necessary to observe whether it is specific. Sexual recognition of EGFR.
  • MC38 cells stably expressing EGFR or HER2 molecules were constructed as follows:
  • MC38 cells expressing HER2 molecule MC38-HER2, a stable cell line established by lentivirus infection
  • MC38 cells expressing EGFR molecule MC38-EGFR, stable by lentivirus infection
  • Cell line 1 ⁇ 10 4 cells/well (96-well plate), overnight;
  • the calculated killing rate is shown in Figure 9, indicating that EGFR CART has a better killing function for cells expressing EGFR and does not recognize HER2 molecules with high homology.
  • Mouse strain is a strain of NSG severely immunodeficient mice (lack of T cell, B cell and NK cell function).
  • A431 cell line (high expression of EGFR) was injected subcutaneously with 3 x 10 6 cells/100 ⁇ L PBS (10 mice).
  • mice 3.4 Every 2 days, the subcutaneous tumor size of the mice was measured until 12 days after the CART cells were injected. The subcutaneous tumors of the control (control) mice were larger, and the tumors in the EGFR CART group were significantly inhibited (Fig. 10).

Abstract

提供了一种EGFR单域抗体改造的CART细胞，所述CART细胞可用于***。还提供了人源化的EGFR单域抗体基因及其编码的单域抗体，EGFR CAR基因及其编码的EGFR CAR嵌合抗原受体，以及含有EGFR CAR基因的慢病毒载体Pre-Lenti-EF1-EGFR CAR。

Description

一种用于***的EGFR单域抗体CART及其应用 技术领域
本发明属于免疫细胞治疗领域，具体地，涉及一种用于***的EGFR单域抗体CART，还涉及慢病毒载体、EGFR CAR基因、EGFR CAR嵌合抗原受体、EGFR单域抗体基因、人源化的EG2h氨基酸，以及它们的制备方法及用途。
背景技术
恶性肿瘤是一种严重威胁人类健康的常见病和多发病，是由于人体内细胞的不正常增生所导致。传统的癌症治疗方式一般包括手术切除、化疗、放射线治疗等。目前癌症治疗方法的研发，均是基于彻底清除癌细胞的设想。手术切除的方式，常因为癌细胞入侵蔓延到邻近组织或远端转移而效果有限。化疗则受限于对体内其他正常组织的毒性。放射治疗也同样会对正常组织造成伤害。另外，癌症的治疗无论是化疗、手术或放疗都对身体有极大伤害，中晚期的肿瘤，无论是何种方式都很难彻底治愈。所以，癌症的治疗仍然是人类面对的巨大挑战。
免疫细胞治疗技术是近几年出现的最新的***的技术，和现有的手术、放疗、化疗相比，具有自身不可替代的优势和发展潜力，因此，被称为肿瘤治疗的最新突破性技术。最近几年，基于嵌合抗原受体(chimeric antigen receptor，CAR)修饰T细胞的过继性细胞免疫治疗(adoptive cellular immunotherapies，ACIs)是目前抗肿瘤研究的热点，CART因不受主要组织相容性复合物(major histocompatibility complex，MHC)的限制，并可克服肿瘤局部免疫抑制微环境和突破宿主免疫耐受状态，使得它在***方面具有独特的优势。
目前，CART细胞在淋巴瘤、白血病的治疗中取得了很大的成功，但是CART细胞作为免疫治疗的一种手段也暴露出一些问题，例如实体瘤治疗的效果不好，主要原因为实体瘤比较致密，使得CART细胞很难进入肿瘤组织，即使进入也很难持续增殖，发挥持久作用。
在CART的技术发展中，主要经过了3代的演化。第一代CART主要使用 了CD3分子的信号，体内增殖能力较弱；第二代CART包括CD3分子的信号和CD28或4-1BB的信号，目前在临床治疗血液***肿瘤中显示了显著的效果；第三代CART相比二代CART，有CD3分子和CD28、4-1BB分子的全部信号，从免疫学理论方面提示三代CART可能比二代CART细胞的活化增殖能力更强，杀瘤能力更强(图1)。
1993年，Hamers-Casterman等研究表明，骆驼抗体中有部分抗体天然缺失轻链，只含有重链，因此又称重链抗体(heavy chain antibodies，HCAbs)。克隆重链抗体的可变区得到只由一个重链可变区组成的单域抗体(single domain antibody，sdAb)，称为VHH(variable domain of heavy chain of heavy-chain antibody，VHH)。VHH晶体直径2.5nm，长4nm，因此又称为纳米抗体(nanobody，Nb)，是自然存在的可与抗原结合的最小片段。纳米粒子具有的颗粒尺寸小、比表面积大、表面能高等特点，以及表面效应、尺寸效应和宏观量子隧道效应使得纳米抗体在诸多方面均优于传统抗体。
单域抗体相比目前CART应用的单链抗体，具有一些显著的优点，更加适合改造为CART用抗体，首先，单域抗体本身为一条链，在筛选过程中，已经证明一条链的单域抗体结合抗原的亲和力较高，因此，单域抗体直接改造为CART用抗体成功率较高；第二，单域抗体的抗原识别部位相比单链抗体较小，有可能克服空间位阻，识别较多的抗原表位，容易接近、进入复杂的抗原分子的内部或接近细胞膜的抗原表位，从而提高CART的杀伤能力。综上，单域抗体技术是制造CART的合适的选择(图2)。然而，迄今为止，单域抗体用于CART的制造，国内外研制、报道的较少，更未见单域抗体改造的三代CART细胞在治疗实体肿瘤方面的研究和报道。
EGFR是erbB受体家族的跨膜蛋白酪氨酸激酶成员。当与生长因子配体结合时，受体可以与附加的EGFR分子发生同源二聚，或者与另一家族成员(如：erbB2(HER2)、erbB3(HER3))发生异源二聚。erbB家族信号传导的失调促进增殖、侵袭、转移、血管生成和肿瘤细胞存活，并且已在许多人类癌症中(如人皮肤鳞癌、肺癌、结直肠癌)得到描述。因此，erbB家族代表抗癌药物开发的合适靶点。目前，有针对EGFR靶点的抗体药物西妥昔单抗用于转移性结直肠癌的治疗，针对突变型EGFR的TKI抑制剂用于肺癌的治疗。
本专利技术针对目前CART较难发挥作用的实体瘤，设计制造了识别实体瘤靶点EGFR的单域抗体改造的三代CART细胞，结合三代CART的优点和单域抗体的优点，本专利的三代单域抗体CART通过细胞实验和动物实验证明了 杀伤肿瘤的能力较强，是一种治疗实体瘤的有效药物。
发明内容
本发明要解决的技术问题(包括但不限于如下)：
1.CART已在血液***肿瘤的治疗方面显示了显著效果，然而，对于实体瘤治疗的效果有待提高；
2.CART使用的scFV，如果来源于传统抗体，需经过基因工程改造，许多抗体改造后亲和力下降，无法满足需要。
因此，本领域殷切需要一种能解决CART在治疗实体肿瘤方面的缺陷以及传统抗体亲和力不高问题的新型CART技术，从而能够提高CART杀伤实体瘤的效果。
为了实现上述目的，本发明提供如下技术方案：
在一个方面，本发明提供一种CART细胞，其特征在于，其为单域抗体改造的CART细胞。
在一个实施方案中，所述单域抗体是EGFR单域抗体。
优选地，所述EGFR单域抗体的基因序列为SEQ ID NO 2。
在又一个实施方案中，所述CART是三代CART。
优选地，所述三代CART的结构序列为SEQ ID NO 3。
在又一个实施方案中，所述CART细胞是EGFR CAR基因修饰的T细胞，所述EGFR CAR基因的序列为SEQ ID NO 4。
在又一个方面，本发明提供一种慢病毒载体Pre-Lenti-EF1-EGFR CAR，其特征在于，其表达EGFR CAR基因，所述EGFR CAR基因的序列为SEQ ID NO4。
在又一个方面，本发明提供一种EGFR CAR基因，其特征在于，所述EGFR CAR基因的序列为SEQ ID NO 4。
在又一个方面，本发明提供一种EGFR CAR嵌合抗原受体，其特征在于，所述EGFR CAR嵌合抗原受体由权利要求6所述的EGFR CAR基因编码。
在又一个方面，本发明提供一种EGFR单域抗体基因，其特征在于，所述EGFR单域抗体基因的序列为SEQ ID NO 2。
在又一个方面，本发明提供一种人源化的EG2h氨基酸，其特征在于，所述人源化的EG2h氨基酸的序列为SEQ ID NO 1。
在又一个方面，本发明提供如上面所述的慢病毒载体、上面所述的EGFR CAR基因、上面所述的EGFR单域抗体基因或上面所述的人源化的EG2h氨基酸用于制备CART细胞的用途。
在又一个方面，本发明提供如上面任一项所述的CART细胞在制备用于***的药物中的用途。
在一个实施方案中，所述肿瘤是实体肿瘤。
在一个实施方案中，所述实体肿瘤是EGFR高表达或异常表达的肿瘤。
优选地，所述肿瘤选自神经胶质细胞瘤、肺癌、食管癌、胃癌、结直肠癌。
在一个实施方案中，所述单域抗体是EGFR单域抗体，所述EGFR单域抗体的基因序列为SEQ ID NO 2。
本发明产生的技术效果(包括但不限于如下)：
1.本发明的三代CART加入了更多的协同刺激信号，CART细胞活化增殖的能力更强；
2.本发明从技术上验证了单域抗体可以改造为杀伤能力较强的CART细胞；
3.本发明研制了一种能够用于实体瘤治疗的、针对EGFR靶点的CART细胞药物。
应该指出，本发明的CART细胞并不局限于利用EGFR的单域抗体来改造三代CART细胞，本领域技术人员可以预见其他单域抗体也可用来改造三代CART细胞并取得期望的效果；本发明也不局限于实体肿瘤，本领域技术人员可以预见，对于非实体肿瘤，本发明也有望取得好的治疗效果。
发明详述
1.依照图3的载体构建方案进行三代慢病毒CAR载体构建。
2.EGFR的人源化单域抗体序列来源。
识别EGFR的单域抗体EG2序列来自Andrea Bell等2010年发表于Cancer Letters杂志上的文章：Differential tumor-targeting abilities of three single-domain antibody formats。基于EGFR-EG2单域抗体的氨基酸序列，按照参考文献(Tomlinson，I.M.，Walter，G.，Marks，J.D.，Llewelyn，M.B.，and Winter，G.(1992)J.Mol.Biol.227，776-798)中人抗体重链的VH-DP-47的氨基酸序列进行人源化。
人源化前EG2氨基酸序列：
FR1(1-30) QVKLEESGGGLVQAGDSLRVSCAASGRDFS
CDR1(31-35) DYVMG
FR2(36-50) WFRQAPGKEREFVAA
CDR2(51-66) ISRNGLTTRYADSVKG
FR3(67-97) RFTISRDNDKNMVYLQMNSLKPEDTAVYYCA
CDR3(98-114) VNSAGTYVSPRSREYDY
FR4(115-125) WGQGTQVTVSS
人源化后EG2h氨基酸序列(SEQ IDNO 1，改变的氨基酸以粗、斜体标示)：
Figure PCTCN2017093652-appb-000001
将此氨基酸序列进行密码子优化后，合成基因。
3.合成两段基因(中美泰和公司)，一段为EGFR单域抗体基因(CD8信号肽+人源化EGFR单域抗体序列EG2h)；
EGFR单域抗体基因(SEQ ID NO 2)：
Figure PCTCN2017093652-appb-000002
一段为三代CAR结构基因(CD8铰链区+CD28跨膜区、胞浆区+4-1BB胞浆区+CD3ζ胞浆区)；
三代CAR结构基因(SEQ IDNO 3)：
Figure PCTCN2017093652-appb-000003
Figure PCTCN2017093652-appb-000004
4.得到合成的基因后，进行分子克隆。首先对两个基因片段进行PCR，然后进行重叠PCR，得到两个片段连接在一起的含有三代CAR结构的EGFR CAR基因，通过酶切慢病毒载体Pre-Lenti-EF1-MCS和EGFR CAR基因，连接、转化、提取质粒、测序，得到序列正确的慢病毒载体Pre-Lenti-EF1-EGFR CAR。
5.包装EGFR CAR慢病毒，感染T细胞，制备成CART，进行表达EGFR的肿瘤细胞的杀伤实验。
6.制备CART，进行动物实验，评估EGFR CART的体内效果。
参考文献：
1).Bell A，Wang ZJ，Arbabi-Ghahroudi M，Chang TA，Durocher Y，Trojahn U，et al.Differential tumor-targeting abilities of three single-domain antibody formats.Cancer Lett.2010；289(1)：81-90.
2).Tomlinson IM，Walter G，Marks JD，Llewelyn MB，Winter G.The repertoire of human germline VH sequences reveals about fifty groups of VH segments with  different hypervariable loops.J Mol Biol.1992；227(3)：776-98.
附图说明
图1为CART载体设计的演化图。
一代载体胞外通过单链抗体(scFv)识别肿瘤标志分子，如CD19，胞内利用CD3分子ζ链传递信号。
二代载体分为两类，一种为协同刺激分子CD28+CD3ζ，一种为协同刺激分子4-1BB+CD3ζ，CD28分子促进T细胞扩增，4-1BB分子不仅促进T细胞扩增，并且延长CART细胞体内存活时间。
三代载体包括协同刺激分子CD28、4-1BB加上CD3ζ分子，基本模拟了T细胞正常发挥作用时受到的刺激信号，和二代载体相比，三代载体理论上将具有更好的效果；识别肿瘤标志分子的抗体可以使用单域抗体。
图2显示单域抗体适合改造为CART用抗体。
CART最常用的单链抗体(scFV)来源于传统抗体，重链和轻链可变区通过连接区链接，改造为一条链的抗体识别部位，后面偶联活化T细胞的元件(参见图1)。
单域抗体在建库、筛选时得到的为VHH，自身为单链，抗原结合能力较强，偶联活化T细胞的元件后，容易改造为CART用分子；并且VHH相比scFV，自身体积较小，容易结合细胞膜表面分子。
CART使用的scFV，如果来源于传统抗体，需经过基因工程改造，许多抗体改造后亲和力下降，无法满足需要。单域抗体的VHH，结合抗原亲和力高，单独即可结合抗原，得到CART用抗体的几率高。
图3为EGFR CAR慢病毒载体构建流程图。
图4为单域抗体基因和三代CAR结构基因PCR产物电泳图。左图为单域抗体基因(438bp)，右图为三代CAR结构基因(939bp)。
图5为重叠PCR产物电泳图。左图为标记物(1000、2000、3000、4000、5000、6000、8000、10000bp)，右图为EGFR CAR基因(1377bp)。
图6为流式细胞术检测CART细胞阳性率。左图为加入对照抗体(ISO-FITC)，右图为EGFR CART细胞的CAR分子阳性率(山羊抗人Fab段FITC标记抗体)。
图7为蛋白质印迹鉴定不同肿瘤细胞中EGFR的表达。
图8为EGFR-CART的杀伤效果。靶细胞为A431细胞，效应细胞为对照 (Control)T细胞和EGFR-CART细胞，从杀伤率可见，EGFR-CART细胞可以杀伤A431细胞，表明CART构建成功。
图9为EGFR-CART特异性杀伤EGFR过表达的细胞。靶细胞为MC38亲代细胞，MC38过表达HER2分子的细胞(MC38-HER2)，MC38过表达EGFR分子的细胞(MC38-EGFR)。由图和杀伤率可见，EGFR-CART特异性杀伤过表达EGFR的细胞，没有杀伤结构相似的表达HER2的MC38细胞，表明EGFR-CART的杀伤特异性较强。
图10显示EGFR-CART有效抑制小鼠皮下肿瘤生长。
A.动物实验设计；
B.尾静脉注射对照和EGFR CART后小鼠皮下肿瘤体积变化；
C.在注射CART后12天，小鼠皮下肿瘤照相，可见注射EGFR CART组小鼠皮下肿瘤生长被抑制。左侧5只小鼠为使用对照CAR-T，右侧5只小鼠为使用3代EGFR CAR-T。
具体实施方式
下面通过具体实施例对本发明进行进一步的阐述，应该理解，下述实施例仅是为了用于说明本发明，并不对发明内容进行限定。
实施例中所用原料和设备均为本领域技术人员熟知，且均为市场上能够购买到或容易获得或制得。
实施例1.Pre-Lenti-EF1-EGFR CAR载体构建
1.1合成EGFR单域抗体基因(SEQ ID NO 2)和三代CAR结构基因(SEQ ID NO 3)，两条基因进行重叠PCR拼接，得到EGFR CAR基因(SEQ ID NO 4)。
EGFR CAR基因(SEQ ID NO 4)：
Figure PCTCN2017093652-appb-000005
Figure PCTCN2017093652-appb-000006
1.2引物设计：
使用序列分析软件pDRAW32分析EGFR CAR基因序列，酶切位点EcoRI、BamHI适合用于克隆基因，能够和Pre-Lenti-EF1-MCS载体的MCS(多克隆位点)匹配，设计两端引物如下，
EcoRI-上游序列如SEQ ID NO 5所示：
Figure PCTCN2017093652-appb-000007
BamHI-下游序列如SEQ ID NO 6所示：
Figure PCTCN2017093652-appb-000008
重叠PCR的引物为：
中间-R，SEQ ID NO 7
Figure PCTCN2017093652-appb-000009
中间-F，SEQ ID NO 8
Figure PCTCN2017093652-appb-000010
1.3 PCR扩增：收到合成的基因和引物后，进行PCR，体系见下(使用Toyobo的KOD Fx酶)：
2×PCR缓冲液，用于KOD Fx 25μL
2mM dNTP 10μL
10mM引物1 1.5μL
10mM引物2 1.5μL
单域抗体基因或三代CAR结构基因 1μL(50ng/μl)
KOD FX 1μL
H2O 10μL
总体积 50μL
PCR程序如下表：
步骤1：94℃ 2分钟
步骤2：98℃ 10秒
步骤3：50℃ 30秒
步骤4：68℃ 1分钟，到步骤2 5个循环
步骤5：98℃ 10秒
步骤6：60℃ 30秒
步骤7：68℃ 1分钟，到步骤5 30个循环
步骤8：68℃ 10分钟
步骤9：4℃ 1小时
取5μL产物电泳，确认产物大小，PCR产物电泳见图4。对剩余PCR产物进行凝胶电泳，胶回收，定量片段浓度后，进行重叠PCR，体系见下：
2×PCR缓冲液，用于KOD Fx 25μL
2mM dNTP 10μL
10mM EcoRI-上游 1.5μL
10mM BamHI-下游 1.5μL
单域抗体基因和三代CAR结构基因PCR片段等摩尔比混合 100ng
KOD FX 1μL
H2O 8-10μL
总体积 50μL
PCR程序为：
步骤1：94℃ 2分钟
步骤2：98℃ 10秒
步骤3：50℃ 30秒
步骤4：68℃，2分钟，到步骤2 5循环
步骤5：98℃ 10秒
步骤6：60℃ 30秒
步骤7：68℃，2分钟，到步骤5 30循环
步骤8：68℃ 10分钟
步骤9：4℃ 1小时
取5μL PCR产物电泳，见图5。剩余PCR产物凝胶电泳，胶回收，进行后续克隆实验。
1.4酶切：
反应体系如下：
Figure PCTCN2017093652-appb-000011
37℃水浴，酶切2个小时。
1.5回收酶切后的产物，使用Biomiga胶回收试剂盒。
1.6连接反应：
根据载体纳摩尔(nmole)数：片段纳摩尔数＝1：3的公式计算，即
Figure PCTCN2017093652-appb-000012
配制连接体系：
Pre-Lenti-EF1-MCS 2.3μL(100ng)
EGFR CAR 1.2μL
H2O 1.5μL
溶液I(Takara公司) 5μL
  10μL
上述内容各组分加至1.5mL离心管中，充分混匀，离心；22℃水浴30分钟。
1.7转化：冰上将上述连接产物加入50μL感受态中，轻弹，静置20分钟；42℃水浴热激90秒；冰上5分钟；加500μL LB，37℃复苏1小时；4000rpm离心5分钟，留适量上清液，用加样枪重悬感受态细菌，涂氨苄抗性LB平板；37℃温箱过夜。
1.8挑克隆，摇菌：连接反应时设置一不加连接片段的阴性对照，第二日观察可见加入EGFR CAR片段的平板内克隆数明显比未加片段的克隆数多2-3倍，判断连接成功。随机挑取6个克隆，摇菌扩增。
1.9质粒提取，使用Biomiga无内毒素质粒提取试剂盒。
1.10测序：选取浓度较高质粒测序，引物为，
Pre-上游-Seq(SEQ ID NO 9)：
Figure PCTCN2017093652-appb-000013
Pre-下游-Seq(SEQ ID NO 10)：
Figure PCTCN2017093652-appb-000014
测序结果比对后，完全正确，得到了表达EGFR CAR基因的慢病毒载体Pre-Lenti-EF1-EGFR CAR。
实施例2.细胞杀伤实验
2.1慢病毒包装
2.1.1包装细胞293T培养于37℃，5％CO2孵箱内，培养基为DMEM/10％ FBS(Hyclone公司)。
2.1.2包装病毒前一天，胰酶消化细胞，1×107细胞/皿种植10cm2培养皿。
2.1.3转染细胞时，除了Pre-Lenti-EF1-MCS-EGFRCAR质粒外，需要和包装质粒psPAX2、pMD2.0G共转染。其中Pre-Lenti-EF1-MCS-EGFR CAR使用5μg，psPAX2使用3.75μg，pMD2.0G使用1.25μg。转染时，将三种质粒的混合物加入500μl opti-MEM培养基内，在另一个微型离心管内将25μl Lipofectamine 2000转染试剂加入500μL opti-MEM培养基内，然后，将稀释的转染试剂逐滴加入稀释的质粒上方，混匀，离心，室温静置20分钟，最后将质粒和转染试剂的混合物加入10cm2培养皿内，轻晃、混匀，放入孵箱。
2.1.4细胞转染3天后，可以收获慢病毒，将10ml含病毒培养基转入10ml离心管内，4℃，1250rpm，离心5分钟，去除死亡的293T细胞，然后，将含病毒培养基浓缩，无菌过滤，分装，-80℃冻存。留存部分病毒测定滴度。
2.2淋巴细胞分离
2.2.1抽血：在无菌的条件下抽取志愿者的静脉血10ml。
2.2.2稀释：将获得的抗凝血液用相同体积的1640培养基稀释。
2.2.3在50ml离心管中加入10ml人淋巴细胞分离液。
2.2.4用电动移液枪将血液贴壁缓慢加入离心管分离液上层。
2.2.5以700g，室温离心25分钟。
2.2.6离心结束后，淋巴细胞在上层血浆层和分离液之间的白膜层中。
2.2.7用巴氏滴管尽量将白膜层吸到另一支离心管中(加入30ml 1640培养基)。
2.2.8 300g，22℃，10分钟。
2.2.9弃掉上层液体，用含IL2的1640培养基重悬，计数。
2.3 T细胞纯化
2.3.1淋巴细胞计数，取1×107个淋巴细胞于微型离心管中。
2.3.2 300g，22℃，10分钟。
2.3.3弃掉上清，80μL磁珠分离缓冲液重悬细胞沉淀，加入20μl的CD3MicroBeads(美天旎公司)。
2.3.4 4℃冰箱中放置1小时。
2.3.5 1小时后，在微型离心管中加入1mL的磁珠分离缓冲液，2500rpm，5℃，离心10分钟。期间准备过滤柱子，将过滤柱安放在磁铁上，用500μL的缓冲液润洗(缓冲液随着重力留下)。
2.3.6离心后细胞弃上清，500μl的分离缓冲液重悬，加入柱子，缓冲液随重力向下流出，500μl的缓冲液洗四次。
2.3.7将柱子从磁铁上卸掉，用1mL分离缓冲液将T细胞冲出，至1.5ml微型离心管。
2.3.8 2500rpm，5℃离心10分钟。
2.3.9含IL2、含10％灭活血清的1640培养基重悬，计数。
2.4 T细胞慢病毒感染
2.4.1纯化T计数，2×106个T细胞/孔(6孔板)，培养过夜后，加入MOI为2的对照病毒液(空载体包装，对照)和Pre-Lenti-EF1-MCS-EGFR CAR病毒液，感染过夜。
2.4.2第二天，补加1ml新鲜培养基。
2.4.3第三天，T细胞充分活化，增殖旺盛，此时将T细胞转入25cm2培养瓶。
2.4.4细胞扩增至一定数量后，利用流式细胞仪鉴定EGFR CART表面识别EGFR的单域抗体的表达阳性率，具体结果如图6所示。取一定数量的CART细胞，分为两管，一管使用山羊抗人Fab段FITC标记抗体和EGFR CART孵育(对应图6右)，另一管加入ISO-FITC，作为阴性对照(对照)(对应图6左)，流式细胞术分析结果，图6可见，约78％的CART细胞表达CAR分子，即CART细胞CAR分子表达的阳性率为78％。
2.5杀伤实验
2.5.1计数高表达EGFR分子的A431细胞(通过Westernblot实验鉴定EGFR表达，见图7)，1×104个靶细胞/孔(96孔板)，过夜；
2.5.2计数病毒CART细胞(对照病毒液感染的对照T细胞和Pre-Lenti-EF1-MCS-EGFR CAR病毒液感染的EGFR CART细胞)，按照效靶比10∶1、5∶1、2.5∶1的比例加入靶细胞上层，实验设计见表1。
表1.杀伤实验设计表(靶细胞：A431)
Figure PCTCN2017093652-appb-000015
Figure PCTCN2017093652-appb-000016
2.6杀伤测量
通过测量死亡细胞释放入细胞培养上清的LDH，来确定CART细胞杀死靶细胞的效果。
2.6.1操作按照Promega试剂盒说明书(CytoTox 96非放射性细胞毒性检测)。
2.6.2步骤2.5中杀伤实验孵育过夜，约18小时后，最大释放孔加入10×细胞裂解液20μL。
2.6.3 2小时后，每孔取50μl上清，转移至96孔板，每孔再加入50μl LDH酶底物，室温静置20分钟后，酶标仪测量吸光度OD492。
2.6.4杀伤率计算，计算公式为
Figure PCTCN2017093652-appb-000017
“实验”为不同效靶比得到的OD492，“效应细胞自发”为对照T细胞或EGFR CART细胞的OD492，“靶细胞自发”为A431细胞最小释放，“靶细胞最大”为A431细胞最大释放。计算得到的杀伤率见图8，表明Pre-Lenti-EF1-MCS-EGFR CART，杀伤功能较好。
2.7 EGFR CART特异性检测
识别EGFR分子的单域抗体来自美洲驼，进行人源化后，需要观察是否特异 性识别EGFR。构建了稳定表达EGFR或HER2分子的MC38细胞，实验如下：
2.7.1计数MC38、表达HER2分子的MC38细胞(MC38-HER2，通过慢病毒感染的方法建立的稳定细胞系)、表达EGFR分子的MC38细胞(MC38-EGFR，通过慢病毒感染的方法建立的稳定细胞系)，1×104个细胞/孔(96孔板)，过夜；
2.7.2计数CART细胞(EGFR CART)，按照效靶比10∶1、5∶1、2.5∶1的比例加入靶细胞上层。
2.7.3通过测量死亡细胞释放入细胞培养上清的LDH，来确定CART细胞杀死MC38、MC38-HER2、MC38-EGFR靶细胞的效果。操作见步骤2.6。
2.7.4杀伤率计算，计算公式同2.6.4。
计算得到的杀伤率见图9，表明EGFR CART，对表达EGFR的细胞特异性杀伤功能较好，没有识别同源性很高的HER2分子。
实施例3.动物实验
3.1小鼠品系：为NSG严重免疫缺陷小鼠品系(缺乏T细胞、B细胞和NK细胞功能)。
3.2肿瘤接种：使用A431细胞系(高表达EGFR)，3×106细胞/100μL PBS皮下注射(10只小鼠)。
3.3 CART注射：A431肿瘤细胞接种8天后，尾静脉注射对照或EGFR CART细胞(1×107细胞/200μL PBS，一组5只小鼠)，隔一天注射，共注射4次(见图10实验设计)。
3.4每隔2天观察，测量小鼠皮下肿瘤大小，直至CART细胞注射12天后，对照组(对照)小鼠皮下肿瘤已较大，照相，可见EGFR CART组肿瘤明显被抑制(图10)。
至此，本领域技术人员应认识到，虽然本文已详尽示出和描述了本发明的多个示例性实施例，但是，在不脱离本发明精神和范围的情况下，仍可根据本发明公开的内容直接确定或推导出符合本发明原理的许多其他变型或修改。因此，本发明的范围应被理解和认定为覆盖了所有这些其他变型或修改。

Claims (13)

  1. 一种CART细胞，其特征在于，其为单域抗体改造的CART细胞。
  2. 如权利要求1所述的CART细胞，其特征在于，所述单域抗体是EGFR单域抗体，优选地，所述EGFR单域抗体的基因序列为SEQ ID NO 2。
  3. 如权利要求1所述的CART细胞，其特征在于，所述CART是三代CART，优选地，所述三代CART的结构序列为SEQ ID NO 3。
  4. 如权利要求1所述的CART细胞，其特征在于，所述CART细胞是EGFR CAR基因修饰的T细胞，所述EGFR CAR基因的序列为SEQ ID NO 4。
  5. 一种慢病毒载体Pre-Lenti-EF1-EGFR CAR，其特征在于，其表达EGFR CAR基因，所述EGFR CAR基因的序列为SEQ ID NO 4。
  6. 一种EGFR CAR基因，其特征在于，所述EGFR CAR基因的序列为SEQ ID NO 4。
  7. 一种EGFR CAR嵌合抗原受体，其特征在于，所述EGFR CAR嵌合抗原受体由权利要求6所述的EGFR CAR基因编码。
  8. 一种EGFR单域抗体基因，其特征在于，所述EGFR单域抗体基因的序列为SEQ ID NO 2。
  9. 一种人源化的EG2h氨基酸，其特征在于，所述人源化的EG2h氨基酸的序列为SEQ ID NO 1。
  10. 如权利要求5所述的慢病毒载体、权利要求6所述的EGFR CAR基因、如权利要求8所述的EGFR单域抗体基因或如权利要求9所述的人源化的EG2h氨基酸用于制备CART细胞的用途。
  11. 如权利要求1-4任一项所述的CART细胞在制备用于***的药物中的用途。
  12. 如权利要求11所述的用途，其特征在于，所述肿瘤是实体肿瘤。
  13. 如权利要求12所述的用途，其特征在于，所述实体肿瘤是EGFR高表达或异常表达的肿瘤，优选地，所述肿瘤选自神经胶质细胞瘤、肺癌、食管癌、胃癌、结直肠癌。
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