WO2019014891A1 - Cart d'anticorps à domaine unique egfr destinée au traitement de tumeur et son application - Google Patents

Cart d'anticorps à domaine unique egfr destinée au traitement de tumeur et son application 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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PCT/CN2017/093652
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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/fr
Publication of WO2019014891A1 publication Critical patent/WO2019014891A1/fr

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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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).

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Abstract

La présente invention concerne une cellule CART modifiée par un anticorps à domaine unique EGFR, la cellule CART pouvant être utilisée pour traiter une tumeur. La présente invention concerne également un gène d'anticorps à domaine unique EGFR humanisé et un anticorps à domaine unique ainsi codé, un gène CAR d'EGFR et un récepteur d'antigène chimère CAR d'EGFR ainsi codé, et un vecteur lentiviral Pre-Lenti-EF1-EGFR CAR contenant le gène CAR d'EGFR.
PCT/CN2017/093652 2017-07-20 2017-07-20 Cart d'anticorps à domaine unique egfr destinée au traitement de tumeur et son application WO2019014891A1 (fr)

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CN201780064335.1A CN109996872A (zh) 2017-07-20 2017-07-20 一种用于治疗肿瘤的egfr单域抗体cart及其应用
PCT/CN2017/093652 WO2019014891A1 (fr) 2017-07-20 2017-07-20 Cart d'anticorps à domaine unique egfr destinée au traitement de tumeur et son application

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101321784A (zh) * 2005-10-11 2008-12-10 埃博灵克斯股份有限公司 针对egfr和igf－ir的纳米抗体tm和多肽
WO2013123061A1 (fr) * 2012-02-13 2013-08-22 Seattle Children's Hospital D/B/A Seattle Children's Research Institute Récepteurs d'antigène chimères bispécifiques et utilisations thérapeutiques de ceux-ci
CN105384825A (zh) * 2015-08-11 2016-03-09 南京传奇生物科技有限公司 一种基于单域抗体的双特异性嵌合抗原受体及其应用
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

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101321784A (zh) * 2005-10-11 2008-12-10 埃博灵克斯股份有限公司 针对egfr和igf－ir的纳米抗体tm和多肽
WO2013123061A1 (fr) * 2012-02-13 2013-08-22 Seattle Children's Hospital D/B/A Seattle Children's Research Institute Récepteurs d'antigène chimères bispécifiques et utilisations thérapeutiques de ceux-ci
CN105384825A (zh) * 2015-08-11 2016-03-09 南京传奇生物科技有限公司 一种基于单域抗体的双特异性嵌合抗原受体及其应用
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

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BELL, A. ET AL.: "Differential Tumor-Targeting Abilities of Three Single-domain Antibody Formats", CANCER LETTERS, vol. 289, 31 December 2010 (2010-12-31), pages 81 - 90, XP026897079, ISSN: 0304-3835 *
ZHANG, MANZE ET AL.: "Research Progress of Chimeric Antigen Receptor-Engineered T Cells in Solid Tumors Therapy", IMMUNOLOGICAL JOURNAL, vol. 33, no. 3, 31 March 2017 (2017-03-31), pages 251 - 257, ISSN: 1000-8861 *

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