WO2021068108A1 - Cellule car-t nkg2d, préparation et utilisation associées - Google Patents

Cellule car-t nkg2d, préparation et utilisation associées Download PDF

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WO2021068108A1
WO2021068108A1 PCT/CN2019/109960 CN2019109960W WO2021068108A1 WO 2021068108 A1 WO2021068108 A1 WO 2021068108A1 CN 2019109960 W CN2019109960 W CN 2019109960W WO 2021068108 A1 WO2021068108 A1 WO 2021068108A1
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nkg2d
cells
fusion protein
car
sequence
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Chinese (zh)
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曾昕
刘江海
李欣檑
刘彬
孔洋
曾顺泽
李玲芳
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成都盛世君联生物技术有限公司
成都盛世锐科生物技术有限公司
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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
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/464—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
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/867—Retroviral vectors

Definitions

  • the present invention relates to the field of biomedicine, in particular to NKG2D CAR-T cells and their preparation and application.
  • Chimeric antigen receptor (CAR) modified immune cells use genetic engineering methods to modify immune cells to express exogenous tumor-specific genes.
  • CAR genes mainly include extracellular recognition domains and intracellular signal transduction domains: the former is used to recognize tumor surface specific molecules and the latter is used to initiate immune cell responses after recognizing tumor surface molecules to exert cytotoxic effects.
  • T cells are a type of lymphocytes and play an important role in cell-mediated immunity. It differs from other lymphocytes (such as B cells and natural killer cells (NK cells)) in the presence of T cell receptors (TCR) on the cell surface.
  • T helper cells also known as CD4 + T or CD4 T cells, express CD4 glycoprotein on their surface. Helper T cells are activated when exposed to peptide antigens presented by MHC (major histocompatibility complex) class II molecules. Once activated, such cells proliferate rapidly and secrete cytokines that can regulate immune responses. Cytotoxic T cells, also known as CD8 + T cells or CD8 T cells, express CD8 glycoprotein on the cell surface.
  • CD8 + T cells are activated when exposed to peptide antigens presented by MHC class I molecules.
  • Memory T cells are a subset of T cells that exist for a long time and respond to related antigens, thus providing the immune system with memories of past infections and/or tumor cells.
  • T cells can produce chimeric antigen receptors on their surface.
  • CAR is a protein that allows T cells to recognize specific proteins (antigens) on tumor cells.
  • Genetically engineered CAR T cells can grow in the laboratory until their number reaches billions. The expanded CART cells can then be infused into the patient.
  • Natural killer (natural killer, abbreviated NK) cells are an important part of the non-specific immune system, and the key mediator cells of the innate immune system.
  • NK cells are a kind of broad-spectrum immune cells, which have the specific function of quickly discovering and destroying abnormal cells (such as cancer or virus-infected cells), and do not need to be sensitized or HLA matching in advance to display a powerful ability to dissolve abnormal cells. active.
  • the use of immune cells (including NK cells) to treat cancer is a new trend in recent years. This new therapy is expected to provide new hopes for curing tumors that are ineffective against traditional surgery, chemotherapy and radiotherapy.
  • NKG2D is an activated receptor of NK cells, which can recognize MHC-I molecules and plays an important role in natural immunity. NKG2D is involved in the recognition of virus-infected cells and the killing of tumor cells by NK. NKG2D belongs to the C-type lectin-like receptor family, because the receptor encoded by this gene exists in the NKG2 (natural killer group 2) complex. The NKG2 gene complex is located on human chromosome 12. NKG2D is a type II transmembrane protein. NKG2D needs to bind some adaptor proteins to complete signal transduction through charged residues in the transmembrane region. Human NKG2D can bind 10kDa DNAX activating protein (DAP10).
  • DAP10 DNAX activating protein
  • DAP10 contains YXXM motif (Tyr-X-X-Meth) in the cytoplasm which can recruit phosphatidylinositol trihydroxy kinase (PI3K) and growth factor receptor binding protein-2. All NK cells, most NKT cells, and macrophages express NKG2D. In addition, NKG2D also exists on the surface of CD8+ T cells. Under normal conditions, human and mouse CD4+ T cells do not express NKG2D. However, in patients, the proportion of CD4+ T cells expressing NKG2D has increased significantly. NKG2D can bind to many different ligands, which belong to major histocompatibility complex class I (MHC-I) related proteins.
  • MHC-I major histocompatibility complex class I
  • NKG2D ligands Another family of human NKG2D ligands are MIC-A and MIC-B. Both MIC-A and MIC-B are polymorphic. Currently, MIC-A has 61 alleles and MIC-B has 30 alleles. The ligands of NKG2D molecules are not expressed or expressed very little in normal cells, but when the cells are infected or become cancerous, the expression of these ligands will increase significantly.
  • NKR-2 NKG2D CAR
  • NKR-2 transfected NK cells or CD8+T cells contact the NKG2D ligand on the tumor cells, they will be activated and kill the tumor cells.
  • NKR-2CART The technical advantages of NKR-2CART include: 1) NKG2D is derived from the human body, and the NKG2D CAR-T formed is very low immunogenic, which prolongs the efficacy cycle of NKR-2; 2) NKG2D receptors can be recognized in most hematomas and Eight different ligands (MICA, MICB and ULBP1-6) expressed on the surface of solid tumor cells give NKR-2 a broad spectrum of tumor treatment; 3) Celyad's Phase I clinical trial results show that NKR-2 is in AML The safety and tolerability of the treatment of patients with MM and MM are good, indicating that although NKG2D has a wide variety of ligands, there is no obvious off-target effect.
  • NKR-2 has a good effect, it also has some shortcomings: 1) NKG2D is mainly expressed on NK cells, but the number of NK cells in the blood of tumor patients is small, and expansion is difficult, and it is difficult to produce NKG2D CAR-NK; 2 ) The expression level of NKG2D on CD8+T is low. Viral transfection can promote its expression on CD8+T. However, whether it is the expression of NKG2D or the activation of T cells after binding with ligand, it depends on intracellular DAP10.
  • NKR-2CAR-T The concentration of NKR-2CAR-T will affect the efficacy of NKR-2CAR-T to a certain extent; 3)
  • the number of CD4+T cells accounts for about 50% of the total number of T cells, but since NKG2D and DAP10 are not expressed on CD4+T, NKR- 2Not applicable to CD4+ T cells.
  • the present invention optimizes and improves NKR-2.
  • the improved NKG2D CAR-T (NKG92) only retains the extracellular 92-216 region (ligand binding region) of the NKG2D receptor, and is connected to the transmembrane domain and costimulatory signal transduction region through its C216 end to form a fusion protein.
  • the present invention forms a fusion protein by connecting the hinge-transmembrane (TM) region of CD8a, the costimulatory region of 4-1BB, and the signal region of CD3zeta.
  • NKG92 transfection can significantly increase the expression of NKG2D 92-216 on the cell membrane surface in both CD4+ T cells (NKG2D negative) and CD4+ T cells (NKG2D low expression); NKG92 transfected T cells can significantly kill A variety of tumor cells died, including hematoma and solid tumors.
  • the present invention provides a fusion protein and NKG2D CAR-T cells capable of expressing the fusion protein, and preparation and application thereof. The cells can specifically recognize and kill tumors and have more efficient tumor killing activity.
  • the fusion protein comprising NKG2D (92-216) or a homologous sequence thereof.
  • the fusion protein is a chimeric antigen receptor (CAR) and comprises an antigen binding domain , Transmembrane domain and intracellular signal transduction domain (preferably costimulatory signal transduction area), the antigen binding domain can specifically bind to tumor-specific antigen NKG2D ligand, and pass the transmembrane domain and intracellular signal
  • the conduction domain preferably a costimulatory signal conduction area
  • the antigen-binding domain therein comprises or is NKG2D (92-216) or its homologous sequence (preferably with at least 80%, at least 81%, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94 %, at least 95%, at
  • the NKG2D ligand is selected from one or more of major histocompatibility complex class I (MHC-I) molecules, MIC-A and MIC-B.
  • MHC-I major histocompatibility complex class I
  • the extracellular 92-216 region of the NKG2D receptor of the present invention is SEQ ID NO: 3 (preferably, its coding sequence is SEQ ID NO: 4), and optionally the extracellular 92-216 region The 216 region was replaced with the homology sequence of the ligand binding region.
  • the transmembrane domain is selected from the group consisting of ⁇ , ⁇ or ⁇ chains of T cell receptors, CD3 ⁇ , CD4, CD5, CD8, CD8 ⁇ , CD9, CD16, CD22, CD28, CD33, CD37, CD45
  • the intracellular signal conduction domain (preferably a costimulatory signal conduction area) is selected from: CD2, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD7, CD22, CD27, CD28, CD30, CD40, CD66d, CD79a, One or a combination of two or more of CD79b, CD83, CD134, CD137, ICOS, CD154, 4-1BB and OX40, LFA-1, LIGHT, NKG2C, and B7-H3; preferably, the total The stimulus signal transduction region contains 4-1BB and CD3 ⁇ intracellular domains.
  • the structure of the fusion protein is NKG2D(92-216)-CD8 ⁇ hinge-CD8TM-4-1BB-CD3 ⁇ , and the fusion protein can specifically recognize NKG2D ligand.
  • a hinge domain between the extracellular antigen binding domain and the transmembrane domain is a hinge domain, and preferably the hinge domain is derived from CD8 ⁇ .
  • residue 216 of NKG2D (92-216) in the fusion protein (preferably CAR) of the present invention is connected as the C-terminus to the hinge domain or the N-terminus of the transmembrane domain.
  • the homologous sequence of NKG2D (92-216) or its uncovered sequence is similar to the N-terminal connection of the hinge domain or transmembrane domain.
  • amino acid sequence of the NKG2D(92-216)-CD8 ⁇ hinge-CD8 TM -4-1BB-CD3 ⁇ fusion protein is shown in SEQ ID NO:1 or its homologous sequence.
  • the homology between the homologous sequence and the original sequence is about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% Or above, 76% or above, 77% or above, 78% or above, 79% or above, 80% or above, 81% or above, 82% or above, 83% or above, 84% or above, 85% Or above, 86% or above, 87% or above, 88% or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% Or above, 96% or above, 97% or above, 98% or above, 99% or above, 99.1 or above, 99.2 or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or above , 99.7% or more, 99.8% or more, or 99.9% or more.
  • Another aspect of the present invention provides a nucleotide sequence expressing the above-mentioned fusion protein.
  • nucleotide sequence is shown in SEQ ID NO: 2 or a degenerate sequence thereof.
  • the homology between the degenerate sequence and the original sequence is about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% Or above, 76% or above, 77% or above, 78% or above, 79% or above, 80% or above, 81% or above, 82% or above, 83% or above, 84% or above, 85% Or above, 86% or above, 87% or above, 88% or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% Or above, 96% or above, 97% or above, 98% or above, 99% or above, 99.1 or above, 99.2 or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or above , 99.7% or more, 99.8% or more, or 99.9% or more.
  • Another aspect of the present invention provides a NKG2D CAR-T cell, which can express the above-mentioned fusion protein.
  • the fusion protein and/or the NKG2D CAR-T cell can effectively kill and/or kill lung cancer cells, breast cancer cells, colon cancer cells, breast cancer cells, and lung cancer cells Wait.
  • the CART cells of the present invention are CD4+ T cells or a cell mixture containing CD4+ T cells and CD8+ T cells.
  • Another aspect of the present invention also provides a method for preparing the above-mentioned NKG2D CAR-T cell, which includes the following steps:
  • step (1) Use the lentiviral packaging plasmid and the lentiviral expression vector plasmid obtained in step (1) to infect 293T cells, package and prepare the lentivirus;
  • step (3) Use the lentivirus obtained in step (2) to infect NK-92 cells to obtain CAR-T cells.
  • the present invention also provides the application of the above-mentioned fusion protein and/or NKG2D CAR-T cells in the preparation of drugs for the treatment and/or prevention of cancer.
  • the application of the fusion protein and/or NKG2D CAR-T cells in the preparation of drugs for the treatment of tumors and related diseases that highly express NKG2D ligands are highly express.
  • the cancer is blood cancer, lymphoma, breast cancer, cervical cancer, colon cancer, or lung cancer.
  • the present invention also provides a pharmaceutical composition, which comprises the above-mentioned NKG2D CAR-T cell, and optionally, pharmaceutically acceptable excipients.
  • the invention provides a fusion protein and NKG2D CAR-T cells capable of expressing the fusion protein.
  • the fusion protein can specifically bind to the tumor-specific antigen NKG2D ligand, and activate the T cell through the transmembrane domain and the costimulatory signal transduction region.
  • the NKG2D CAR-T cells are constructed by using NKG2D receptors for CAR-T cells.
  • the fusion protein and NKG2D CAR-T cells capable of expressing the fusion protein use NKG2D ligand as the target antigen, which can specifically kill tumor cells. It can be used as a therapeutic drug for tumor diseases and is used for tumors with high expression of NKG2D ligand. Treatment provides a new method for the prevention and treatment of tumors.
  • Figure 1 shows a schematic diagram of the CAR in the NKG92-CART structure provided by an embodiment of the present invention, where A is a structure containing a CD8a hinge-transmembrane (TM) domain and a 4-1BB co-stimulatory domain fused with the CD3 ⁇ signaling region
  • TM hinge-transmembrane
  • B is the structure of the NKG2D 92-216 homodimer
  • C is formed on the surface of transduced T cells through the conserved cysteine in the hinge domain of CD8a NKG92 homodimer.
  • Fig. 2 shows the results of NKG2D CAR-NK flow cytometric detection of the positive rate of CAR cells provided by an embodiment of the present invention, wherein Fig. 2A is the control group; Fig. 2B is the experimental group.
  • Figure 3 is a FACS analysis of NKG2D expression on the cell surface 5 days after the Lentiviral vector encoded by NKG2D-CAR was used to transduce T cells, where A is a graph and B is a histogram.
  • Figure 4 is a diagram of the experimental results of NKG2D CAR-T cells killing different tumor cells provided by the embodiments of the present invention, in which (from left to right) are respectively directed against myeloid leukemia cells K562, lymphoma raji, and breast cancer cells MDA-MB- 231.
  • the nucleotide sequence encoding the NKG2D-CD8 ⁇ hinge-CD8 TM -4-1BB-CD3 ⁇ fusion protein of the present invention is any DNA sequence capable of encoding the fusion protein, preferably, the sequence is SEQ ID NO: 2 or its complementary sequence.
  • the nucleotide sequence encoding the NKG2D-CD8 ⁇ hinge-CD8 TM -4-1BB-CD3 ⁇ fusion protein of the present invention can be hybridized with the nucleotide sequence of SEQ ID NO: 2 under stringent conditions.
  • stringent conditions may be any of low stringency conditions, medium stringency conditions, and high stringency conditions, and preferably high stringency conditions.
  • low stringency conditions may be 30°C, 5 ⁇ SSC, 5 ⁇ Denhardt solution, 0.5% SDS, 52% formamide
  • medium stringency conditions may be 40°C, 5 ⁇ SSC, 5 ⁇ The conditions of Denhardt solution, 0.5% SDS, 52% formamide
  • high stringency conditions can be 50°C, 5 ⁇ SSC, 5 ⁇ Denhardt solution, 0.5% SDS, 52% formamide.
  • those skilled in the art should understand that the higher the temperature, the more highly homologous polynucleotides can be obtained.
  • those skilled in the art can select a comprehensive result formed by multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration that affect the stringency of hybridization to achieve the corresponding stringency.
  • hybridizable polynucleotide can also be calculated by FASTA, BLAST and other homology search software with default parameters set by the system, which has about 60% or more of the polynucleotide encoding sequence number 6 , About 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or Above, 80% or above, 81% or above, 82% or above, 83% or above, 84% or above, 85% or above, 86% or above, 87% or above, 88% or above, 89% or Above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% or above, 96% or above, 97% or above, 98% or above, 99% or Polynucleosides of above, 99.1 or above, 99.2 or above, 99.3% or above, 99.4% or above, 99.5% or above, 99.6% or
  • encoding nucleotides include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.
  • the nucleotide sequence encoding the protein may include introns.
  • lentivirus refers to the genus of the Retroviridae family, which can effectively infect acyclic and post-mitotic cells; they can transmit a significant amount of genetic information into the host cell's DNA, so that they are the best gene delivery vector. One of the effective methods.
  • promoter is defined as a DNA sequence that is required to initiate specific transcription of a polynucleotide sequence and is recognized or guided by the synthetic machinery of the cell.
  • the term "specifically binds" refers to recognizing a specific antigen but not substantially recognizing or binding other molecules in the sample.
  • carrier is a composition of matter, which includes an isolated nucleic acid, and which can be used to deliver the isolated nucleic acid to the inside of a cell.
  • vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides related to ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.
  • cancer is defined as a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, colorectal cancer, liver cancer, lung cancer, and so on.
  • NKG2D (92-216) or its homologous sequence as the antigen binding domain in the examples of CAR
  • those skilled in the art will understand that NKG2D (92-216) or its homologous sequence can be further truncated Source sequence to prepare CAR-T cells that can kill tumors more efficiently.
  • 1-20 amino acid residues can be deleted in the amino acid sequence of NKG2D (92-216), or some amino acid residues can be replaced and modified.
  • NKG2D CAR-T and nkg2D-car T have the same meaning, which means CAR-T cells expressing NKG2D molecules; CD8 TM means transmembrane domain.
  • NKG2D-CD8 TM -4-1BB-CD3 ⁇ fusion gene sequence (its amino acid sequence is shown in SEQ ID NO: 1, the gene sequence is shown in SEQ ID NO: 2, and its structure diagram is shown in Figure 1a, Figure 1b and Figures 1c)
  • the NKG2D-CD8 TM -4-1BB-CD3 ⁇ fusion gene sequence was transformed and connected to the PWPXLD-kana vector by restriction digestion, and the upstream of the gene was the EF-1 ⁇ promoter.
  • the vector is transformed into E.
  • the lentiviral packaging helper plasmids psPax2 and PMD2.0G were transformed into DH5 ⁇ , ampicillin screened, and plasmids were extracted.
  • BES 50mM BES, 280mM NaCl, 1.5mM Na 2 HPO4;
  • Plasmid system PWPXLD plasmid vector containing CAR gene fragment: 9 ⁇ g; psPax2: 9 ⁇ g; and PMD2.0G: 4.5 ⁇ g.
  • the mixed liquid was added to the medium, gently mix the medium to make the transfection system evenly distributed, and replace the 2% FBS DMEM medium after 18 hours. After 48 hours, the culture supernatant was collected, and the supernatant was centrifuged at 1000 xg for 10 min to remove cell debris, and filtered with a 0.45 ⁇ m filter. The filtered virus solution was added to one-third volume of the virus solution TAKR lentivirus concentration reagent concentrator, inverted and mixed, and then allowed to stand overnight at 4°C. After overnight, the virus solution was centrifuged at 4°C at 1500g 45min, the supernatant was discarded, resuspended in PBS and aliquoted, and stored at -80°C.
  • Configure sorting buffer 40mM EDTA (1ml), PBS (38ml), 20% BSA (1ml); pre-cool at 4°C;
  • T cell culture medium preparation culture medium (MACS); cytokine (IL-2 (200U/ml)); plasma (5% inactivated);
  • T cell stimulating factor dynabeads magnetic beads with buffer removed
  • cell: magnetic beads 1:2;
  • T cells Take T cells, add 1x10 6 /mL to the well plate; culture at 37°C, 5% CO 2;
  • Detection of maximum release add 20 microliters of LDH maximum release reagent to each well in the maximum release group and volume correction group, respectively, at 37°C, 5% CO 2 , and incubate for 45 min;
  • kill rate (experimental group-spontaneous group A-spontaneous group B + blank group) / (maximum release value-volume correction value-spontaneous group B + blank group)
  • the CART of the present invention can have a significant curative effect or inhibitory effect on myeloid leukemia, breast cancer, lung cancer, breast cancer and cervical cancer.

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Abstract

La présente invention concerne une protéine de fusion, comprenant un domaine de liaison à l'antigène, un domaine transmembranaire et une région de signalisation costimulatrice. Le domaine de liaison à l'antigène peut se lier de manière spécifique à un antigène spécifique à une tumeur, qui est un ligand NKG2D, et activer des cellules T au moyen du domaine transmembranaire et de la région de signalisation costimulatrice. La protéine de fusion et une cellule CAR-T NKG2D capable d'exprimer la protéine de fusion selon la présente invention peuvent éliminer de manière spécifique des cellules tumorales à l'aide de la cellule CAR-T NKG2D comprenant le ligand NKG2D en tant qu'antigène cible. La protéine de fusion et la cellule CAR-T NKG2D peuvent être utilisées en tant que médicament pour le traitement de maladies tumorales et peuvent être utilisées pour traiter des tumeurs dans lesquelles un ligand d'une molécule NKG2D est hautement exprimé, ce qui permet de fournir une nouvelle méthode pour la prévention ou le traitement de tumeurs.
PCT/CN2019/109960 2019-10-08 2019-10-08 Cellule car-t nkg2d, préparation et utilisation associées WO2021068108A1 (fr)

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