WO2023001256A1 - Nanoparticle and use thereof in detection of car-positive expression rate - Google Patents

Nanoparticle and use thereof in detection of car-positive expression rate Download PDF

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WO2023001256A1
WO2023001256A1 PCT/CN2022/107197 CN2022107197W WO2023001256A1 WO 2023001256 A1 WO2023001256 A1 WO 2023001256A1 CN 2022107197 W CN2022107197 W CN 2022107197W WO 2023001256 A1 WO2023001256 A1 WO 2023001256A1
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protein
cells
car
amino acid
acid sequence
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PCT/CN2022/107197
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French (fr)
Chinese (zh)
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秦丽丽
胡旭乐
孙丽芹
陈宜顶
苗景赟
古万超
侯洋
张雪瑶
范华
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北京百普赛斯生物科技股份有限公司
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Priority to CN202280020744.2A priority Critical patent/CN116981684A/en
Publication of WO2023001256A1 publication Critical patent/WO2023001256A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the disclosure relates to the field of biotechnology, in particular to a nanoparticle and its application for detecting the positive expression rate of CAR.
  • T cells In the immune system, T cells rely on antigen-presenting cells (APCs) to kill abnormal cells.
  • APCs antigen-presenting cells
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • T cells can eventually recognize abnormal cells and kill them. But tumor cells destroy these "fingerprints" of themselves, so that APC cells cannot recognize it.
  • Chimeric Antigen Receptor is an artificial receptor molecule manufactured by genetic engineering technology, which can endow immune effector cells (such as T lymphocytes) with specificity for a target epitope, thereby Enhance the function of T lymphocyte recognition antigen signal and activation (Sadelain, M., R. Brentjens, and I. Riviere. The promise and potential pitfalls of chimeric antigen receptors. Curr Opin Immunol, 2009.21 (2): p.215-23 .). CAR-T cells can recognize specific target antigens in an MHC-unrestricted manner, and continuously activate expanded T cells. CAR-T cell therapy is essentially a kind of adoptive cell transfer (ACT).
  • ACT adoptive cell transfer
  • ACT was first proposed as a process in which donor lymphocytes are infused into the recipient during organ transplantation. This process antigen-mediated rejection of transplanted organs Then it gradually developed into a therapy for malignant tumors (Mitchison,N.A..Studies on the immunological response to foreign tumor transplants in the mouse.I.The role of lymph node cells in conferring immunity by adoptive transfer. The Journal of experimental medicine , 1955.102(2): p.157-177.).
  • CAR-T cells the active ingredient to play a tumor-killing role is the T cells with positive CAR-expression rate.
  • the packaging specifications and clinical dosage of CAR-T cell products are expressed by the number of CAR-T positive cells.
  • the detection of CAR positive expression rate is an important quality control step in the production process of CAR-T cells.
  • the positive expression rate of CAR is mainly detected by the Anti-Fab antibody or Protein L protein of the light chain or hinge region, but the above methods all have the defect of non-specific binding, and cannot prove whether the CAR can bind the target antigen. Therefore, the target protein used to detect the positive expression rate of CAR has the advantage of strong specificity and is favored by the industry. However, the preparation of most target proteins is relatively difficult, and the obtained target proteins may also have adverse effects on the detection of CAR positive expression rate.
  • the transmembrane region of the target protein due to the hydrophobicity of the transmembrane region of the target protein, it is necessary to introduce detergent to dissolve the target protein during the purification process, so that the prepared target protein will contain detergent, and the presence of detergent will lead to T cells undergo membrane lysis, which makes the target protein in the detergent system unable to be used for the detection of CAR positive rate.
  • using the soluble extracellular loop region of the target protein to detect the positive expression rate of CAR although the preparation of the target protein is difficult, but because there is no limit to the result of the transmembrane region, it cannot guarantee that its conformation is consistent with the natural conformation, and there are problems that cannot be correctly identified Flaws of CAR.
  • VLP Virus-like particles
  • CLP core-like particles
  • CP viral capsid protein
  • Patent Application US20110189159A1 is based on the discovery that proteins of interest can be delivered to cells as fused or unfused proteins; in particular, by making VLPs with two Gag fusions (Gag-protein of interest and Gag-protease), the The protein of interest is delivered to cells as an unfused protein for therapy.
  • the vaccine disclosed in PCT patent application WO2021022008A1 comprises VLP, and pharmaceutically acceptable excipients, carriers and/or adjuvants; wherein, the VLP comprises (a) a synthetic or natural lipid bilayer, (b) embedded in a lipid bilayer The anchor molecule in (c) the antigen bound to the anchor molecule.
  • eVLP has shown unique research and application value in many fields such as vaccine development, targeted drug delivery, biomedical imaging and sensing, and tissue engineering. However, studies on the positive expression rate of eVLP in CAR are rarely reported.
  • the present disclosure provides a nanoparticle and its application for detecting the positive expression rate of CAR.
  • the present disclosure solves the technical problems to be solved by the present disclosure through the following technical solutions.
  • nanoparticle comprising:
  • enveloped virus-like particle eVLP
  • eVLP enveloped virus-like particle
  • the enveloped virus-like particle skeleton protein comprises a viral core protein or a functional fragment thereof, and the enveloped virus-like particle skeleton protein or a functional fragment thereof assembles to form an enveloped virus-like particle, and the target protein and/or its Functional fragments are displayed on enveloped virus-like particles to form target protein-eVLP nanoparticles.
  • the target protein is a CAR target protein; the CAR target protein and/or its functional fragments are displayed on enveloped virus-like particles to form CAR target protein-eVLP nanoparticles .
  • the virus is selected from the group consisting of retrovirus, baculovirus, filovirus, coronavirus, influenza virus, paramyxovirus, respiratory syncytial virus, arenavirus, Newcastle disease virus, para Influenza virus, bunya virus, hepatitis C virus, hepatitis B virus.
  • the retrovirus is selected from the group consisting of human immunodeficiency virus, simian immunodeficiency virus, murine leukemia virus, and bovine leukemia virus.
  • the baculovirus is vesicular stomatitis virus.
  • the filovirus is Ebola virus.
  • the coronavirus is selected from the group consisting of novel coronavirus pneumonia virus (SARS-CoV-2), SARS-CoV, MERS-Cov, 229E, NL63, OC43 and HKU1.
  • SARS-CoV-2 novel coronavirus pneumonia virus
  • SARS-CoV SARS-CoV
  • MERS-Cov MERS-Cov
  • 229E NL63
  • OC43 OC43
  • HKU1 novel coronavirus pneumonia virus
  • the virus core protein is selected from: retrovirus Gag protein, baculovirus matrix protein M protein, filovirus core protein, coronavirus M, E and NP proteins, influenza virus M1 protein, paramyxovirus M protein, respiratory syncytial virus (RSV) M protein, arenavirus Z protein, Newcastle disease virus M protein, parainfluenza virus M protein, bunya virus N protein, hepatitis C virus core protein C , hepatitis B virus core protein C and its composition.
  • the retroviral Gag protein is selected from the group consisting of human immunodeficiency virus Gag protein, simian immunodeficiency virus Gag protein, mouse leukemia virus Gag protein and bovine leukemia virus Gag protein. In one or more preferred embodiments, said retroviral Gag protein is said human immunodeficiency virus Gag protein. In one or more preferred embodiments, the human immunodeficiency virus Gag protein comprises an amino acid sequence with at least 80% or more identity to SEQ ID No.1, preferably 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences. In one or more preferred embodiments, the amino acid sequence of the human immunodeficiency virus Gag protein is shown in SEQ ID No.1.
  • the baculovirus matrix protein M protein is vesicular stomatitis virus M virus core protein.
  • the filovirus core protein is Ebola virus VP40 virus core protein.
  • the core protein of the coronavirus is selected from core proteins of novel coronavirus pneumonia virus (SARS-CoV-2), SARS-CoV, MERS-Cov, 229E, NL63, OC43 and HKU1.
  • SARS-CoV-2 novel coronavirus pneumonia virus
  • SARS-CoV SARS-CoV
  • MERS-Cov MERS-Cov
  • 229E NL63, OC43 and HKU1.
  • the virus is a virus that acquires an envelope when budding from the cell membrane of the host cell.
  • the CAR target protein comprises a membrane protein.
  • the membrane protein comprises a transmembrane protein.
  • the transmembrane protein comprises a multispanin.
  • the CAR target protein is selected from CD20, Claudin18.1, Claudin18.2, CD133, GPRC5D, CCR5, CCR8, BCMA, GPCR, CD147, CD19, CD123, CD138, CD22, CD30 , CD33, CD38, CD70, CAIX, EGFR, EGFRVIII, FOLR1, GPC3, HER2, HGFR, Anti-FMC63Ab, CLL-1, SLAMF7, CD4, CD5, CD8A&CD8B, FAP, IL13RA2, GPC3, GUCY2C, Her3, PSMA, ROR1 , SLAMF7, B7-H3, CD147, CEA, MUC16, Nectin-4, VEGFR2, Anti-RTX Ab, B7-H3, CAIX, CD7, CEA, MUC1, NKG2D, PSCA, uPAR, GD2, FR, PMEL, CA9, CD171/L1-CAM, IL-13R ⁇ 2, M
  • EGP-40 FBP, GD3, FSA, PSA, HMGA2, fetal acetylcholine receptor, LeY, EpCAM, mesothelin, IGFR1, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, Any of CYFRA21-1, SCC, AFU, EBV-VCA, POA, ⁇ 2-MG, PROGRP, or MSLN.
  • the CAR target protein comprises a multispanning protein.
  • the CAR target protein is selected from any of CD20, Claudin18.2, CD133, GPRC5D, CCR5, CCR8, CD19, BCMA, GPC3, CD30, CD22, EGFR, EGFRVIII, HER2 or GPCR A sort of.
  • the CAR target protein is Claudin18.2.
  • the amino acid sequence of Claudin18.2 comprises an amino acid sequence with at least 80% or more identity to SEQ ID No.4, preferably 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences.
  • the amino acid sequence of the Claudin18.2 is shown in SEQ ID No.4.
  • the CAR target protein is CD20.
  • the amino acid sequence of CD20 comprises an amino acid sequence with at least 80% or more identity to SEQ ID No.15, preferably 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99%, more preferably an amino acid sequence with an identity of 98% or more: more preferably, the amino acid sequence of the CD20 is shown in SEQ ID No.15.
  • the CAR target protein and/or its functional fragment and/or the viral core protein and/or its functional fragment are linked to a marker.
  • the N-terminal and/or C-terminal of the CAR target protein and/or its functional fragment and/or the N-terminal and/or C-terminal of the viral core protein and/or its functional fragment are connected to markers are linked.
  • the C-terminus of Claudin 18.2 or the C-terminus of Gag is labeled with a label.
  • the C-terminus of CD20 or the C-terminus of Gag is labeled with a label.
  • the label is selected from a detectable label, a purification tag, a reporter tag, and combinations thereof.
  • the detectable label is selected from a fluorescent group, a chemiluminescent label, an electrochemiluminescent label, and combinations thereof.
  • the fluorophore is selected from FITC, GFP, RFP, YFP, TRITC, PE, FAM, RRX, TR, Cy2, Cy3, Cy5, ECD, PC5.5, PC7, APC, Any of APC-A70, APC-A75, Pac-Blue, Alexa488, mBBr, 5-IAF, E-118, DTAF, rhodamine green, and KrO.
  • the chemiluminescent label is acridinium ester, isoluminol, horseradish peroxidase, or alkaline phosphatase.
  • the electrochemiluminescent label is ruthenium terpyridine or a derivative N-hydroxysuccinamide ester thereof.
  • the purification tag is selected from HIS-Tag, GST-Tag, MBP-Tag, NusA-Tag, FLAG-Tag, SUMO, Avi-Tag, Halo-Tag and SNAP-Tag any kind.
  • the purification tag is detected by a secondary antibody.
  • the reporter tag is selected from any one of c-Myc, HA, or luciferase.
  • the CAR target protein is displayed on the eVLP, and at the same time, the C-terminal of the CAR target protein or the C-terminal of Gag is labeled with a marker.
  • it relates to a labeled Claudin 18.2-eVLP, the Claudin 18.2-eVLP being linked to a label.
  • the CAR target protein-eVLP nanoparticles are Claudin 18.2-eVLP nanoparticles, the Claudin18.2 and/or its functional fragments and/or the viral core protein and/or its Functional fragments are linked to markers.
  • the N-terminal and/or C-terminal of the Claudin 18.2 and/or its functional fragment and/or the N-terminal and/or C-terminal of the viral core protein and/or its functional fragment are combined with a marker connected.
  • the enveloped virus-like particle backbone protein is Gag protein.
  • the C-terminus of the Claudin 18.2 or the C-terminus of the Gag protein is linked to a label.
  • the present disclosure relates to a labeled CD20-eVLP, the CD20-eVLP is linked to the label.
  • the CAR target protein-eVLP nanoparticles are CD20-eVLP nanoparticles, and the CD20 and/or its functional fragments and/or the viral core protein and/or its functional fragments are combined with markers are linked.
  • the N-terminal and/or C-terminal of the CD20 and/or its functional fragments and/or the N-terminal and/or C-terminal of the viral core protein and/or its functional fragments are compatible with the label connect.
  • the enveloped virus-like particle backbone protein is Gag protein.
  • the C-terminus of the CD20 or the C-terminus of the Gag protein is linked to a label.
  • the marker is GPF.
  • the amino acid sequence of the GFP comprises an amino acid sequence with at least 80% or more identity to SEQ ID No.2, preferably 85%, 90%, 95%, 96%, 97% , 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences. In one or more preferred embodiments, the amino acid sequence of the GFP is shown in SEQ ID No.2.
  • the C-terminus of the Gag protein is connected to the marker GFP, which is denoted as Gag-GFP, and the amino acid sequence of the Gag-GFP contains at least 80% or more of the same sequence as SEQ ID No.3.
  • An amino acid sequence with identity preferably an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity.
  • the amino acid sequence of the Gag-GFP is shown in SEQ ID No.3.
  • the C-terminus of the Claudin 18.2 is linked to the marker GFP, denoted as Claudin 18.2-GFP, and the amino acid sequence of the Claudin 18.2-GFP has at least 80% or more of SEQ ID No.5
  • the amino acid sequence with identity is preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more, more preferably 98% or 99% or more.
  • the amino acid sequence of the Claudin 18.2-GFP is shown in SEQ ID No.5.
  • nanoparticles which includes:
  • a recombinant plasmid comprising enveloped virus-like particle (eVLP) skeleton protein and/or its functional fragment gene and a recombinant plasmid of target protein and/or its functional fragment gene; ) a recombinant plasmid of the backbone protein and/or its functional fragment gene and the target protein and/or its functional fragment gene; and
  • eVLP enveloped virus-like particle
  • eVLP enveloped virus-like particle
  • target protein and/or its functional fragment recombinant plasmid Transfect the enveloped virus-like particle (eVLP) skeleton protein and/or its functional fragment recombinant plasmid and target protein and/or its functional fragment recombinant plasmid into the host cell to express the enveloped virus-like particle skeleton protein and/or functional fragments thereof and target protein and/or functional fragments thereof; the enveloped virus-like particle skeleton protein or functional fragments thereof are assembled to form enveloped virus-like particles, and the target protein and/or functional fragments thereof are displayed On the enveloped virus-like particle, forming target protein-eVLP nanoparticles; and, optionally,
  • the nucleic acid sequence encoding GAG comprises a nucleotide sequence with at least 80% or more identity to SEQ ID No.7, preferably 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical nucleotide sequences, more preferably 98% or 99% or more identical nucleotide sequences: more preferably, the amino acid sequence of the GFP is as SEQ ID No. 7.
  • the Gag-GFP encoding nucleic acid sequence comprises a nucleotide sequence with at least 80% or more identity to SEQ ID No.8, preferably 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical nucleotide sequences, more preferably 98% or 99% or more identical nucleotide sequences: more preferably, the amino acid sequence of the GFP is as SEQ ID No .8 shown.
  • the nucleic acid sequence encoding Claudin 18.2 comprises a nucleotide sequence with at least 80% or more identity to SEQ ID No.9, preferably 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical nucleotide sequences, more preferably 98% or 99% or more identical nucleotide sequences: more preferably, the amino acid sequence of the GFP is as SEQ ID No .9 shown.
  • the coding nucleic acid sequence of Claudin 18.2-GFP comprises a nucleotide sequence having at least 80% or more identity with SEQ ID No.10, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical nucleotide sequences, more preferably 98% or 99% or more identical nucleotide sequences: more preferably, the amino acid sequence of the GFP is as SEQ ID No.10 is shown.
  • the nucleic acid sequence encoding CD20 comprises a nucleotide sequence with at least 80% or more identity to SEQ ID No.16, preferably 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical nucleotide sequences, more preferably 98% or 99% or more identical nucleotide sequences: more preferably, the amino acid sequence of the GFP is as SEQ ID No. 16.
  • the host cell is a prokaryotic host cell or a eukaryotic host cell.
  • the prokaryotic host cell is selected from bacterial cells such as E. coli, B. subtilis and mycobacteria.
  • the eukaryotic host cell is selected from any one of animal cells, plant cells or fungi.
  • the eukaryotic host cell is selected from any one of yeast, insect, avian, plant, Caenorhabditis elegans, and mammalian host cells.
  • Non-limiting examples of such insect cells are: Spodoptera frugiperda (Sf) cells, such as Sf9, Sf21, Trichoplusia ni cells, such as High Five cells, and Drosophila S2 cells.
  • Sf Spodoptera frugiperda
  • yeast host cells examples include S. cerevisiae, Kluy veromyces lactis (K. lactis), Candida species including Candida albicans (C .albicans) and C. glabrata, Aspergillus nidulans, Schizosaccharomyces pombe (S. pombe), Pichia pastoris, and lipolytica Yarrowia lipolytica.
  • mammalian cells examples include COS cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells (e.g. HEK293 cells), 633 cells, Vero, BHK cells, African green Monkey cells, CV1 cells, HeLa cells, MDCK cells and Hep-2 cells.
  • CHO Chinese hamster ovary
  • HEK human embryonic kidney
  • the host cell is selected from any one of HEK293 cells, 633 cells, Vero cells, BHK cells, prokaryotic cells, yeast cells, plant cells, insect cells and mammalian cells.
  • it relates to a method for detecting the positive expression rate of CAR, comprising incubating the nanoparticle or the nanoparticle prepared by the method with CAR-modified cells, and then performing detection.
  • the cells are washed after incubation of the cells prior to performing the assay.
  • the CAR-modified cells are selected from CAR-like (HEK293) cells, CAR-T cells, CAR-NK cells, CAR-M cells, CAR-NKT cells, CAR-Treg Any of cells and CAR- ⁇ T cells.
  • the method for detecting the positive expression rate of CAR includes incubating the aforementioned labeled Claudin 18.2-eVLP with CAR-modified cells, washing and then detecting.
  • the method for detecting the positive expression rate of CAR includes incubating the aforementioned labeled Claudin 18.2-eVLP with CAR-T cells, washing and then detecting.
  • the Claudin 18.2-eVLP ensures the natural complete conformation of the CAR target protein, which improves the success rate of isolating antibodies that can recognize the natural membrane protein structure.
  • the method for detecting the positive expression rate of CAR includes incubating the aforementioned labeled CD20-eVLP with CAR-modified cells, washing and then detecting.
  • the method for detecting the positive expression rate of CAR includes incubating the aforementioned labeled CD20-eVLP with CAR-T cells, washing and then detecting.
  • the CD20-eVLP ensures the natural complete conformation of the CAR target protein, which improves the success rate of isolating antibodies that can recognize the natural membrane protein structure.
  • the incubation temperature is 2°C-40°C, preferably 37°C, and/or, the incubation time is 15min-2 hours, preferably 1 hour; and/or, the The concentration of CO2 for incubation is 2%-8%, preferably 5%.
  • the number of washings is 1-6 times, preferably 3 times.
  • the wash buffer is 0.5%-5% BSA, preferably 2% BSA.
  • the detection is any one of flow cytometry detection, immunoassay, ELISA, SPR, BLI.
  • the enveloped virus-like particle eVLP backbone protein is Gag protein.
  • the C-terminus of the Claudin 18.2 or the C-terminus of the Gag protein is linked to a label.
  • the C-terminus of the CD20 or the C-terminus of the Gag protein is linked to a label.
  • the ability of HIV-1 Gag to self-assemble into eVLPs is utilized to display the CAR target protein on the membrane surface ( Figure 1).
  • the tetraspanning protein Claudin 18.2 is displayed on the HIV-1 Gag envelope VLP, and the C-terminus of Claudin 18.2 or the C-terminus of Gag is labeled with a marker.
  • Claudin 18.2-eVLP was used to evaluate the positive expression rate of CAR by flow cytometry. All the Claudin 18.2-eVLPs produced in this disclosure can be evaluated for the expression of target antigen-binding CAR.
  • the nanoparticles are CAR target protein-eVLP nanoparticles.
  • the kit contains the aforementioned labeled CAR target protein-eVLP.
  • the kit contains the aforementioned labeled Claudin 18.2-eVLP.
  • the kit contains the aforementioned labeled CD20-eVLP.
  • the kit is an ELISA kit, an SPR kit or a BLI kit.
  • the ELISA kit contains the aforementioned CAR target protein-eVLP, microtiter plate, blocking solution, sample diluent, enzyme conjugate, concentrated washing solution, enzyme substrate solution and stop solution.
  • the SPR kit comprises the aforementioned CAR target protein-eVLP nanoparticles.
  • the BLI kit comprises the aforementioned CAR target protein-eVLP nanoparticles.
  • the kit described in the present disclosure can be used not only to detect the positive expression rate of CAR, but also to prove that CAR can specifically bind to a target protein.
  • the kit may include reagents for preparing the CAR target protein-eVLP and/or administering the CAR target protein-eVLP.
  • the kit may further include reagents for evaluating the activity of the CAR target protein-eVLP in vitro and in vivo.
  • the kit can include reagents and/or devices for administration, such as an inhaler or nebulizer, and the kit can also include one or more buffers, and the like.
  • the liquid solutions are aqueous solutions, especially preferably sterile aqueous solutions.
  • the components of the kit may also be provided in dry powder form.
  • liquid solutions can be reconstituted by the addition of suitable solvents.
  • Fig. 1 is a schematic diagram of the principle of HIV-Gag enveloped VLP displaying multiple transmembrane target antigens.
  • Figure 2 shows the electrophoresis results of crude samples after sucrose density gradient centrifugation, wherein lane A is the crude sample of R1242, lane B is the crude sample of RG288, and lane C is the crude sample of RG341.
  • Figure 3 shows the electrophoresis detection results of the samples separated by Sepharose 6FF, where Figure A is R1242, Figure B is RG288, and Figure C is RG341.
  • Figure 4 shows the detection results of R1242-210521F1 by transmission electron microscope.
  • Figure 5 shows the ELISA method to detect the binding activity of Claudin 18.2-eVLP and its specific antibody IMAB362, wherein Figure A is the ELISA detection result of the combination of RG288-210503F1 and IMAB362; Figure B is the ELISA detection result of the combination of RG341-210521F1 and IMAB362.
  • Figure 6 is a schematic diagram of the IMAB361 CAR-like structure.
  • Figure 7 shows that Claudin18.2-eVLP of the present disclosure is used to evaluate the expression of IMAB362 CAR-like: where Figure A is when the FITC-anti mFab Ab is diluted 200 times, it evaluates the expression of IMAB362 CAR-like; Figure B is when When FITC-Protein L is used at a concentration of 10 ⁇ g/mL, it evaluates the expression of IMAB362 CAR-like; Figure C is when Claudin-18.2 Protein, His Tag TM is used at a concentration of 10 ⁇ g/mL, it evaluates the expression of IMAB362 CAR-like; Figure D is the evaluation of IMAB362 CAR-like expression when RG288-210503F1 is used at a concentration of 10 ⁇ g/mL; Figure E is the evaluation of IMAB362 CAR-like expression when RG341-210521F1 is used at a concentration of 10 ⁇ g/mL.
  • Figure 8 shows that the CD20-eVLP of the present disclosure is used to evaluate the expression of Ofatumumab CAR-like: when RG344-210628F1 is used at a concentration of 20 ⁇ g/mL, it evaluates the expression of Ofatumumab CAR-like.
  • the present disclosure discloses an eVLP and its application for detecting the positive expression rate of CAR.
  • Those skilled in the art can refer to the content herein to obtain the eVLP and realize its application.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present disclosure.
  • the preparation method and application of the present disclosure have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the preparation method and application herein without departing from the content, spirit and scope of the present disclosure to realize and Apply the disclosed technology.
  • virus-like particle refers to a structure that resembles a virus in at least one property but has been shown to be non-infectious. In general, virus-like particles lack the viral genome and are unable to replicate. In addition, virus-like particles can often be produced in large quantities by heterologous expression and can be easily purified.
  • enveloped VLP or alternatively “eVLP” refers to an enveloped virus-like particle formed by wrapping a virus-like particle with a lipid envelope derived from a host cell.
  • CAR target protein refers to a protein that can specifically bind to CAR.
  • eVLP backbone protein in the present disclosure can be understood as a protein forming the backbone structure of an eVLP, which may mainly consist of viral core protein or include viral core protein as well as other proteins.
  • viral core protein refers to the envelope protein, which in some cases is also capable of driving budding and release of particles from the host cell.
  • the term "functional fragment” corresponds to a fragment of a full-length protein that has a truncated structure, but still maintains all or part of the function of the full-length protein; for example, for the core protein, its corresponding functional fragment can be understood as a truncated core A fragment of a protein that still has the ability to form at least a partial capsid of the virus or to drive budding and the release of particles from the host cell; for the target protein, its corresponding functional fragment can be understood as a fragment of the truncated target protein, It still has the ability to specifically bind to CAR.
  • chimeric antigen receptor or alternatively “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain and a functional signal derived from a stimulatory molecule The cytoplasmic signaling domain of the transduction domain.
  • CAR-like refers to chimeric antigen receptor (CAR) modified HEK293 cells, that is, HEK293 is edited by CAR means, and CAR is displayed on the surface of HEK293.
  • CAR-NK refers to chimeric antigen receptor (CAR) modified NK cells, wherein NK cells are natural killer cells (natural killer cells), NK cells are important immune cells of the body, not only with anti-tumor, anti-virus Infection is related to immune regulation, and in some cases participates in the occurrence of hypersensitivity and autoimmune diseases, and can recognize target cells and killing mediators.
  • NK cells are regarded as effector cells that also have the potential to enhance their anti-tumor ability through CAR modification because of their special mechanism for recognizing target cells, short physiological cycle, and extensive tumor killing ability.
  • CAR-M refers to chimeric antigen receptor (CAR)-modified macrophages (CAR macrophages), that is, the use of CAR means to edit human macrophages so that they can directly phagocytize tumors.
  • CAR-NKT refers to chimeric antigen receptor (CAR) modified NKT cells (Natural killer T cells), wherein NKT cells are a kind of T cell receptor TCR and NK cell receptors on the cell surface Special T cell subsets.
  • CAR-NKT cells combine the original advantages of NKT cells and the specificity of CAR therapy to better achieve tumor killing effects.
  • CAR-Treg refers to chimeric antigen receptor (CAR) modified regulatory T cells (Tregs), where Tregs are a type of T cell subset that controls autoimmune reactivity in the body, also known as inhibitory T cells in the early days Cells (suppressor T cells).
  • Regulatory T cells can be divided into naturally occurring natural regulatory T cells (n T-regs) and induced adaptive regulatory T cells (a T-regs or i T-regs), such as Th3, Tr1, and There are CD8 Treg, NKT cells, etc., which are closely related to the occurrence of autoimmune diseases, and their abnormal expression can lead to autoimmune diseases.
  • CAR- ⁇ T refers to chimeric antigen receptor (CAR)-modified ⁇ T cells, where ⁇ T cells are a type of T cell between adaptive immunity and innate immunity, accounting for 1% of peripheral blood T lymphocytes -5%, mainly distributed in mucosa and epithelial tissues.
  • ⁇ T cells recognize antigens without MHC restriction, not only can kill tumor cells in various ways, but also present antigens as antigen-presenting cells (APC).
  • APC antigen-presenting cells
  • Transforming ⁇ T cells into CAR- ⁇ T cells can accurately recognize specific antigens and kill tumor cells efficiently. Using the characteristics of ⁇ T cells may challenge the treatment of solid tumors.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are separated by a short Flexible polypeptide linkers are contiguously linked and are capable of being expressed as single chain polypeptides, and wherein the scFv retains the specificity of the intact antibody from which it was derived.
  • antigen refers to a molecule that elicits an immune response.
  • the immune response may involve antibody production or activation of specific immunocompetent cells or both.
  • any macromolecule including virtually all proteins or peptides, can serve as an antigen.
  • antigens can be derived from recombinant or genomic DNA.
  • any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response thus encodes an "antigen".
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form.
  • ELISA Enzyme-Linked Immunosorbnent Assay. It combines the known antigen or antibody on the surface of the solid phase carrier, and then uses the enzyme-labeled (coupled) antibody or antigen to incubate with it, and then develops the color through the chromogenic substance, and the color depth is related to the content of the substance to be tested In direct proportion, it can be observed with the naked eye.
  • ELISA experiments there are three necessary reagents: known antigens or antibodies (for binding to solid-phase supports); enzyme-labeled antibodies or antigens (markers); chromogenic reagents (for color development). There are four common ELISA experiments: direct ELISA, indirect ELISA, sandwich ELISA and competition ELISA.
  • SPR surface plasmon resonance
  • BLI Bio-Layer Interferometry
  • FACS Fluorescence activated Cell Sorting.
  • the working principle of the flow cytometer is to put the cells to be tested into the sample tube after being stained with a specific fluorescent dye. down into the flow chamber filled with sheath fluid. Under the confinement of the sheath fluid, the cells are arranged in a single row and ejected from the nozzle of the flow chamber to form a cell column, which intersects the incident laser beam perpendicularly, and the cells in the liquid column are excited by the laser to generate fluorescence.
  • a series of optical systems (lenses, apertures, filters, detectors, etc.) in the instrument collect signals such as fluorescence, light scattering, light absorption, or cell electrical impedance, and the computer system collects, stores, displays, and analyzes the various signals that are measured , to make statistical analysis on various indicators.
  • HIS-Tag consists of 6-10 histidine residues, with a molecular weight of less than 0.84KD, and is usually inserted at the C-terminal or N-terminal of the target protein.
  • HIS-Tag is currently the most commonly used tag for prokaryotic expression. After protein purification, this tag does not need to be removed, and it will not affect the function of the protein. At the same time, the protein purification steps are simple, the purification conditions are mild, and the protein will not be greatly affected.
  • GST-Tag is a glutathione thiol transferase tag with a relatively large molecular mass of about 26KD, which is inserted at the C-terminus or N-terminus of the target protein, and is commonly used at the N-terminus in Escherichia coli.
  • the GST (glutathione thiol transferase) protein itself is a transferase that plays an important role in the detoxification process.
  • there are two purposes for choosing GST tags one is to improve the solubility of protein expression, and the other is to increase the expression level of protein. After protein expression and purification, it is necessary to determine whether to remove the tag according to different protein applications. If the tag is large, whether to remove it should be considered according to the downstream application. If the GST fusion portion is to be removed, it can be excised with a site-specific protease.
  • MBP-Tag is a maltose-binding protein tag with 346 amino acid residues and a molecular weight of 42.5KDa, encoded by the malE gene of Escherichia coli K12, which can be placed at the N-terminus during construction to improve solubility (especially for eukaryotic proteins) .
  • the folding of MBP requires the help of two molecular chaperone systems, DnaK-DnaJ-GrpE and GroEL-GeoES, which can make these molecular chaperones gather near the target protein to help it fold correctly.
  • maltose-binding protein in the form of tagged protein can reduce the degradation of the target protein, improve the water solubility of the expressed product, and also provide a basis for the purification of the target protein in the future.
  • Maltose-binding protein can be adsorbed by polysaccharide resin, so the fusion protein can be separated from other protein components when passing through the column.
  • NusA-Tag is a transcription termination/anti-termination protein tag.
  • NusA is a protein of Escherichia coli itself, that is, a transcription anti-termination factor, with 495 amino acid residues and a molecular weight of 54.87KDa. Screened from the Escherichia coli protein library. NusA does not have an independent purification tag function, so it should be used in conjunction with other tags (such as His tags).
  • FLAG-Tag is a fusion polypeptide of 8 amino acids (DYKDDDDK, SEQ ID No.12), and the Kozak sequence constructed in the vector enables the fusion protein with FLAG to express more efficiently in the eukaryotic expression system.
  • SUMO refers to a SUMO-tagged protein, which is a small molecule ubiquitin-related modification protein and a class of large proteins that are highly conserved in eukaryotes and participate in protein small ubiquitination-related modification.
  • SUMO can not only be used as a fusion tag for recombinant protein expression, but also has the function of a molecular chaperone, which can promote the correct folding of the protein, is resistant to heat and protease, and is more helpful in maintaining the stability of the target protein. stability.
  • Avi-Tag is a short peptide of 15 amino acids with a single biotinylated lysine site, which is completely different from the known natural biotinylated sequence, and can be added to the N-terminal and C-terminal of the target protein . After fusion expression, it can be biotinylated by biotin ligase. In order to purify the recombinant protein, low-affinity monomeric avidin or avidin derivatives are selected.
  • Halo-Tag refers to a genetically modified derivative of a dehalogenase that can be efficiently covalently bound to a variety of synthetic HaloTag ligands. This monomeric protein with a molecular weight of 33KDa can be fused to the N-terminal or C-terminal of the recombinant protein and expressed in prokaryotic and eukaryotic systems.
  • HaloTag ligands are small molecule chemicals that can covalently bind to HaloTag proteins in vitro or in vivo.
  • SNAP-Tag is derived from human O6-methylguanine-DNA methyltransferase (O6-alkylguanine-DNA-alkyltransferase).
  • O6-alkylguanine-DNA-alkyltransferase O6-alkylguanine-DNA-alkyltransferase
  • SNAP-Tag can be covalently combined with substrates with high specificity, so that proteins can be labeled with biotin or fluorescent groups (such as fluorescein and rhodamine).
  • biotin or fluorescent groups such as fluorescein and rhodamine.
  • the active sulfhydryl site of SNAP accepts the side chain benzyl group carried by benzylguanine and releases guanine.
  • This new covalent combination of thioether bonds makes the target protein carried by SNAP carry the label carried by the benzyl group.
  • Benzylguanine is stable under biochemical conditions
  • c-Myc tagged protein is a small tag containing 10 amino acids (EQKLISEEDL, SEQ ID No.13). These 10 amino acids are expressed as antigenic epitopes and can still recognize their corresponding antibodies in different protein frameworks. C-Myc tag has been successfully applied in Western-blot hybridization, immunoprecipitation and flow cytometry, and can be used to detect the expression of recombinant proteins in target cells. Common vectors include pCMV-MYC, pcDNA3.1(+)_myc-hisA, pCMV-RFP-C-Myc, pCMV-Myc, etc.
  • HA tag protein the tag sequence YPYDVPDYA (SEQ ID No.14), derived from the hemagglutinin surface epitope of influenza virus, 9 amino acids, has little effect on the spatial structure of the foreign target protein, and is easy to construct into a tag protein Fused to the N-terminus or C-terminus. Anti-HA antibody detection and ELISA detection are commonly used.
  • luciferase is derived from luciferin in living organisms, common ones are firefly luciferase, Renilla luciferase and Guassia luciferase.
  • the eVLP backbone used in this example is HIV-1 SF2 p 55 Gag (GenBank accession no. K02007) protein (SEQ ID No. 1), and a single Gag protein has the ability to self-assemble into enveloped VLP particles.
  • the recombinant plasmid R1221 containing the Gag-encoding gene and the recombinant plasmid R1242 containing the Gag-GFP encoding gene were respectively constructed: the polynucleotide SEQ ID No. Restriction endonuclease BamHI cutting site and Kozak sequence were added at 7 and 8, 5' end; stop codon TAA and restriction endonuclease XhoI cutting site were added at 3' end.
  • the synthetic product was double-digested with restriction endonucleases BamHI and XhoI, then subjected to agarose gel electrophoresis, and the target fragment was recovered by cutting the gel.
  • the target fragments were respectively ligated with pcNDA3.1(+) which had also been double-digested with restriction enzymes BamHI and XhoI to construct recombinant eukaryotic expression vectors, ie recombinant plasmids R1221 and R1242. Then, the recombinant plasmids R1221 and R1242 were respectively transformed into Escherichia coli E.coli DH5 ⁇ , cultured at 37°C for 16 hours, and then a single colony was picked for plasmid extraction. Glycogel electrophoresis identification, the positive clones were sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the clones with correct sequencing were selected for amplification and extraction of plasmids. After the extracted plasmids were sterile filtered, they were stored at -20°C for later use.
  • the recombinant plasmid R1353 containing the gene encoding Claudin 18.2 and the recombinant plasmid R1303 containing the gene encoding Claudin 18.2-GFP were respectively constructed: the polynucleotide SEQ ID encoding the amino acid sequence shown in SEQ ID No.4 and 5 was synthesized by Shanghai Bioengineering Co., Ltd. No. 9 and 10, 5' end added restriction endonuclease BamHI site, Kozak sequence; 3' end added stop codon TAA and restriction endonuclease XhoI site.
  • the synthetic product was double-digested with restriction endonucleases BamHI and XhoI, then subjected to agarose gel electrophoresis, and the target fragment was recovered by cutting the gel.
  • the target fragments were respectively ligated with pcNDA3.1(+) which had also been double-digested with restriction endonucleases BamHI and XhoI to construct recombinant eukaryotic expression vectors, ie recombinant plasmids R1353 and R1303.
  • the recombinant plasmids R1353 and R1303 were respectively transformed into Escherichia coli DH5 ⁇ , cultured at 37°C for 16 hours, and single colonies were picked for plasmid extraction, and the extracted plasmids were double-enzymatically digested with restriction endonucleases BamHI and XhoI, and carried out on agar Glycogel electrophoresis identification, the positive clones were sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the clones with correct sequencing were selected for amplification and extraction of plasmids. After the extracted plasmids were sterile filtered, they were stored at -20°C for later use.
  • the constructed recombinant plasmid R1242 was transfected into HEK293 cells (corresponding to cell ID R1242) using PEI transfection reagent, and the blank control eVLP was expressed and assembled.
  • HEK293 cells were subcultured at 1 ⁇ 10 ⁇ 6 cells/mL and cultured at 37°C. On the day of transfection, counting was performed, and the cell density was adjusted to 2 ⁇ 10 ⁇ 6 cells/mL, and the viability was over 95%.
  • the corresponding relationship between the dose of plasmid and the number of cells is 1 ⁇ 10 ⁇ 6 cells corresponding to 0.6 ⁇ g of plasmid
  • the corresponding relationship between the dose of PEI and the dose of plasmid is: the quality of PEI is 3 times the quality of DNA.
  • the day before transfection HEK293 cells were subcultured at 1 ⁇ 10 ⁇ 6 cells/mL and cultured at 37°C. Count the cells on the day of transfection, adjust the cell density to 2 ⁇ 10 ⁇ 6 cells/mL, and the viability rate is above 95%.
  • the corresponding relationship between the dose of plasmid and the number of cells is 1 ⁇ 10 ⁇ 6 cells corresponding to 0.6 ⁇ g of plasmid
  • the corresponding relationship between the dose of PEI and the dose of plasmid is: the quality of PEI is 3 times the quality of DNA.
  • the supernatants of the harvested R1242, RG288 and RG341 cells were centrifuged at 2000rpm at 4°C for 20min to remove cell debris and filtered with a 0.22 ⁇ m filter membrane, then subjected to sucrose density gradient centrifugation, and the supernatant was subjected to 30% sucrose density ultracentrifugation (2,6000rpm /min, 1.5h at 4°C), and the precipitate was resuspended in PBS to obtain crude pure samples of R1242, RG288 and RG341eVLP respectively.
  • lane A is the crude pure sample of R1242
  • lane B is the crude pure sample of RG288,
  • lane C is the crude pure sample of RG341.
  • the target bands of Gag (about 55kDa) and Gag-GFP (about 80kDa) can be clearly seen from the electrophoresis results, which proves that a large number of VLPs can be harvested after the supernatant is ultracentrifuged at a density of 30% sucrose cushion.
  • the crude pure samples of R1242, RG288 and RG341 were respectively treated with Benzonase (ACROBiosystems) at an enzyme concentration of 200 U/mL for 1 hour at room temperature.
  • the samples after nucleic acid removal were used separately
  • the ExplorerTM 100 low-pressure liquid chromatography system (GE Healthcare) was used for SEC experiments, and Sepharose 6FF resin was loaded into an XK 16/70 chromatographic column (GE Healthcare) with a final bed volume of 130 mL.
  • the void volume of the column was determined using Blue Dextran 2000 (HMW calibration kit, GE Healthcare).
  • the column was washed with 3 column volumes of degassed Milli-Q ultrapure water, and the column was equilibrated with 1 column volume of PBS, pH 7.4 at a rate of 2 mL/min (60 cm/h).
  • Example 4 In order to further confirm that the method in Example 4 can successfully prepare eVLP, R1242-210521F1 was subjected to a transmission electron microscope morphology test (entrusted to Beijing Zhongke Baice Technology Service Co., Ltd.), and the results are shown in Figure 4. It can be seen that R1242-210521F1 appears as a hollow sphere with a diameter of about 150 nm under the electron microscope. The results of electron micrographs have proved macroscopically that the method of Example 4 can successfully prepare eVLP with HIV-1 Gag as the backbone protein.
  • ELISA enzyme-linked immunosorbent assay
  • Coating use 0.5 ⁇ g/well (5 ⁇ g/ml, 100 ⁇ l/well) of RG288-210503F1 or RG341-210521F1 to coat a 96-well plate (Corning Company, Cat. ).
  • the coating buffer used for dilution of RG288-210503F1 or RG341-210521F1 is 15mM Na 2 CO 3 , 35mM NaHCO 3 , 7.7mM NaN 3 , pH 9.6.
  • washing buffer TBS, 0.05% Tween-20, pH 7.4
  • Blocking each well was blocked with 300 ⁇ l blocking buffer (TBS, 2% BSA, pH 7.4) for 1.5 h at 37° C.
  • Add detection antibody add 100 ⁇ l anti-human IgG antibody (Jackson, catalog number: 109-035-098) to each well, and incubate at 37°C for 1 hour.
  • the antibody was diluted with dilution buffer (TBS buffer containing 0.5% BSA, pH 7.4) at a ratio of 1:20000 in advance.
  • OD450-ODBlank is the final OD value.
  • the well corresponding to ODBlank is the measurement result in step 5 where no sample is added but only an equal volume of dilution buffer is added, which is used as a blank control.
  • FIG. 5 The ELISA test results are shown in Figure 5, where Figure A is the ELISA test result for the combination of RG288-210503F1 and IMAB362; Figure B is the ELISA test result for the combination of RG341-210521F1 and IMAB362.
  • the EC50 values of the combination of Claudin 18.2-eVLP RG288-210503F1 and RG341-210521F1 of the present disclosure with IMAB362 are 1.64ng/mL and 1.94ng/mL respectively, indicating that the Claudin 18.2-eVLP of the present disclosure has good binding activity to IMAB362, thus proving that Claudin 18.2 tetraspanin was successfully displayed on eVLP in the correct conformation.
  • IMAB362 CAR-like (HEK293) cells were prepared to verify the application of Claudin 18.2-eVLP in evaluating the positive expression rate of CAR.
  • the IMAB362 CAR structure is shown in Figure 6.
  • the corresponding amino acid sequence is 6, and the polynucleotide sequence is SEQ ID No.11.
  • Example 3 for the process of transfecting HEK293 cells with the IMAB362 CAR recombinant plasmid.
  • the difference is that in this example, IMAB362 CAR-like (HEK293) monoclonal cells need to be screened out through G418 resistance, and the marker ID is C633, which is used in the CAR positive expression rate evaluation experiment.
  • Example 8 Claudin-18.2-eVLP is used to evaluate the positive expression rate of CAR
  • anti-Fab antibodies FITC-anti mFab Ab (Thermo Scientific, Cat. No. 31543)), FITC-Protein L (ACROBiosystems, Cat. No. RPL-PF141), and Claudin 18.2 extracellular loop region protein were used respectively: Claudin-18.2 Protein, His Tag TM (ACROBiosystems, Cat.No.CL2-H51H6) and R1242-210521F1, RG288-210503F1 and RG341-210521F1 of the present disclosure were used to evaluate the expression of IMAB362 CAR. Specific steps are as follows:
  • C633 cells were cultured in DMEM medium containing 10% fetal bovine serum, and placed in a CO 2 incubator (37° C., 5% CO 2 ).
  • FITC-anti mFab Ab FITC-Protein L, Claudin-18.2 Protein, His Tag TM , R1242-210521F1, RG288-210503F1 and RG341-210521F1 were diluted with FACS buffer (2% BSA) respectively, and then The diluted sample solution was added separately to the tubes containing the cells. Mix well and incubate at 4°C for 60 min. (For Claudin-18.2 Protein, His Tag TM , wash the cells once with FACS buffer (2% BSA) after incubation, add PE-anti His antibody (Biolegend, Cat. No. 362603), and incubate at 4°C for 60 min in the dark. )
  • Figure 7 shows the evaluation of IMAB362 CAR-like expression when FITC-anti mFab Ab is diluted 200 times (positive rate is 95.93%);
  • Figure B shows when FITC-Protein L is used at a concentration of 10 ⁇ g /mL, it evaluates the expression of IMAB362 CAR-like (positive rate is 64.70%);
  • Figure C is when Claudin-18.2 Protein, His Tag TM is used at a concentration of 10 ⁇ g/mL, it evaluates the expression of IMAB362 CAR-like (positive rate is 64.70%); The ratio was 1.17%), indicating that only the extracellular loop region of Claudin 18.2 was expressed, and the IMAB362 CAR-like structure could not be recognized;
  • Figure D shows the evaluation of IMAB362 CAR-like expression (positive The rate is 99.52%);
  • Figure E shows the evaluation of IMAB362 CAR-like expression when RG341-210521F1 is used at a concentration of 10
  • Example 9 CD20-eVLP is used to evaluate the positive expression rate of CAR
  • this example focuses on another multi-spanning protein CD20 , using the nanoparticles of the present disclosure, the positive expression rate of CAR was evaluated. Specific steps are as follows:
  • the preparation method refers to Example 7; wherein, the amino acid sequence of Ofatumumab CAR is SEQ ID No.17, and the polynucleotide sequence is SEQ ID No.18; the prepared Ofatumumab CAR -like (HEK293) monoclonal cells, labeled ID is RC539b, used for CAR positive expression rate evaluation experiment;
  • CD20-eVLP is used for evaluating and verifying the positive expression rate of CAR. For specific methods, refer to Example 8.
  • the conformation of the CD20 target antigen displayed on the CD20-eVLP of the present disclosure is correct, and can specifically bind to Ofatumumab CAR-like cell clones.
  • the positive expression rate of CAR is detected by FACS detection of GFP, it shows >99% positive rate effect.
  • the technical means of the present disclosure can be widely applied to the preparation of multiple transmembrane protein-eVLP, and then applied to the evaluation of CAR positive rate expression.

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Abstract

A nanoparticle and the use thereof in the detection of a CAR-positive expression rate. The nanoparticle comprises: (i) an enveloped virus-like particle (eVLP) skeleton protein and/or a functional fragment thereof; and (ii) a target protein and/or a functional fragment thereof. The nanoparticle can be used for detecting a CAR-positive expression rate, and can also be used for proving that a CAR can specifically bind to a target protein.

Description

一种纳米颗粒及其检测CAR阳性表达率的应用A kind of nanoparticle and its application in detecting the positive expression rate of CAR 技术领域technical field
本公开涉及生物技术领域，具体地涉及一种纳米颗粒及其检测CAR阳性表达率的应用。The disclosure relates to the field of biotechnology, in particular to a nanoparticle and its application for detecting the positive expression rate of CAR.
背景技术Background technique
在免疫***中，T细胞需要借助抗原递呈细胞(APC)来杀死异常细胞。APC表面有许多用于识别细胞表面抗原的MHC(主要组织相容性复合体)分子，在APC识别到异常细胞之后，MHC就会和T细胞受体(TCR)结合，把信号传递给T细胞。在CD3、CD4和CD8等分子的帮助下，T细胞才能最终识别异常细胞并将其杀死。但是肿瘤细胞却把自己的这些“指纹”给毁了，导致APC细胞就无法识别它。In the immune system, T cells rely on antigen-presenting cells (APCs) to kill abnormal cells. There are many MHC (major histocompatibility complex) molecules on the surface of the APC that are used to recognize cell surface antigens. After the APC recognizes abnormal cells, the MHC will bind to the T cell receptor (TCR) and transmit the signal to the T cell. . With the help of molecules such as CD3, CD4 and CD8, T cells can eventually recognize abnormal cells and kill them. But tumor cells destroy these "fingerprints" of themselves, so that APC cells cannot recognize it.
嵌合抗原受体(Chimeric Antigen Receptor，CAR)是一种基因工程技术制造的人工受体分子，它可以赋予免疫效应细胞(如T淋巴细胞)针对某个靶点抗原表位的特异性，从而增强T淋巴细胞识别抗原信号与活化的功能(Sadelain,M.,R.Brentjens,and I.Riviere.The promise and potential pitfalls of chimeric antigen receptors.Curr Opin Immunol,2009.21(2):p.215-23.)。CAR-T细胞能以MHC非限制性方式识别特定目的抗原，并且持续活化扩增的T细胞。CAR-T细胞疗法本质上是一种过继细胞转移(ACT)，ACT最早被提出是在器官移植过程中，供体的淋巴细胞被输入受体体内的过程，该过程抗原介导移植器官排斥反应的发生，后逐渐发展为治疗恶性肿瘤的疗法(Mitchison,N.A..Studies on the immunological response to foreign tumor transplants in the mouse.I.The role of lymph node cells in conferring immunity by adoptive transfer.The Journal of experimental medicine,1955.102(2):p.157-177.)。Chimeric Antigen Receptor (CAR) is an artificial receptor molecule manufactured by genetic engineering technology, which can endow immune effector cells (such as T lymphocytes) with specificity for a target epitope, thereby Enhance the function of T lymphocyte recognition antigen signal and activation (Sadelain, M., R. Brentjens, and I. Riviere. The promise and potential pitfalls of chimeric antigen receptors. Curr Opin Immunol, 2009.21 (2): p.215-23 .). CAR-T cells can recognize specific target antigens in an MHC-unrestricted manner, and continuously activate expanded T cells. CAR-T cell therapy is essentially a kind of adoptive cell transfer (ACT). ACT was first proposed as a process in which donor lymphocytes are infused into the recipient during organ transplantation. This process antigen-mediated rejection of transplanted organs Then it gradually developed into a therapy for malignant tumors (Mitchison,N.A..Studies on the immunological response to foreign tumor transplants in the mouse.I.The role of lymph node cells in conferring immunity by adoptive transfer.The Journal of experimental medicine , 1955.102(2): p.157-177.).
对于CAR-T细胞来说，发挥肿瘤杀伤作用的有效成分是CAR阳性表达率的T细胞。CAR-T细胞产品的包装规格及临床使用剂量是以CAR-T阳性细胞数表示的，CAR阳性表达率的检测是CAR-T细胞生产过程中的重要质控步骤。For CAR-T cells, the active ingredient to play a tumor-killing role is the T cells with positive CAR-expression rate. The packaging specifications and clinical dosage of CAR-T cell products are expressed by the number of CAR-T positive cells. The detection of CAR positive expression rate is an important quality control step in the production process of CAR-T cells.
现有技术中主要针对轻链或铰链区的Anti-Fab抗体或Protein L蛋白来检测CAR阳性表达率，但上述方法均存在非特异结合的缺陷，而无法证明CAR能否结合靶抗原。因此，靶点蛋白用于检测CAR阳性表达率具有特异性强的优势而备受业界人士的青睐。然而，大多数靶点蛋白的制备比较困难，所得的靶点蛋白也有可能给CAR阳性表达率的检测带来不良影响。例如，由于靶点蛋白跨膜区的疏水特性，在纯化过程中需要引入去垢剂来溶解靶点蛋白，从而造成制备的靶点蛋白中会含有去垢剂，而去垢剂的存在会导致T细胞发生膜溶解，这使得在去垢剂体系中的靶点蛋白无法用于CAR阳性率的检测。此外，使用靶点蛋白可溶性的胞外loop区检测CAR阳性表达率，虽然靶点蛋白制备难度低，但因为没有跨膜区的结果限制，无法保证其构象与天然构象一致，而存在无法正确识别CAR的缺陷。In the prior art, the positive expression rate of CAR is mainly detected by the Anti-Fab antibody or Protein L protein of the light chain or hinge region, but the above methods all have the defect of non-specific binding, and cannot prove whether the CAR can bind the target antigen. Therefore, the target protein used to detect the positive expression rate of CAR has the advantage of strong specificity and is favored by the industry. However, the preparation of most target proteins is relatively difficult, and the obtained target proteins may also have adverse effects on the detection of CAR positive expression rate. For example, due to the hydrophobicity of the transmembrane region of the target protein, it is necessary to introduce detergent to dissolve the target protein during the purification process, so that the prepared target protein will contain detergent, and the presence of detergent will lead to T cells undergo membrane lysis, which makes the target protein in the detergent system unable to be used for the detection of CAR positive rate. In addition, using the soluble extracellular loop region of the target protein to detect the positive expression rate of CAR, although the preparation of the target protein is difficult, but because there is no limit to the result of the transmembrane region, it cannot guarantee that its conformation is consistent with the natural conformation, and there are problems that cannot be correctly identified Flaws of CAR.
为了解决这些问题，需要开发一种CAR靶点蛋白，其既保持了完整的天然构象，且能特异性的结合CAR。In order to solve these problems, it is necessary to develop a CAR target protein that maintains a complete natural conformation and can specifically bind CAR.
病毒样颗粒(virus-like particles,VLP)，也称为核心样颗粒(Core-like Particle，CLP)，是由病毒衣壳蛋白(capsid protein,CP)自组装形成的球形或管状蛋白纳米结构，具有与天然病毒衣壳类似的结构，不含基因组、无感染性。VLP主要分为eVLP和非包膜VLP。在VLP来自包膜病毒的情况时，这个过程是通过从宿主细胞出芽，这造成VLP被脂质双分子层包裹(中国专利申请CN101146522A)。美国专利申请US20110189159A1基于发现感兴趣的蛋白质可以作为融合的或未融合的蛋白质被递送到细胞；特别是，用两种Gag融合(感兴趣的Gag-蛋白和Gag-蛋白酶)制成VLP，可以将感兴趣的蛋白作为未融合的蛋白传递给细胞用于治疗。PCT专利申请WO2021022008A1公开的疫苗包含VLP，以及药学上可接受的赋形剂、载体和/或佐剂；其中，VLP包含(a)合成或天然脂质双层，(b)嵌入脂质双层中的锚分子，(c)与锚分子结合的抗原。近年来eVLP在疫苗开发、药物靶向递送、生物医学成像与传感、组织工程等众多领域展现出独特的研究与应用价值。然而，eVLP在CAR阳性表达率的研究却罕有报道。Virus-like particles (VLP), also known as core-like particles (CLP), are spherical or tubular protein nanostructures formed by the self-assembly of viral capsid protein (CP), It has a structure similar to the capsid of natural viruses, does not contain a genome, and is non-infectious. VLPs are mainly divided into eVLPs and non-enveloped VLPs. In the case of the VLP from an enveloped virus, this process is by budding from the host cell, which results in the VLP being encapsulated by a lipid bilayer (Chinese patent application CN101146522A). U.S. Patent Application US20110189159A1 is based on the discovery that proteins of interest can be delivered to cells as fused or unfused proteins; in particular, by making VLPs with two Gag fusions (Gag-protein of interest and Gag-protease), the The protein of interest is delivered to cells as an unfused protein for therapy. The vaccine disclosed in PCT patent application WO2021022008A1 comprises VLP, and pharmaceutically acceptable excipients, carriers and/or adjuvants; wherein, the VLP comprises (a) a synthetic or natural lipid bilayer, (b) embedded in a lipid bilayer The anchor molecule in (c) the antigen bound to the anchor molecule. In recent years, eVLP has shown unique research and application value in many fields such as vaccine development, targeted drug delivery, biomedical imaging and sensing, and tissue engineering. However, studies on the positive expression rate of eVLP in CAR are rarely reported.
发明内容Contents of the invention
为了解决现有技术中存在的问题，本公开提供了一种纳米颗粒及其检测CAR阳性表达率的应用。In order to solve the problems existing in the prior art, the present disclosure provides a nanoparticle and its application for detecting the positive expression rate of CAR.
具体地，本公开通过以下技术方案来解决本公开所要解决的技术问题。Specifically, the present disclosure solves the technical problems to be solved by the present disclosure through the following technical solutions.
在本公开的一个方面，涉及一种纳米颗粒，其包含：In one aspect of the present disclosure, it relates to a nanoparticle comprising:
(i)包膜病毒样颗粒(eVLP)骨架蛋白和/或其功能片段；和(i) enveloped virus-like particle (eVLP) backbone proteins and/or functional fragments thereof; and
(ii)靶点蛋白和/或其功能片段，(ii) target protein and/or functional fragment thereof,
其中，所述包膜病毒样颗粒骨架蛋白包含病毒核心蛋白或其功能片段，所述包膜病毒样颗粒骨架蛋白或其功能片段组装形成包膜病毒样颗粒，所述靶点蛋白和/或其功能片段展示在包膜病毒样颗粒上，形成靶点蛋白-eVLP纳米颗粒。Wherein, the enveloped virus-like particle skeleton protein comprises a viral core protein or a functional fragment thereof, and the enveloped virus-like particle skeleton protein or a functional fragment thereof assembles to form an enveloped virus-like particle, and the target protein and/or its Functional fragments are displayed on enveloped virus-like particles to form target protein-eVLP nanoparticles.
在一个或多个实施方案中，所述靶点蛋白为CAR靶点蛋白；所述CAR靶点蛋白和/或其功能片段展示在包膜病毒样颗粒上，形成CAR靶点蛋白-eVLP纳米颗粒。In one or more embodiments, the target protein is a CAR target protein; the CAR target protein and/or its functional fragments are displayed on enveloped virus-like particles to form CAR target protein-eVLP nanoparticles .
在一个或多个实施方案中，所述病毒选自逆转录病毒、杆状病毒、线状病毒、冠状病毒、流感病毒、副粘病 毒、呼吸道合胞病毒、沙粒病毒、新城疫病毒、副流感病毒、布尼亚病毒、丙型肝炎病毒、乙型肝炎病毒。In one or more embodiments, the virus is selected from the group consisting of retrovirus, baculovirus, filovirus, coronavirus, influenza virus, paramyxovirus, respiratory syncytial virus, arenavirus, Newcastle disease virus, para Influenza virus, bunya virus, hepatitis C virus, hepatitis B virus.
在一个或多个实施方案中，所述逆转录病毒选自人类免疫缺陷病毒、猴免疫缺陷病毒、小鼠白血病病毒和牛白血病病毒。In one or more embodiments, the retrovirus is selected from the group consisting of human immunodeficiency virus, simian immunodeficiency virus, murine leukemia virus, and bovine leukemia virus.
在一个或多个实施方案中，所述杆状病毒为水疱性口炎病毒。In one or more embodiments, the baculovirus is vesicular stomatitis virus.
在一个或多个实施方案中，所述线状病毒为埃博拉病毒。In one or more embodiments, the filovirus is Ebola virus.
在一个或多个实施方案中，所述冠状病毒选自新型冠状肺炎病毒(SARS-CoV-2)、SARS-CoV、MERS-Cov、229E、NL63、OC43和HKU1。In one or more embodiments, the coronavirus is selected from the group consisting of novel coronavirus pneumonia virus (SARS-CoV-2), SARS-CoV, MERS-Cov, 229E, NL63, OC43 and HKU1.
在一个或多个实施方案中，所述的病毒核心蛋白选自：逆转录病毒Gag蛋白、杆状病毒基质蛋白M蛋白、线状病毒核心蛋白、冠状病毒M、E和NP蛋白、流感病毒M1蛋白、副粘病毒M蛋白、呼吸道合胞病毒(RSV)M蛋白、沙粒病毒Z蛋白、新城疫病毒M蛋白、副流感病毒M蛋白、布尼亚病毒N蛋白、丙型肝炎病毒核心蛋白C、乙型肝炎病毒核心蛋白C和其组合物。In one or more embodiments, the virus core protein is selected from: retrovirus Gag protein, baculovirus matrix protein M protein, filovirus core protein, coronavirus M, E and NP proteins, influenza virus M1 protein, paramyxovirus M protein, respiratory syncytial virus (RSV) M protein, arenavirus Z protein, Newcastle disease virus M protein, parainfluenza virus M protein, bunya virus N protein, hepatitis C virus core protein C , hepatitis B virus core protein C and its composition.
在一个或多个实施方案中，所述逆转录病毒Gag蛋白选自：人类免疫缺陷病毒Gag蛋白、猴免疫缺陷病毒Gag蛋白、小鼠白血病病毒Gag蛋白和牛白血病病毒Gag蛋白。在一个或多个优选的实施方案中，所述逆转录病毒Gag蛋白为所述人类免疫缺陷病毒Gag蛋白。在一个或多个优选的实施方案中，所述人类免疫缺陷病毒Gag蛋白包含与SEQ ID No.1具有至少80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列。在一个或多个优选的实施方案中，所述人类免疫缺陷病毒Gag蛋白的氨基酸序如SEQ ID No.1所示。In one or more embodiments, the retroviral Gag protein is selected from the group consisting of human immunodeficiency virus Gag protein, simian immunodeficiency virus Gag protein, mouse leukemia virus Gag protein and bovine leukemia virus Gag protein. In one or more preferred embodiments, said retroviral Gag protein is said human immunodeficiency virus Gag protein. In one or more preferred embodiments, the human immunodeficiency virus Gag protein comprises an amino acid sequence with at least 80% or more identity to SEQ ID No.1, preferably 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences. In one or more preferred embodiments, the amino acid sequence of the human immunodeficiency virus Gag protein is shown in SEQ ID No.1.
在一个或多个实施方案中，所述杆状病毒基质蛋白M蛋白为水疱性口炎病毒M病毒核心蛋白。In one or more embodiments, the baculovirus matrix protein M protein is vesicular stomatitis virus M virus core protein.
在一个或多个实施方案中，所述线状病毒核心蛋白为埃博拉病毒VP40病毒核心蛋白。In one or more embodiments, the filovirus core protein is Ebola virus VP40 virus core protein.
在一个或多个实施方案中，所述冠状病毒的核心蛋白选自新型冠状肺炎病毒(SARS-CoV-2)、SARS-CoV、MERS-Cov、229E、NL63、OC43和HKU1的核心蛋白。In one or more embodiments, the core protein of the coronavirus is selected from core proteins of novel coronavirus pneumonia virus (SARS-CoV-2), SARS-CoV, MERS-Cov, 229E, NL63, OC43 and HKU1.
在一个或多个实施方案中，所述病毒为从宿主细胞的细胞膜上芽生时获得包膜的病毒。In one or more embodiments, the virus is a virus that acquires an envelope when budding from the cell membrane of the host cell.
在一个或多个实施方案中，所述CAR靶点蛋白包括膜蛋白。In one or more embodiments, the CAR target protein comprises a membrane protein.
在一个或多个实施方案中，所述膜蛋白包括跨膜蛋白。In one or more embodiments, the membrane protein comprises a transmembrane protein.
在一个或多个实施方案中，所述跨膜蛋白包括多次跨膜蛋白。In one or more embodiments, the transmembrane protein comprises a multispanin.
在一个或多个实施方案中，所述CAR靶点蛋白选自CD20、Claudin18.1、Claudin18.2、CD133、GPRC5D、CCR5、CCR8、BCMA、GPCR、CD147、CD19、CD123、CD138、CD22、CD30、CD33、CD38、CD70、CAIX、EGFR、EGFRVIII、FOLR1、GPC3、HER2、HGFR、Anti-FMC63Ab、CLL-1、SLAMF7、CD4、CD5、CD8A&CD8B、FAP、IL13RA2、GPC3、GUCY2C、Her3、PSMA、ROR1、SLAMF7、B7-H3、CD147、CEA、MUC16、Nectin-4、VEGFR2、Anti-RTX Ab、B7-H3、CAIX、CD7、CEA、MUC1、NKG2D、PSCA、uPAR、GD2、FR、PMEL、CA9、CD171/L1-CAM、IL-13Rα2、MART-1、ERBB2、ERBB3、ERBB4、NY-ESO-1、MAGE家族蛋白、BAGE家族蛋白、GAGE家族蛋白、AFP、CD44v7/8、IL-11Rα、EGP-2、EGP-40、FBP、GD3、FSA、PSA、HMGA2、胎儿型乙酰胆碱受体、LeY、EpCAM、间皮素、IGFR1、CA125、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、SCC、AFU、EBV-VCA、POA、β2-MG、PROGRP或MSLN中任一种。In one or more embodiments, the CAR target protein is selected from CD20, Claudin18.1, Claudin18.2, CD133, GPRC5D, CCR5, CCR8, BCMA, GPCR, CD147, CD19, CD123, CD138, CD22, CD30 , CD33, CD38, CD70, CAIX, EGFR, EGFRVIII, FOLR1, GPC3, HER2, HGFR, Anti-FMC63Ab, CLL-1, SLAMF7, CD4, CD5, CD8A&CD8B, FAP, IL13RA2, GPC3, GUCY2C, Her3, PSMA, ROR1 , SLAMF7, B7-H3, CD147, CEA, MUC16, Nectin-4, VEGFR2, Anti-RTX Ab, B7-H3, CAIX, CD7, CEA, MUC1, NKG2D, PSCA, uPAR, GD2, FR, PMEL, CA9, CD171/L1-CAM, IL-13Rα2, MART-1, ERBB2, ERBB3, ERBB4, NY-ESO-1, MAGE family proteins, BAGE family proteins, GAGE family proteins, AFP, CD44v7/8, IL-11Rα, EGP- 2. EGP-40, FBP, GD3, FSA, PSA, HMGA2, fetal acetylcholine receptor, LeY, EpCAM, mesothelin, IGFR1, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, Any of CYFRA21-1, SCC, AFU, EBV-VCA, POA, β2-MG, PROGRP, or MSLN.
在一个或多个实施方案中，所述CAR靶点蛋白包括多次跨膜蛋白。In one or more embodiments, the CAR target protein comprises a multispanning protein.
在一个或多个实施方案中，所述CAR靶点蛋白选自CD20、Claudin18.2、CD133、GPRC5D、CCR5、CCR8、CD19、BCMA、GPC3、CD30、CD22、EGFR、EGFRVIII、HER2或GPCR中任一种。In one or more embodiments, the CAR target protein is selected from any of CD20, Claudin18.2, CD133, GPRC5D, CCR5, CCR8, CD19, BCMA, GPC3, CD30, CD22, EGFR, EGFRVIII, HER2 or GPCR A sort of.
在一个或多个实施方案中，所述CAR靶点蛋白为Claudin18.2。在一个或多个优选的实施方案中，所述Claudin18.2的氨基酸序列包含与SEQ ID No.4具有至少80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列。在一个或多个优选的实施方案中，所述Claudin18.2的氨基酸序列如SEQ ID No.4所示。In one or more embodiments, the CAR target protein is Claudin18.2. In one or more preferred embodiments, the amino acid sequence of Claudin18.2 comprises an amino acid sequence with at least 80% or more identity to SEQ ID No.4, preferably 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences. In one or more preferred embodiments, the amino acid sequence of the Claudin18.2 is shown in SEQ ID No.4.
在一个或多个实施方案中，所述CAR靶点蛋白为CD20。在一个或多个优选的实施方案中，所述CD20的氨基酸序列包含与SEQ ID No.15具有至少80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列：更优选地，所述CD20的氨基酸序列如SEQ ID No.15所示。In one or more embodiments, the CAR target protein is CD20. In one or more preferred embodiments, the amino acid sequence of CD20 comprises an amino acid sequence with at least 80% or more identity to SEQ ID No.15, preferably 85%, 90%, 95%, 96%, An amino acid sequence with an identity of 97%, 98%, or 99%, more preferably an amino acid sequence with an identity of 98% or more: more preferably, the amino acid sequence of the CD20 is shown in SEQ ID No.15.
在一个或多个实施方案中，所述CAR靶点蛋白和/或其功能片段和/或所述病毒核心蛋白和/或其功能片段与标记物相连接。In one or more embodiments, the CAR target protein and/or its functional fragment and/or the viral core protein and/or its functional fragment are linked to a marker.
在一个或多个实施方案中，所述CAR靶点蛋白和/或其功能片段的N末端和/或C末端和/或病毒核心蛋白和/或其功能片段的N末端和/或C末端与标记物相连接。在一个或多个优选的实施方案中，在Claudin 18.2的C末端或Gag的C末端用标记物进行标记。在另一个或多个优选的实施方案中，在CD20的C末端或Gag的C末端用标记物进行标记。In one or more embodiments, the N-terminal and/or C-terminal of the CAR target protein and/or its functional fragment and/or the N-terminal and/or C-terminal of the viral core protein and/or its functional fragment are connected to markers are linked. In one or more preferred embodiments, the C-terminus of Claudin 18.2 or the C-terminus of Gag is labeled with a label. In another or more preferred embodiments, the C-terminus of CD20 or the C-terminus of Gag is labeled with a label.
在一个或多个实施方案中，所述标记物选自可检测标记物、纯化标签、报告标签和其组合物。In one or more embodiments, the label is selected from a detectable label, a purification tag, a reporter tag, and combinations thereof.
在一个或多个实施方案中，所述可检测标记物选自荧光基团、化学发光标记物、电化学发光标记物和其组合物。In one or more embodiments, the detectable label is selected from a fluorescent group, a chemiluminescent label, an electrochemiluminescent label, and combinations thereof.
在一个或多个实施方案中，所述荧光基团选自FITC、GFP、RFP、YFP、TRITC、PE、FAM、RRX、TR、Cy2、Cy3、Cy5、ECD、PC5.5、PC7、APC、APC-A70、APC-A75、Pac-Blue、Alexa488、mBBr、5-IAF、E-118、DTAF、 罗丹明绿和KrO中的任一种。In one or more embodiments, the fluorophore is selected from FITC, GFP, RFP, YFP, TRITC, PE, FAM, RRX, TR, Cy2, Cy3, Cy5, ECD, PC5.5, PC7, APC, Any of APC-A70, APC-A75, Pac-Blue, Alexa488, mBBr, 5-IAF, E-118, DTAF, rhodamine green, and KrO.
在一个或多个实施方案中，所述化学发光标记物为吖啶酯、异鲁米诺、辣根过氧化物酶或碱性磷酸酶。In one or more embodiments, the chemiluminescent label is acridinium ester, isoluminol, horseradish peroxidase, or alkaline phosphatase.
在一个或多个实施方案中，所述电化学发光标记物为三联吡啶钌或其衍生N-羟基琥珀酰胺酯。In one or more embodiments, the electrochemiluminescent label is ruthenium terpyridine or a derivative N-hydroxysuccinamide ester thereof.
在一个或多个实施方案中，所述纯化标签选自HIS-Tag、GST-Tag、MBP-Tag、NusA-Tag、FLAG-Tag、SUMO、Avi-Tag、Halo-Tag和SNAP-Tag中的任一种。In one or more embodiments, the purification tag is selected from HIS-Tag, GST-Tag, MBP-Tag, NusA-Tag, FLAG-Tag, SUMO, Avi-Tag, Halo-Tag and SNAP-Tag any kind.
在一个或多个实施方案中，所述纯化标签通过二抗检测。In one or more embodiments, the purification tag is detected by a secondary antibody.
在一个或多个实施方案中，所述报告标签选自c-Myc、HA或荧光素酶中的任一种。In one or more embodiments, the reporter tag is selected from any one of c-Myc, HA, or luciferase.
在一个或多个实施方案中，将CAR靶点蛋白展示在eVLP上，同时在CAR靶点蛋白的C末端或Gag的C末端使用标记物进行标记。In one or more embodiments, the CAR target protein is displayed on the eVLP, and at the same time, the C-terminal of the CAR target protein or the C-terminal of Gag is labeled with a marker.
在本公开的另一方面，涉及一种带标记物的Claudin 18.2-eVLP，所述Claudin 18.2-eVLP和标记物相连接。In another aspect of the present disclosure, it relates to a labeled Claudin 18.2-eVLP, the Claudin 18.2-eVLP being linked to a label.
在一个或多个实施方案中，所述CAR靶点蛋白-eVLP纳米颗粒为Claudin 18.2-eVLP纳米颗粒，所述Claudin18.2和/或其功能片段和/或所述病毒核心蛋白和/或其功能片段与标记物相连接。In one or more embodiments, the CAR target protein-eVLP nanoparticles are Claudin 18.2-eVLP nanoparticles, the Claudin18.2 and/or its functional fragments and/or the viral core protein and/or its Functional fragments are linked to markers.
在一个或多个实施方案中，所述Claudin 18.2和/或其功能片段的N末端和/或C末端和/或病毒核心蛋白和/或其功能片段的N末端和/或C末端与标记物相连接。In one or more embodiments, the N-terminal and/or C-terminal of the Claudin 18.2 and/or its functional fragment and/or the N-terminal and/or C-terminal of the viral core protein and/or its functional fragment are combined with a marker connected.
在一个或多个实施方案中，所述包膜病毒样颗粒骨架蛋白为Gag蛋白。In one or more embodiments, the enveloped virus-like particle backbone protein is Gag protein.
在一个或多个实施方案中，所述Claudin 18.2的C末端或所述Gag蛋白的C末端与标记物相连。In one or more embodiments, the C-terminus of the Claudin 18.2 or the C-terminus of the Gag protein is linked to a label.
在本公开的另一方面，涉及一种带标记物的CD20-eVLP，所述CD20-eVLP和标记物相连接。In another aspect of the present disclosure, it relates to a labeled CD20-eVLP, the CD20-eVLP is linked to the label.
在一个或多个实施方案中，所述CAR靶点蛋白-eVLP纳米颗粒为CD20-eVLP纳米颗粒，所述CD20和/或其功能片段和/或所述病毒核心蛋白和/或其功能片段与标记物相连接。In one or more embodiments, the CAR target protein-eVLP nanoparticles are CD20-eVLP nanoparticles, and the CD20 and/or its functional fragments and/or the viral core protein and/or its functional fragments are combined with markers are linked.
在一个或多个实施方案中，所述CD20和/或其功能片段的N末端和/或C末端和/或病毒核心蛋白和/或其功能片段的N末端和/或C末端与标记物相连接。In one or more embodiments, the N-terminal and/or C-terminal of the CD20 and/or its functional fragments and/or the N-terminal and/or C-terminal of the viral core protein and/or its functional fragments are compatible with the label connect.
在一个或多个实施方案中，所述包膜病毒样颗粒骨架蛋白为Gag蛋白。In one or more embodiments, the enveloped virus-like particle backbone protein is Gag protein.
在一个或多个实施方案中，所述CD20的C末端或所述Gag蛋白的C末端与标记物相连。In one or more embodiments, the C-terminus of the CD20 or the C-terminus of the Gag protein is linked to a label.
在一个或多个实施方案中，所述标记物为GPF。In one or more embodiments, the marker is GPF.
在一个或多个实施方案中，所述GFP的氨基酸序列包含与SEQ ID No.2具有至少80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列。在一个或多个优选的实施方案中，所述GFP的氨基酸序列如SEQ ID No.2所示。In one or more embodiments, the amino acid sequence of the GFP comprises an amino acid sequence with at least 80% or more identity to SEQ ID No.2, preferably 85%, 90%, 95%, 96%, 97% , 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences. In one or more preferred embodiments, the amino acid sequence of the GFP is shown in SEQ ID No.2.
在一个或多个实施方案中，所述Gag蛋白的C末端与标记物GFP相连，记为Gag-GFP，所述Gag-GFP的氨基酸序列包含与SEQ ID No.3具有至少80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列。在一个或多个优选的实施方案中，所述Gag-GFP的氨基酸序列如SEQ ID No.3所示。In one or more embodiments, the C-terminus of the Gag protein is connected to the marker GFP, which is denoted as Gag-GFP, and the amino acid sequence of the Gag-GFP contains at least 80% or more of the same sequence as SEQ ID No.3. An amino acid sequence with identity, preferably an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence with 98% or more 99% identity. In one or more preferred embodiments, the amino acid sequence of the Gag-GFP is shown in SEQ ID No.3.
在一个或多个实施方案中，所述Claudin 18.2的C末端与标记物GFP相连，记为Claudin 18.2-GFP，所述Claudin 18.2-GFP的氨基酸序列与SEQ ID No.5具有至少80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列。在一个或多个优选的实施方案中，所述Claudin 18.2-GFP的氨基酸序列如SEQ ID No.5所示。In one or more embodiments, the C-terminus of the Claudin 18.2 is linked to the marker GFP, denoted as Claudin 18.2-GFP, and the amino acid sequence of the Claudin 18.2-GFP has at least 80% or more of SEQ ID No.5 The amino acid sequence with identity is preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more, more preferably 98% or 99% or more. In one or more preferred embodiments, the amino acid sequence of the Claudin 18.2-GFP is shown in SEQ ID No.5.
在本公开的另一方面，涉及一种纳米颗粒的制备方法，其包括：In another aspect of the present disclosure, it relates to a method for preparing nanoparticles, which includes:
(1)重组质粒的构建：(1) Construction of recombinant plasmids:
分别构建包含包膜病毒样颗粒(eVLP)骨架蛋白和/或其功能片段基因的重组质粒和靶点蛋白和/或其功能片段基因的重组质粒；或，构建同时包含包膜病毒样颗粒(eVLP)骨架蛋白和/或其功能片段基因和靶点蛋白和/或其功能片段基因的重组质粒；和Respectively constructing a recombinant plasmid comprising enveloped virus-like particle (eVLP) skeleton protein and/or its functional fragment gene and a recombinant plasmid of target protein and/or its functional fragment gene; ) a recombinant plasmid of the backbone protein and/or its functional fragment gene and the target protein and/or its functional fragment gene; and
(2)宿主细胞转染、蛋白表达和颗粒组装：(2) Host cell transfection, protein expression and particle assembly:
将步骤(1)构建的包膜病毒样颗粒(eVLP)骨架蛋白和/或其功能片段重组质粒和靶点蛋白和/或其功能片段重组质粒转染宿主细胞，表达包膜病毒样颗粒骨架蛋白和/或其功能片段和靶点蛋白和/或其功能片段；所述包膜病毒样颗粒骨架蛋白或其功能片段组装形成包膜病毒样颗粒，所述靶点蛋白和/或其功能片段展示在包膜病毒样颗粒上，形成靶点蛋白-eVLP纳米颗粒；以及，任选地，Transfect the enveloped virus-like particle (eVLP) skeleton protein and/or its functional fragment recombinant plasmid and target protein and/or its functional fragment recombinant plasmid into the host cell to express the enveloped virus-like particle skeleton protein and/or functional fragments thereof and target protein and/or functional fragments thereof; the enveloped virus-like particle skeleton protein or functional fragments thereof are assembled to form enveloped virus-like particles, and the target protein and/or functional fragments thereof are displayed On the enveloped virus-like particle, forming target protein-eVLP nanoparticles; and, optionally,
(3)纯化靶点蛋白-eVLP纳米颗粒。(3) Purify target protein-eVLP nanoparticles.
在一个或多个实施方案中，所述GAG的编码核酸序列包含与SEQ ID No.7具有至少80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的核苷酸序列：更优选地，所述GFP的氨基酸序列如SEQ ID No.7所示。In one or more embodiments, the nucleic acid sequence encoding GAG comprises a nucleotide sequence with at least 80% or more identity to SEQ ID No.7, preferably 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical nucleotide sequences, more preferably 98% or 99% or more identical nucleotide sequences: more preferably, the amino acid sequence of the GFP is as SEQ ID No. 7.
在一个或多个实施方案中，所述Gag-GFP编码核酸序列包含与SEQ ID No.8具有至少80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的核苷酸序列：更优选地，所述GFP的氨基酸序列如SEQ ID No.8所示。In one or more embodiments, the Gag-GFP encoding nucleic acid sequence comprises a nucleotide sequence with at least 80% or more identity to SEQ ID No.8, preferably 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical nucleotide sequences, more preferably 98% or 99% or more identical nucleotide sequences: more preferably, the amino acid sequence of the GFP is as SEQ ID No .8 shown.
在一个或多个实施方案中，所述Claudin 18.2的编码核酸序列包含与SEQ ID No.9具有至少80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的核苷酸序列：更优选地，所述GFP的氨基酸序列如SEQ ID No.9所示。In one or more embodiments, the nucleic acid sequence encoding Claudin 18.2 comprises a nucleotide sequence with at least 80% or more identity to SEQ ID No.9, preferably 85%, 90%, 95%, 96% %, 97%, 98%, 99% or more identical nucleotide sequences, more preferably 98% or 99% or more identical nucleotide sequences: more preferably, the amino acid sequence of the GFP is as SEQ ID No .9 shown.
在一个或多个实施方案中，所述Claudin 18.2-GFP的编码核酸序列包含与SEQ ID No.10具有至少80％或以上 同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的核苷酸序列：更优选地，所述GFP的氨基酸序列如SEQ ID No.10所示。In one or more embodiments, the coding nucleic acid sequence of Claudin 18.2-GFP comprises a nucleotide sequence having at least 80% or more identity with SEQ ID No.10, preferably 85%, 90%, 95% , 96%, 97%, 98%, 99% or more identical nucleotide sequences, more preferably 98% or 99% or more identical nucleotide sequences: more preferably, the amino acid sequence of the GFP is as SEQ ID No.10 is shown.
在一个或多个实施方案中，所述CD20的编码核酸序列包含与SEQ ID No.16具有至少80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的核苷酸序列：更优选地，所述GFP的氨基酸序列如SEQ ID No.16所示。In one or more embodiments, the nucleic acid sequence encoding CD20 comprises a nucleotide sequence with at least 80% or more identity to SEQ ID No.16, preferably 85%, 90%, 95%, 96% , 97%, 98%, 99% or more identical nucleotide sequences, more preferably 98% or 99% or more identical nucleotide sequences: more preferably, the amino acid sequence of the GFP is as SEQ ID No. 16.
在一个或多个实施方案中，所述宿主细胞为原核宿主细胞或真核宿主细胞。In one or more embodiments, the host cell is a prokaryotic host cell or a eukaryotic host cell.
在一个或多个实施方案中，所述原核宿主细胞选自细菌细胞，例如大肠杆菌、枯草芽孢杆菌(B.subtilis)和分枝杆菌。In one or more embodiments, the prokaryotic host cell is selected from bacterial cells such as E. coli, B. subtilis and mycobacteria.
在一个或多个实施方案中，所述真核宿主细胞选自动物细胞、植物细胞或真菌中的任一种。In one or more embodiments, the eukaryotic host cell is selected from any one of animal cells, plant cells or fungi.
在一个或多个实施方案中，所述真核宿主细胞选自酵母、昆虫、禽类、植物、秀丽隐杆线虫和哺乳动物宿主细胞中的任一种。In one or more embodiments, the eukaryotic host cell is selected from any one of yeast, insect, avian, plant, Caenorhabditis elegans, and mammalian host cells.
所述昆虫细胞的非限制性例子有：草地夜蛾(Spodoptera frugiperda)(Sf)细胞、例如Sf9、Sf21，粉纹夜蛾(Trichoplusia ni)细胞，例如High Five细胞，和果蝇S2细胞。Non-limiting examples of such insect cells are: Spodoptera frugiperda (Sf) cells, such as Sf9, Sf21, Trichoplusia ni cells, such as High Five cells, and Drosophila S2 cells.
所述真菌(包括酵母)宿主细胞的例子有酿酒酵母(S.cerevisiae)、乳克鲁维酵母(Kluy veromyces lactis；K.lactis)、假丝酵母属(Candida)物种包括白色假丝酵母(C.albicans)和光滑假丝酵母(C.glabrata)、构巢曲霉(Aspergillus nidulans)、粟酒裂殖酵母(Schizosaccharomyces pombe；S.pombe)、巴斯德毕赤酵母(Pichia pastoris)、和解脂耶罗酵母(Yarrowia lipolytica)。Examples of such fungal (including yeast) host cells are S. cerevisiae, Kluy veromyces lactis (K. lactis), Candida species including Candida albicans (C .albicans) and C. glabrata, Aspergillus nidulans, Schizosaccharomyces pombe (S. pombe), Pichia pastoris, and lipolytica Yarrowia lipolytica.
所述哺乳动物细胞的例子有COS细胞、小鼠L细胞、LNCaP细胞、中国仓鼠卵巢(CHO)细胞、人胚肾(HEK)细胞(例如HEK293细胞)、633细胞、Vero、BHK细胞、非洲绿猴细胞、CV1细胞、HeLa细胞、MDCK细胞和Hep-2细胞。Examples of such mammalian cells are COS cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells (e.g. HEK293 cells), 633 cells, Vero, BHK cells, African green Monkey cells, CV1 cells, HeLa cells, MDCK cells and Hep-2 cells.
在一个或多个实施方案中，所述宿主细胞选自HEK293细胞、633细胞、Vero细胞、BHK细胞、原核细胞、酵母细胞、植物细胞、昆虫细胞和哺乳动物细胞中的任一种。In one or more embodiments, the host cell is selected from any one of HEK293 cells, 633 cells, Vero cells, BHK cells, prokaryotic cells, yeast cells, plant cells, insect cells and mammalian cells.
在本公开的另一方面，涉及检测CAR阳性表达率的方法，包括将所述的纳米颗粒或所述方法制备的纳米颗粒与CAR修饰的细胞孵育，然后进行检测。In another aspect of the present disclosure, it relates to a method for detecting the positive expression rate of CAR, comprising incubating the nanoparticle or the nanoparticle prepared by the method with CAR-modified cells, and then performing detection.
在一个或多个实施方案中，在细胞孵育后、进行检测前洗涤细胞。In one or more embodiments, the cells are washed after incubation of the cells prior to performing the assay.
在一个或多个优选的实施方案中，所述CAR修饰的细胞选自CAR-like(HEK293)细胞、CAR-T细胞、CAR-NK细胞、CAR-M细胞、CAR-NKT细胞、CAR-Treg细胞及CAR-γδT细胞中的任一种。In one or more preferred embodiments, the CAR-modified cells are selected from CAR-like (HEK293) cells, CAR-T cells, CAR-NK cells, CAR-M cells, CAR-NKT cells, CAR-Treg Any of cells and CAR-γδT cells.
在一个或多个实施方案中，涉及检测CAR阳性表达率的方法，包括将前述带标记物的Claudin 18.2-eVLP与CAR修饰的细胞孵育、洗涤后进行检测。In one or more embodiments, the method for detecting the positive expression rate of CAR includes incubating the aforementioned labeled Claudin 18.2-eVLP with CAR-modified cells, washing and then detecting.
优选地，涉及检测CAR阳性表达率的方法，包括将前述带标记物的Claudin 18.2-eVLP与CAR-T细胞孵育、洗涤后进行检测。Preferably, the method for detecting the positive expression rate of CAR includes incubating the aforementioned labeled Claudin 18.2-eVLP with CAR-T cells, washing and then detecting.
在一个或多个实施方案中，所述Claudin 18.2-eVLP保证了CAR靶点蛋白的天然完整构象，提高了分离出可识别天然膜蛋白结构的抗体的成功率。In one or more embodiments, the Claudin 18.2-eVLP ensures the natural complete conformation of the CAR target protein, which improves the success rate of isolating antibodies that can recognize the natural membrane protein structure.
在一个或多个实施方案中，涉及检测CAR阳性表达率的方法，包括将前述带标记物的CD20-eVLP与CAR修饰的细胞孵育、洗涤后进行检测。In one or more embodiments, the method for detecting the positive expression rate of CAR includes incubating the aforementioned labeled CD20-eVLP with CAR-modified cells, washing and then detecting.
优选地，涉及检测CAR阳性表达率的方法，包括将前述带标记物的CD20-eVLP与CAR-T细胞孵育、洗涤后进行检测。Preferably, the method for detecting the positive expression rate of CAR includes incubating the aforementioned labeled CD20-eVLP with CAR-T cells, washing and then detecting.
在一个或多个实施方案中，所述CD20-eVLP保证了CAR靶点蛋白的天然完整构象，提高了分离出可识别天然膜蛋白结构的抗体的成功率。In one or more embodiments, the CD20-eVLP ensures the natural complete conformation of the CAR target protein, which improves the success rate of isolating antibodies that can recognize the natural membrane protein structure.
在一个或多个实施方案中，所述孵育的温度为2℃-40℃，优选37℃，和/或，所述孵育的时间为15min-2小时，优选1小时；和/或，所述孵育的CO 2的浓度为2％-8％，优选5％。 In one or more embodiments, the incubation temperature is 2°C-40°C, preferably 37°C, and/or, the incubation time is 15min-2 hours, preferably 1 hour; and/or, the The concentration of CO2 for incubation is 2%-8%, preferably 5%.
在一个或多个实施方案中，所述洗涤的次数为1-6次，优选3次。In one or more embodiments, the number of washings is 1-6 times, preferably 3 times.
在一个或多个实施方案中，所述洗涤缓冲液为0.5％-5％BSA，优选2％BSA。In one or more embodiments, the wash buffer is 0.5%-5% BSA, preferably 2% BSA.
在一个或多个实施方案中，所述检测为流式细胞术检测、免疫检测、ELISA、SPR、BLI中的任一种。In one or more embodiments, the detection is any one of flow cytometry detection, immunoassay, ELISA, SPR, BLI.
在一个或多个实施方案中，所述包膜病毒样颗粒eVLP骨架蛋白为Gag蛋白。In one or more embodiments, the enveloped virus-like particle eVLP backbone protein is Gag protein.
在一个或多个实施方案中，所述Claudin 18.2的C末端或所述Gag蛋白的C末端与标记物相连接。In one or more embodiments, the C-terminus of the Claudin 18.2 or the C-terminus of the Gag protein is linked to a label.
在一个或多个实施方案中，所述CD20的C末端或所述Gag蛋白的C末端与标记物相连接。In one or more embodiments, the C-terminus of the CD20 or the C-terminus of the Gag protein is linked to a label.
在一个或多个实施方案中，利用HIV-1 Gag能够自组装成eVLP的能力，将CAR靶点蛋白展示在膜表面(图1)。优选地，将四次跨膜蛋白Claudin 18.2展示在HIV-1 Gag包膜VLP上，同时在Claudin 18.2的C末端或Gag的C末端用标记物进行标记。利用流式细胞术检测使用Claudin 18.2-eVLP来评估CAR阳性表达率。本公开所产生的Claudin 18.2-eVLP均可以进行靶抗原结合CAR的表达评估。In one or more embodiments, the ability of HIV-1 Gag to self-assemble into eVLPs is utilized to display the CAR target protein on the membrane surface (Figure 1). Preferably, the tetraspanning protein Claudin 18.2 is displayed on the HIV-1 Gag envelope VLP, and the C-terminus of Claudin 18.2 or the C-terminus of Gag is labeled with a marker. Claudin 18.2-eVLP was used to evaluate the positive expression rate of CAR by flow cytometry. All the Claudin 18.2-eVLPs produced in this disclosure can be evaluated for the expression of target antigen-binding CAR.
在本公开的另一方面，涉及一种试剂盒，其含有前述纳米颗粒。In another aspect of the present disclosure, it relates to a kit comprising the aforementioned nanoparticles.
在一个或多个实施方案中，所述纳米颗粒是CAR靶点蛋白-eVLP纳米颗粒。In one or more embodiments, the nanoparticles are CAR target protein-eVLP nanoparticles.
在一个或多个实施方案中，所述试剂盒含有前述带标记物的CAR靶点蛋白-eVLP。In one or more embodiments, the kit contains the aforementioned labeled CAR target protein-eVLP.
在一个或多个实施方案中，所述试剂盒含有前述带标记物的Claudin 18.2-eVLP。In one or more embodiments, the kit contains the aforementioned labeled Claudin 18.2-eVLP.
在一个或多个实施方案中，所述试剂盒含有前述带标记物的CD20-eVLP。In one or more embodiments, the kit contains the aforementioned labeled CD20-eVLP.
在一个或多个实施方案中，所述试剂盒为ELISA试剂盒、SPR试剂盒或BLI试剂盒。In one or more embodiments, the kit is an ELISA kit, an SPR kit or a BLI kit.
在一个或多个实施方案中，所述ELISA试剂盒含有前述CAR靶点蛋白-eVLP、酶标板、封闭液、样品稀释液、酶结合物、浓缩洗涤液、酶底物溶液和终止液。In one or more embodiments, the ELISA kit contains the aforementioned CAR target protein-eVLP, microtiter plate, blocking solution, sample diluent, enzyme conjugate, concentrated washing solution, enzyme substrate solution and stop solution.
在一个或多个实施方案中，所述SPR试剂盒包含前述CAR靶点蛋白-eVLP纳米颗粒。In one or more embodiments, the SPR kit comprises the aforementioned CAR target protein-eVLP nanoparticles.
在一个或多个实施方案中，所述BLI试剂盒包含前述CAR靶点蛋白-eVLP纳米颗粒。In one or more embodiments, the BLI kit comprises the aforementioned CAR target protein-eVLP nanoparticles.
本公开所述试剂盒既可用于检测CAR阳性表达率，也能证明CAR能特异性结合靶蛋白。The kit described in the present disclosure can be used not only to detect the positive expression rate of CAR, but also to prove that CAR can specifically bind to a target protein.
在非限制性实例中，所述试剂盒中可包括用于制备CAR靶点蛋白-eVLP和/或施用CAR靶点蛋白-eVLP的试剂。试剂盒可进一步包括用于在体外和体内评估CAR靶点蛋白-eVLP活性的试剂。在某些方面，试剂盒可以包括用于给药的试剂和/或装置，例如吸入器或喷雾器，试剂盒还可以包括一种或多种缓冲液等。In a non-limiting example, the kit may include reagents for preparing the CAR target protein-eVLP and/or administering the CAR target protein-eVLP. The kit may further include reagents for evaluating the activity of the CAR target protein-eVLP in vitro and in vivo. In certain aspects, the kit can include reagents and/or devices for administration, such as an inhaler or nebulizer, and the kit can also include one or more buffers, and the like.
当试剂盒的组分以一种和/或多种液体溶液形式提供时，液体溶液是水溶液，尤其优选无菌水溶液。此外，试剂盒的组分也可以干燥粉末的形式提供。当试剂和/或组分以干粉形式提供时，可以通过添加合适的溶剂来重新构建液体溶液。When the components of the kit are provided in one and/or more liquid solutions, the liquid solutions are aqueous solutions, especially preferably sterile aqueous solutions. In addition, the components of the kit may also be provided in dry powder form. When reagents and/or components are provided as dry powders, liquid solutions can be reconstituted by the addition of suitable solvents.
应理解，在本公开范围内中，本公开的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present disclosure, the above-mentioned technical features of the present disclosure and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.
附图说明Description of drawings
图1为HIV-Gag包膜VLP展示多次跨膜靶抗原的原理示意图。Fig. 1 is a schematic diagram of the principle of HIV-Gag enveloped VLP displaying multiple transmembrane target antigens.
图2示出了蔗糖密度梯度离心后粗纯样品电泳结果，其中泳道A为R1242粗纯样品，泳道B为RG288粗纯样品，泳道C为RG341粗纯样品。Figure 2 shows the electrophoresis results of crude samples after sucrose density gradient centrifugation, wherein lane A is the crude sample of R1242, lane B is the crude sample of RG288, and lane C is the crude sample of RG341.
图3示出了Sepharose 6FF分离后样品的电泳检测结果，其中图A为R1242，图B为RG288，图C为RG341。Figure 3 shows the electrophoresis detection results of the samples separated by Sepharose 6FF, where Figure A is R1242, Figure B is RG288, and Figure C is RG341.
图4示出了R1242-210521F1透射电镜检测结果。Figure 4 shows the detection results of R1242-210521F1 by transmission electron microscope.
图5示出了ELISA方法检测Claudin 18.2-eVLP与其特异抗体IMAB362的结合活性，其中图A为RG288-210503F1和IMAB362结合的ELISA检测结果；图B为RG341-210521F1和IMAB362结合的ELISA检测结果。Figure 5 shows the ELISA method to detect the binding activity of Claudin 18.2-eVLP and its specific antibody IMAB362, wherein Figure A is the ELISA detection result of the combination of RG288-210503F1 and IMAB362; Figure B is the ELISA detection result of the combination of RG341-210521F1 and IMAB362.
图6为IMAB361 CAR-like结构示意图。Figure 6 is a schematic diagram of the IMAB361 CAR-like structure.
图7示出了本公开Claudin18.2-eVLP用于评估IMAB362 CAR-like表达情况：其中图A为当FITC-anti mFab Ab稀释200倍时，其评估IMAB362 CAR-like表达情况；图B为当FITC-Protein L使用浓度为10μg/mL时，其评估IMAB362 CAR-like表达情况；图C为当Claudin-18.2 Protein,His Tag TM使用浓度为10μg/mL时，其评估IMAB362 CAR-like表达情；图D为当RG288-210503F1使用浓度为10μg/mL时，其评估IMAB362 CAR-like表达情况；图E为当RG341-210521F1使用浓度为10μg/mL时，其评估IMAB362 CAR-like表达情况。 Figure 7 shows that Claudin18.2-eVLP of the present disclosure is used to evaluate the expression of IMAB362 CAR-like: where Figure A is when the FITC-anti mFab Ab is diluted 200 times, it evaluates the expression of IMAB362 CAR-like; Figure B is when When FITC-Protein L is used at a concentration of 10 μg/mL, it evaluates the expression of IMAB362 CAR-like; Figure C is when Claudin-18.2 Protein, His Tag TM is used at a concentration of 10 μg/mL, it evaluates the expression of IMAB362 CAR-like; Figure D is the evaluation of IMAB362 CAR-like expression when RG288-210503F1 is used at a concentration of 10 μg/mL; Figure E is the evaluation of IMAB362 CAR-like expression when RG341-210521F1 is used at a concentration of 10 μg/mL.
图8示出了本公开CD20-eVLP用于评估Ofatumumab CAR-like表达情况：当RG344-210628F1使用浓度为20μg/mL时，其评估Ofatumumab CAR-like表达情况。Figure 8 shows that the CD20-eVLP of the present disclosure is used to evaluate the expression of Ofatumumab CAR-like: when RG344-210628F1 is used at a concentration of 20 μg/mL, it evaluates the expression of Ofatumumab CAR-like.
具体实施方式detailed description
本公开公开了一种eVLP及其检测CAR阳性表达率的应用。本领域技术人员可以参考本文内容，获得该eVLP，实现其应用，特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本公开内。本公开的制备方法及应用已经通过较佳的实施例进行了描述，相关人员明显能在不脱离本公开内容、精神和范围内对本文制备方法和应用进行改动或适当变更与组合，来实现和应用本公开技术。The present disclosure discloses an eVLP and its application for detecting the positive expression rate of CAR. Those skilled in the art can refer to the content herein to obtain the eVLP and realize its application. In particular, it should be noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present disclosure. The preparation method and application of the present disclosure have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the preparation method and application herein without departing from the content, spirit and scope of the present disclosure to realize and Apply the disclosed technology.
定义definition
除非另外定义，否则本文使用的所有技术和科学术语具有本公开所属领域的普通技术人员通常所理解的相同的含义。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
术语“病毒样颗粒”(VLP)是指在至少一种属性上类似病毒但是已经证明其没有感染性的结构。一般而言，病毒样颗粒缺乏病毒基因组，并且不能复制。另外，病毒样颗粒通常可以通过异源表达来大量生产，而且可以容易地纯化。The term "virus-like particle" (VLP) refers to a structure that resembles a virus in at least one property but has been shown to be non-infectious. In general, virus-like particles lack the viral genome and are unable to replicate. In addition, virus-like particles can often be produced in large quantities by heterologous expression and can be easily purified.
术语“包膜VLP”或可替代的“eVLP”是指病毒样颗粒被源于宿主细胞的脂质包膜包裹，形成的包膜病毒样颗粒。The term "enveloped VLP" or alternatively "eVLP" refers to an enveloped virus-like particle formed by wrapping a virus-like particle with a lipid envelope derived from a host cell.
术语“CAR靶点蛋白”是指能与CAR特异性结合的蛋白。The term "CAR target protein" refers to a protein that can specifically bind to CAR.
术语“eVLP骨架蛋白”在本公开中可以理解为形成eVLP的骨架结构的蛋白质，所述eVLP的骨架结构可以主要由病毒核心蛋白构成或包括病毒核心蛋白以及其他蛋白。The term "eVLP backbone protein" in the present disclosure can be understood as a protein forming the backbone structure of an eVLP, which may mainly consist of viral core protein or include viral core protein as well as other proteins.
术语“病毒核心蛋白”是指包膜蛋白，在一些情况下，其还能够驱动出芽和颗粒从宿主细胞的释放。The term "viral core protein" refers to the envelope protein, which in some cases is also capable of driving budding and release of particles from the host cell.
术语“功能片段”对应于全长的蛋白质具有截短的结构，但仍然保持全长蛋白质的全部或部分功能的片段；例如对于核心蛋白而言，其对应的功能片段可以理解为截短的核心蛋白的片段，其仍具有形成病毒至少部分衣壳或驱动出芽和颗粒从宿主细胞的释放的能力；对于靶点蛋白而言，其对应的功能片段可以理解为截短的靶点蛋白的片段，其仍具备与CAR特异性结合的能力。The term "functional fragment" corresponds to a fragment of a full-length protein that has a truncated structure, but still maintains all or part of the function of the full-length protein; for example, for the core protein, its corresponding functional fragment can be understood as a truncated core A fragment of a protein that still has the ability to form at least a partial capsid of the virus or to drive budding and the release of particles from the host cell; for the target protein, its corresponding functional fragment can be understood as a fragment of the truncated target protein, It still has the ability to specifically bind to CAR.
术语“嵌合抗原受体”或可替代的“CAR”是指重组多肽构建体，该重组多肽构建体至少包含细胞外抗原结合结构域、跨膜结构域和包含源自刺激分子的功能性信号传导结构域的细胞质信号传导结构域。The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain and a functional signal derived from a stimulatory molecule The cytoplasmic signaling domain of the transduction domain.
术语“CAR-like(HEK293)”是指嵌合抗原受体(CAR)修饰的HEK293细胞，即用CAR手段编辑HEK293， 在HEK293的表面展示CAR。The term "CAR-like (HEK293)" refers to chimeric antigen receptor (CAR) modified HEK293 cells, that is, HEK293 is edited by CAR means, and CAR is displayed on the surface of HEK293.
术语“CAR-NK”是指嵌合抗原受体(CAR)修饰的NK细胞，其中NK细胞是自然杀伤细胞(natural killer cell)，NK细胞是机体重要的免疫细胞，不仅与抗肿瘤、抗病毒感染和免疫调节有关，而且在某些情况下参与超敏反应和自身免疫性疾病的发生，能够识别靶细胞、杀伤介质。NK细胞因其特殊的识别靶细胞的机制、短暂的生理周期、广泛的肿瘤杀伤能力等优势，被视为同样有潜力通过CAR修饰增强其抗肿瘤能力的效应细胞。The term "CAR-NK" refers to chimeric antigen receptor (CAR) modified NK cells, wherein NK cells are natural killer cells (natural killer cells), NK cells are important immune cells of the body, not only with anti-tumor, anti-virus Infection is related to immune regulation, and in some cases participates in the occurrence of hypersensitivity and autoimmune diseases, and can recognize target cells and killing mediators. NK cells are regarded as effector cells that also have the potential to enhance their anti-tumor ability through CAR modification because of their special mechanism for recognizing target cells, short physiological cycle, and extensive tumor killing ability.
术语“CAR-M”是指嵌合抗原受体(CAR)修饰的巨噬细胞(CAR macrophages)，即用CAR手段编辑人体巨噬细胞，使其可以直接吞噬肿瘤。The term "CAR-M" refers to chimeric antigen receptor (CAR)-modified macrophages (CAR macrophages), that is, the use of CAR means to edit human macrophages so that they can directly phagocytize tumors.
术语“CAR-NKT”是指嵌合抗原受体(CAR)修饰的NKT细胞(Natural killer T cell)，其中NKT细胞是一种细胞表面既有T细胞受体TCR，又有NK细胞受体的特殊T细胞亚群。CAR-NKT细胞结合NKT细胞原本的优势和CAR疗法的特异性，更好的实现肿瘤杀伤效果。The term "CAR-NKT" refers to chimeric antigen receptor (CAR) modified NKT cells (Natural killer T cells), wherein NKT cells are a kind of T cell receptor TCR and NK cell receptors on the cell surface Special T cell subsets. CAR-NKT cells combine the original advantages of NKT cells and the specificity of CAR therapy to better achieve tumor killing effects.
术语“CAR-Treg”是指嵌合抗原受体(CAR)修饰的调节性T细胞(Tregs)，其中Tregs是一类控制体内自身免疫反应性的T细胞亚群，早期亦称作抑制性T细胞(suppressor T cells)。调节性T细胞可分为天然产生的自然调节性T细胞(n T-regs)和诱导产生的适应性调节性T细胞(a T-regs或i T-regs)，如Th3、Tr1，另外尚有CD8 Treg、NKT细胞等，与自身免疫性疾病的***密切，其异常表达可导致自身免疫性疾病。The term "CAR-Treg" refers to chimeric antigen receptor (CAR) modified regulatory T cells (Tregs), where Tregs are a type of T cell subset that controls autoimmune reactivity in the body, also known as inhibitory T cells in the early days Cells (suppressor T cells). Regulatory T cells can be divided into naturally occurring natural regulatory T cells (n T-regs) and induced adaptive regulatory T cells (a T-regs or i T-regs), such as Th3, Tr1, and There are CD8 Treg, NKT cells, etc., which are closely related to the occurrence of autoimmune diseases, and their abnormal expression can lead to autoimmune diseases.
术语“CAR-γδT”是指嵌合抗原受体(CAR)修饰的γδT细胞，其中γδT细胞是介于适应性免疫和固有免疫之间的一类T细胞，占外周血T淋巴细胞的1％-5％，主要分布在黏膜和上皮组织中。γδT细胞识别抗原无MHC限制，不仅可以通过多种方式杀伤肿瘤细胞，而且作为抗原提呈细胞(APC)发挥提呈抗原作用。将γδT细胞改造成CAR-γδT细胞，使其能够精准的识别特异性抗原，高效杀死肿瘤细胞，利用γδT细胞特性或许可以挑战实体瘤的治疗。The term "CAR-γδT" refers to chimeric antigen receptor (CAR)-modified γδT cells, where γδT cells are a type of T cell between adaptive immunity and innate immunity, accounting for 1% of peripheral blood T lymphocytes -5%, mainly distributed in mucosa and epithelial tissues. γδT cells recognize antigens without MHC restriction, not only can kill tumor cells in various ways, but also present antigens as antigen-presenting cells (APC). Transforming γδT cells into CAR-γδT cells can accurately recognize specific antigens and kill tumor cells efficiently. Using the characteristics of γδT cells may challenge the treatment of solid tumors.
术语“scFv”是指融合蛋白，该融合蛋白包含至少一个包含轻链可变区的抗体片段和至少一个包含重链可变区的抗体片段，其中该轻链和重链可变区通过短的柔性多肽接头连续地连接，并且能够表达为单链多肽，并且其中该scFv保留了衍生其的完整抗体的特异性。The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are separated by a short Flexible polypeptide linkers are contiguously linked and are capable of being expressed as single chain polypeptides, and wherein the scFv retains the specificity of the intact antibody from which it was derived.
术语“抗原”或“Ag”是指引起免疫响应的分子。免疫响应可以涉及抗体产生或特定免疫活性细胞的活化或两者。技术人员将理解实际上包括所有蛋白质或肽的任何大分子都可以充当抗原。此外，抗原可以衍生自重组或基因组DNA。技术人员将理解包含编码引发免疫响应的蛋白质的核苷酸序列或部分核苷酸序列的任何DNA都因此编码“抗原”。The term "antigen" or "Ag" refers to a molecule that elicits an immune response. The immune response may involve antibody production or activation of specific immunocompetent cells or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. The skilled artisan will understand that any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response thus encodes an "antigen".
术语“核酸”或“多核苷酸”是指单链或双链形式的脱氧核糖核酸(DNA)或核糖核酸(RNA)及其聚合物。The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form.
术语“ELISA”是酶联接免疫吸附剂测定(Enzyme-Linked Immunosorbnent Assay)的简称。它是将已知的抗原或抗体结合在固相载体表面，然后利用酶标记(偶联)的抗体或抗原与之孵育，在通过显色物显色，其显色深浅与待测物质的含量成正比，用肉眼即可观察。在ELISA实验中，有三种必需的试剂：已知的抗原或抗体(用于结合到固相载体上)；酶标的抗体或抗原(标记物)；显色剂(用于显色)。常见的ELISA实验有四种：直接ELISA、间接ELISA、夹心ELISA和竞争ELISA。The term "ELISA" is an abbreviation for Enzyme-Linked Immunosorbnent Assay. It combines the known antigen or antibody on the surface of the solid phase carrier, and then uses the enzyme-labeled (coupled) antibody or antigen to incubate with it, and then develops the color through the chromogenic substance, and the color depth is related to the content of the substance to be tested In direct proportion, it can be observed with the naked eye. In ELISA experiments, there are three necessary reagents: known antigens or antibodies (for binding to solid-phase supports); enzyme-labeled antibodies or antigens (markers); chromogenic reagents (for color development). There are four common ELISA experiments: direct ELISA, indirect ELISA, sandwich ELISA and competition ELISA.
术语“SPR”是表面等离子体共振(Surface plasmon resonance)的简称，其应用SPR原理检测生物传感芯片(biosensor chip)上配位体与分析物之间的相互作用情况，作为一个通用检测平台，被广泛应用在药物筛选、科学研究等领域，用于生物分子间亲和力、结合特异性、浓度定量等分析。The term "SPR" is the abbreviation of surface plasmon resonance (Surface plasmon resonance), which uses the SPR principle to detect the interaction between ligands and analytes on a biosensor chip (biosensor chip), as a general detection platform, It is widely used in drug screening, scientific research and other fields for the analysis of biomolecular affinity, binding specificity, concentration quantification, etc.
术语“BLI”是生物膜干涉技术(Bio-Layer Interferometry)的简称，是基于干涉光谱图的位移变化来检测生物分子间相互作用的一种实验方法。The term "BLI" is the abbreviation of Bio-Layer Interferometry, which is an experimental method to detect the interaction between biomolecules based on the shift change of the interference spectrum.
术语“FACS”是流式细胞荧光分选技术(Fluorescence activated Cell Sorting)的简称，流式细胞仪的工作原理是将待测细胞经特异性荧光染料染色后放入样品管中，在气体的压力下进入充满鞘液的流动室。在鞘液的约束下细胞排成单列由流动室的喷嘴喷出，形成细胞柱，后者与入射的激光束垂直相交，液柱中的细胞被激光激发产生荧光。仪器中一系列光学***(透镜、光阑、滤片和检测器等)收集荧光、光散射、光吸收或细胞电阻抗等信号，计算机***进行收集、储存、显示并分析被测定的各种信号，对各种指标做出统计分析。The term "FACS" is the abbreviation of Fluorescence activated Cell Sorting. The working principle of the flow cytometer is to put the cells to be tested into the sample tube after being stained with a specific fluorescent dye. down into the flow chamber filled with sheath fluid. Under the confinement of the sheath fluid, the cells are arranged in a single row and ejected from the nozzle of the flow chamber to form a cell column, which intersects the incident laser beam perpendicularly, and the cells in the liquid column are excited by the laser to generate fluorescence. A series of optical systems (lenses, apertures, filters, detectors, etc.) in the instrument collect signals such as fluorescence, light scattering, light absorption, or cell electrical impedance, and the computer system collects, stores, displays, and analyzes the various signals that are measured , to make statistical analysis on various indicators.
术语“HIS-Tag”由6-10个组氨酸残基组成，分子量不到0.84KD，通常***在目的蛋白的C末端或N末端。HIS-Tag是目前原核表达最常用的标签，蛋白纯化完之后可以不需切除此标签，也不会对蛋白产生功能影响。同时，蛋白纯化步骤简便，纯化条件温和，对蛋白也不会产生太大影响。The term "HIS-Tag" consists of 6-10 histidine residues, with a molecular weight of less than 0.84KD, and is usually inserted at the C-terminal or N-terminal of the target protein. HIS-Tag is currently the most commonly used tag for prokaryotic expression. After protein purification, this tag does not need to be removed, and it will not affect the function of the protein. At the same time, the protein purification steps are simple, the purification conditions are mild, and the protein will not be greatly affected.
术语“GST-Tag”为谷胱甘肽巯基转移酶标签，相对分子质量较大，约为26KD，***在目的蛋白的C末端或N末端，大肠杆菌中常用在N端。GST(谷胱甘肽巯基转移酶)蛋白本身是一个在解毒过程中起到重要作用的转移酶。一般选择GST标签的目的有两个，一是提高蛋白表达的可溶性，二是提高蛋白的表达量。蛋白表达纯化结束后需根据不同的蛋白应用而确定是否切除标签，标签较大，切除与否需根据下游应用考虑。如果要去除GST融合部分，可用位点特异性蛋白酶切除。The term "GST-Tag" is a glutathione thiol transferase tag with a relatively large molecular mass of about 26KD, which is inserted at the C-terminus or N-terminus of the target protein, and is commonly used at the N-terminus in Escherichia coli. The GST (glutathione thiol transferase) protein itself is a transferase that plays an important role in the detoxification process. Generally, there are two purposes for choosing GST tags, one is to improve the solubility of protein expression, and the other is to increase the expression level of protein. After protein expression and purification, it is necessary to determine whether to remove the tag according to different protein applications. If the tag is large, whether to remove it should be considered according to the downstream application. If the GST fusion portion is to be removed, it can be excised with a site-specific protease.
术语“MBP-Tag”为麦芽糖结合蛋白标签，氨基酸残基数346，分子量42.5KDa，由大肠杆菌K12的malE基因编码，构建时可放在N端，用来提高可溶性(尤其是真核蛋白)。MBP的折叠需要DnaK-DnaJ-GrpE和GroEL-GeoES两个分子伴侣***的帮助，这可以使这些分子伴侣聚集到目的蛋白的附近帮助其正确折叠。另外，以标签蛋白形式存在的麦芽糖结合蛋白可以减少目的蛋白的降解，提高表达产物的水溶性，也为以后对目的蛋白的纯化提供了基础。麦芽糖结合蛋白能够被多糖树脂吸附，因此在过柱时，能够使融合蛋白与其它蛋白成份分离。The term "MBP-Tag" is a maltose-binding protein tag with 346 amino acid residues and a molecular weight of 42.5KDa, encoded by the malE gene of Escherichia coli K12, which can be placed at the N-terminus during construction to improve solubility (especially for eukaryotic proteins) . The folding of MBP requires the help of two molecular chaperone systems, DnaK-DnaJ-GrpE and GroEL-GeoES, which can make these molecular chaperones gather near the target protein to help it fold correctly. In addition, maltose-binding protein in the form of tagged protein can reduce the degradation of the target protein, improve the water solubility of the expressed product, and also provide a basis for the purification of the target protein in the future. Maltose-binding protein can be adsorbed by polysaccharide resin, so the fusion protein can be separated from other protein components when passing through the column.
术语“NusA-Tag”为转录终止/抗终止蛋白标签，NusA是大肠杆菌自身的一种蛋白，即转录抗终止因子，氨基 酸残基数495，分子量：54.87KDa，由1999年Davia将NusA从4000种大肠杆菌蛋白库中筛得。NusA不具有独立的纯化标签功能，所以要与其它标签(如His标签)联用。The term "NusA-Tag" is a transcription termination/anti-termination protein tag. NusA is a protein of Escherichia coli itself, that is, a transcription anti-termination factor, with 495 amino acid residues and a molecular weight of 54.87KDa. Screened from the Escherichia coli protein library. NusA does not have an independent purification tag function, so it should be used in conjunction with other tags (such as His tags).
术语“FLAG-Tag”为8个氨基酸(DYKDDDDK，SEQ ID No.12)的融合多肽，同时载体中构建的Kozak序列使得带有FLAG的融合蛋白在真核表达***中表达效率更高。The term "FLAG-Tag" is a fusion polypeptide of 8 amino acids (DYKDDDDK, SEQ ID No.12), and the Kozak sequence constructed in the vector enables the fusion protein with FLAG to express more efficiently in the eukaryotic expression system.
术语“SUMO”为SUMO标签蛋白，是一种小分子泛素相关修饰蛋白，是存在于真核生物中高度保守的参与蛋白质小泛素化相关修饰的一类大蛋白。与GST、MBP或NusA相比，SUMO不仅可以作为重组蛋白表达的融合标签还具备分子伴侣的功能，能促进蛋白的正确折叠，对热和蛋白酶具有耐受性，更有助于保持目的蛋白的稳定性。The term "SUMO" refers to a SUMO-tagged protein, which is a small molecule ubiquitin-related modification protein and a class of large proteins that are highly conserved in eukaryotes and participate in protein small ubiquitination-related modification. Compared with GST, MBP or NusA, SUMO can not only be used as a fusion tag for recombinant protein expression, but also has the function of a molecular chaperone, which can promote the correct folding of the protein, is resistant to heat and protease, and is more helpful in maintaining the stability of the target protein. stability.
术语“Avi-Tag”是一个15个氨基酸的短肽，具有一个单生物素化赖氨酸位点，与已知天然可生物素化序列完全不同，可以加在目标蛋白的N端和C端。融合表达后，可被生物素连接酶生物素化，为了纯化重组蛋白选用低亲和性的单体抗生物素蛋白或抗生物素蛋白衍生物。The term "Avi-Tag" is a short peptide of 15 amino acids with a single biotinylated lysine site, which is completely different from the known natural biotinylated sequence, and can be added to the N-terminal and C-terminal of the target protein . After fusion expression, it can be biotinylated by biotin ligase. In order to purify the recombinant protein, low-affinity monomeric avidin or avidin derivatives are selected.
术语“Halo-Tag”是一种脱卤素酶的遗传修饰衍生物，可与多种合成的HaloTag配基有效地共价结合。这个分子量为33KDa的单体蛋白能融合在重组蛋白的N端或C端，并在原核和真核***中表达。HaloTag配基是小分子化学物，能够在体外或体内与HaloTag蛋白共价结合。The term "Halo-Tag" refers to a genetically modified derivative of a dehalogenase that can be efficiently covalently bound to a variety of synthetic HaloTag ligands. This monomeric protein with a molecular weight of 33KDa can be fused to the N-terminal or C-terminal of the recombinant protein and expressed in prokaryotic and eukaryotic systems. HaloTag ligands are small molecule chemicals that can covalently bind to HaloTag proteins in vitro or in vivo.
术语“SNAP-Tag”是从人的O6-甲基鸟嘌呤-DNA甲基转移(O6-alkylguanine-DNA-alkyltransferase)获得。无论体内还是体外，SNAP-Tag都能与底物高特异性地共价结合，使蛋白标记上生物素或荧光基团(如荧光素和若丹明)。SNAP所带的活性巯基位点接受了苯甲基鸟嘌呤所携带的侧链苯甲基基团，释放出了鸟嘌呤。这种新的硫醚键共价结合使SNAP所带的目的蛋白携带上了苯甲基基团所带的标记物。苯甲基鸟嘌呤在生化条件下稳定，并且没有其他蛋白会和这类物质作用，所以SNAP标签反应是高特异的。The term "SNAP-Tag" is derived from human O6-methylguanine-DNA methyltransferase (O6-alkylguanine-DNA-alkyltransferase). No matter in vivo or in vitro, SNAP-Tag can be covalently combined with substrates with high specificity, so that proteins can be labeled with biotin or fluorescent groups (such as fluorescein and rhodamine). The active sulfhydryl site of SNAP accepts the side chain benzyl group carried by benzylguanine and releases guanine. This new covalent combination of thioether bonds makes the target protein carried by SNAP carry the label carried by the benzyl group. Benzylguanine is stable under biochemical conditions, and no other protein will interact with this kind of substance, so the SNAP tagging reaction is highly specific.
术语“c-Myc”标签蛋白，是一个含10个氨基酸(EQKLISEEDL，SEQ ID No.13)的小标签，这10个氨基酸作为抗原表位表达在不同的蛋白质框架中仍可识别其相应抗体。C-Myc tag已成功应用在Western-blot杂交技术、免疫沉淀和流式细胞计量术中,可用于检测重组蛋白质在靶细胞中的表达。常见的载体有pCMV-MYC、pcDNA3.1(+)_myc-hisA、pCMV-RFP-C-Myc、pCMV-Myc等。The term "c-Myc" tagged protein is a small tag containing 10 amino acids (EQKLISEEDL, SEQ ID No.13). These 10 amino acids are expressed as antigenic epitopes and can still recognize their corresponding antibodies in different protein frameworks. C-Myc tag has been successfully applied in Western-blot hybridization, immunoprecipitation and flow cytometry, and can be used to detect the expression of recombinant proteins in target cells. Common vectors include pCMV-MYC, pcDNA3.1(+)_myc-hisA, pCMV-RFP-C-Myc, pCMV-Myc, etc.
术语“HA”标签蛋白，标签序列YPYDVPDYA(SEQ ID No.14)，源于流感病毒的红细胞凝集素表面抗原决定簇，9个氨基酸，对外源靶蛋白的空间结构影响小，容易构建成标签蛋白融合到N端或者C端。常用Anti－HA抗体检测和ELISA检测。The term "HA" tag protein, the tag sequence YPYDVPDYA (SEQ ID No.14), derived from the hemagglutinin surface epitope of influenza virus, 9 amino acids, has little effect on the spatial structure of the foreign target protein, and is easy to construct into a tag protein Fused to the N-terminus or C-terminus. Anti-HA antibody detection and ELISA detection are commonly used.
术语“荧光素酶”来源于生物体内的荧光素，常见的有萤火虫荧光素酶、海肾荧光素酶和Guassia荧光素酶。The term "luciferase" is derived from luciferin in living organisms, common ones are firefly luciferase, Renilla luciferase and Guassia luciferase.
实施例Example
实施例1 eVLP骨架质粒的构建The construction of embodiment 1 eVLP backbone plasmid
本实施例中使用的eVLP骨架为HIV-1 SF2p 55Gag(GenBank accession no.K02007)蛋白(SEQ ID No.1)，单独的Gag蛋白具备可以自组装成包膜VLP颗粒的能力。 The eVLP backbone used in this example is HIV-1 SF2 p 55 Gag (GenBank accession no. K02007) protein (SEQ ID No. 1), and a single Gag protein has the ability to self-assemble into enveloped VLP particles.
首先，分别构建含有Gag编码基因的重组质粒R1221和Gag-GFP编码基因的重组质粒R1242：由上海生物工程有限公司合成编码SEQ ID No.1和3所示氨基酸序列的多核苷酸SEQ ID No.7和8，5’末端添加限制性内切酶BamHⅠ酶切位点、Kozak序列；3’末端添加终止密码子TAA和限制性内切酶XhoⅠ酶切位点。合成产物用限制性内切酶BamHⅠ和XhoⅠ双酶切，然后进行琼脂糖凝胶电泳，切胶回收目的片段。将目的片段分别与同样经限制性内切酶BamHⅠ和XhoⅠ双酶切的pcNDA3.1(+)连接，构建重组真核表达载体，即重组质粒R1221和R1242。然后，将重组质粒R1221和R1242分别转化大肠杆菌E.coli DH5α，37℃培养16h后挑取单菌落进行质粒提取，将提取后的质粒用限制性内切酶BamHⅠ和XhoⅠ双酶切后进行琼脂糖凝胶电泳鉴定，将阳性克隆送上海生物工程有限公司进行测序，选择测序正确的克隆进行质粒的放大提取，提取后的质粒无菌过滤后，-20℃保存备用。First, the recombinant plasmid R1221 containing the Gag-encoding gene and the recombinant plasmid R1242 containing the Gag-GFP encoding gene were respectively constructed: the polynucleotide SEQ ID No. Restriction endonuclease BamHI cutting site and Kozak sequence were added at 7 and 8, 5' end; stop codon TAA and restriction endonuclease XhoI cutting site were added at 3' end. The synthetic product was double-digested with restriction endonucleases BamHI and XhoI, then subjected to agarose gel electrophoresis, and the target fragment was recovered by cutting the gel. The target fragments were respectively ligated with pcNDA3.1(+) which had also been double-digested with restriction enzymes BamHI and XhoI to construct recombinant eukaryotic expression vectors, ie recombinant plasmids R1221 and R1242. Then, the recombinant plasmids R1221 and R1242 were respectively transformed into Escherichia coli E.coli DH5α, cultured at 37°C for 16 hours, and then a single colony was picked for plasmid extraction. Glycogel electrophoresis identification, the positive clones were sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the clones with correct sequencing were selected for amplification and extraction of plasmids. After the extracted plasmids were sterile filtered, they were stored at -20°C for later use.
实施例2 Claudin 18.2重组表达质粒的构建Example 2 Construction of Claudin 18.2 recombinant expression plasmid
首先，分别构建含有Claudin 18.2编码基因的重组质粒R1353和Claudin 18.2-GFP编码基因的重组质粒R1303：由上海生物工程有限公司合成编码SEQ ID No.4和5所示氨基酸序列的多核苷酸SEQ ID No.9和10，5’末端添加限制性内切酶BamHⅠ酶切位点、Kozak序列；3’末端添加终止密码子TAA和限制性内切酶XhoⅠ酶切位点。合成产物用限制性内切酶BamHⅠ和XhoⅠ双酶切，然后进行琼脂糖凝胶电泳，切胶回收目的片段。将目的片段分别与同样经限制性内切酶BamHⅠ和XhoⅠ双酶切的pcNDA3.1(+)连接，构建重组真核表达载体，即重组质粒R1353和R1303。然后，将重组质粒R1353和R1303分别转化大肠杆菌E.coli DH5α，37℃培养16h后挑取单菌落进行质粒提取，将提取后的质粒用限制性内切酶BamHⅠ和XhoⅠ双酶切后进行琼脂糖凝胶电泳鉴定，将阳性克隆送上海生物工程有限公司进行测序，选择测序正确的克隆进行质粒的放大提取，提取后的质粒无菌过滤后，-20℃保存备用。First, the recombinant plasmid R1353 containing the gene encoding Claudin 18.2 and the recombinant plasmid R1303 containing the gene encoding Claudin 18.2-GFP were respectively constructed: the polynucleotide SEQ ID encoding the amino acid sequence shown in SEQ ID No.4 and 5 was synthesized by Shanghai Bioengineering Co., Ltd. No. 9 and 10, 5' end added restriction endonuclease BamHI site, Kozak sequence; 3' end added stop codon TAA and restriction endonuclease XhoI site. The synthetic product was double-digested with restriction endonucleases BamHI and XhoI, then subjected to agarose gel electrophoresis, and the target fragment was recovered by cutting the gel. The target fragments were respectively ligated with pcNDA3.1(+) which had also been double-digested with restriction endonucleases BamHI and XhoI to construct recombinant eukaryotic expression vectors, ie recombinant plasmids R1353 and R1303. Then, the recombinant plasmids R1353 and R1303 were respectively transformed into Escherichia coli DH5α, cultured at 37°C for 16 hours, and single colonies were picked for plasmid extraction, and the extracted plasmids were double-enzymatically digested with restriction endonucleases BamHI and XhoⅠ, and carried out on agar Glycogel electrophoresis identification, the positive clones were sent to Shanghai Bioengineering Co., Ltd. for sequencing, and the clones with correct sequencing were selected for amplification and extraction of plasmids. After the extracted plasmids were sterile filtered, they were stored at -20°C for later use.
实施例3空白HIV-1 Gag-eVLP及Claudin 18.2-eVLP在HEK293细胞中的表达Example 3 Expression of blank HIV-1 Gag-eVLP and Claudin 18.2-eVLP in HEK293 cells
3.1空白HIV-1 Gag-eVLP在HEK293细胞中的表达3.1 Expression of blank HIV-1 Gag-eVLP in HEK293 cells
使用PEI转染试剂将构建好的重组质粒R1242转染HEK293细胞(对应细胞ID为R1242)，进行空白对照eVLP的表达和组装。转染前一天将HEK293细胞按照1×10^6个细胞/mL传代，37℃培养。转染当天进行计数， 调整细胞密度为2×10^6个细胞/mL，活率95％以上。The constructed recombinant plasmid R1242 was transfected into HEK293 cells (corresponding to cell ID R1242) using PEI transfection reagent, and the blank control eVLP was expressed and assembled. The day before transfection, HEK293 cells were subcultured at 1×10^6 cells/mL and cultured at 37°C. On the day of transfection, counting was performed, and the cell density was adjusted to 2×10^6 cells/mL, and the viability was over 95%.
根据细胞密度计算转染复合物中各组分的用量：质粒剂量与细胞的数量对应关系为1×10^6个细胞对应0.6μg质粒，PEI剂量与质粒剂量的对应关系为：PEI的质量为DNA质量的3倍。Calculate the dosage of each component in the transfection complex according to the cell density: the corresponding relationship between the dose of plasmid and the number of cells is 1×10^6 cells corresponding to 0.6 μg of plasmid, and the corresponding relationship between the dose of PEI and the dose of plasmid is: the quality of PEI is 3 times the quality of DNA.
根据上述计算得到的组分用量配置转染复合物：将1.2mg浓度为200μg/mL的重组质粒加入CD 293TGE培养基(Acrobiosystems；货号CM-1156-11)至50mL(A液)，3.6mg浓度为1mg/mL的PEI转染试剂加入CD 293TGE培养基至50ml(B液)，分别混匀后，将B液缓慢加入至A液中，混匀，室温静置10-15min后，将转染混合液缓慢滴加入1000mL HEK293细胞中。37℃，5％CO 2，135rpm培养48h后，收集R1242的培养上清。 Prepare the transfection complex according to the amount of components calculated above: add 1.2 mg of the recombinant plasmid at a concentration of 200 μg/mL to CD 293TGE medium (Acrobiosystems; Cat. No. CM-1156-11) to 50 mL (solution A), with a concentration of 3.6 mg Add CD 293TGE medium to 50ml of PEI transfection reagent at 1mg/mL (solution B), mix them separately, slowly add solution B to solution A, mix well, let stand at room temperature for 10-15min, and then transfect The mixture was slowly added dropwise into 1000mL HEK293 cells. After culturing at 37° C., 5% CO 2 , and 135 rpm for 48 hours, the culture supernatant of R1242 was collected.
3.2 Claudin 18.2-eVLP在HEK293细胞中的表达3.2 Expression of Claudin 18.2-eVLP in HEK293 cells
使用PEI转染试剂将构建好的重组质粒按照比例R1303:R1221＝2:1、R1353:R1242＝2:1分别转染HEK293细胞(获得对应细胞ID分别为RG288(转染有R1303和R1221)和RG341(转染有R1353和R1242))，进行eVLP的表达和组装。转染前一天将HEK293细胞按照1×10^6个细胞/mL传代，37℃培养。转染当天进行计数，调整细胞密度为2×10^6个细胞/mL，活率95％以上。Using PEI transfection reagent, the constructed recombinant plasmids were transfected into HEK293 cells according to the ratios R1303:R1221=2:1 and R1353:R1242=2:1 (the corresponding cell IDs obtained were respectively RG288 (transfected with R1303 and R1221) and RG341 (transfected with R1353 and R1242)) for eVLP expression and assembly. The day before transfection, HEK293 cells were subcultured at 1×10^6 cells/mL and cultured at 37°C. Count the cells on the day of transfection, adjust the cell density to 2×10^6 cells/mL, and the viability rate is above 95%.
根据细胞密度计算转染复合物中各组分的用量：质粒剂量与细胞的数量对应关系为1×10^6个细胞对应0.6μg质粒，PEI剂量与质粒剂量的对应关系为：PEI的质量为DNA质量的3倍。Calculate the dosage of each component in the transfection complex according to the cell density: the corresponding relationship between the dose of plasmid and the number of cells is 1×10^6 cells corresponding to 0.6 μg of plasmid, and the corresponding relationship between the dose of PEI and the dose of plasmid is: the quality of PEI is 3 times the quality of DNA.
根据上述计算得到的组分用量配置转染复合物：将1.2mg浓度为200μg/mL的重组质粒加入CD 293TGE培养基至50mL(A液)，3.6mg浓度为1mg/mL的PEI转染试剂加入CD 293TGE培养基至50ml(B液)，分别混匀后，将B液缓慢加入至A液中，混匀室温静置10-15min后，将转染混合液缓慢滴加入1000mL HEK293细胞中。37℃，5％CO 2，135rpm培养48h后，分别收集RG288和RG341的培养上清。 Prepare the transfection complex according to the amount of components calculated above: add 1.2mg of recombinant plasmid with a concentration of 200μg/mL to CD 293TGE medium to 50mL (solution A), add 3.6mg of PEI transfection reagent with a concentration of 1mg/mL CD 293TGE medium to 50ml (solution B), mix well, slowly add solution B to solution A, mix well and let stand at room temperature for 10-15min, then slowly add the transfection mixture dropwise into 1000mL HEK293 cells. After culturing at 37° C., 5% CO 2 , and 135 rpm for 48 hours, the culture supernatants of RG288 and RG341 were collected respectively.
实施例4空白eVLP和Claudin 18.2-eVLP的分离纯化Example 4 Separation and Purification of Blank eVLP and Claudin 18.2-eVLP
将收获的R1242、RG288和RG341细胞上清液，分别进行2000rpm，4℃离心20min去除细胞碎片并用0.22μm滤膜过滤，进行蔗糖密度梯度离心，上清经30％蔗糖密度超速离心(2,6000rpm/min，4℃1.5h)，沉淀用PBS重悬，分别得到R1242、RG288和RG341eVLP粗纯样品。分别取样进行SDS-PAGE检测，结果如图2所示，其中泳道A为R1242粗纯样品，泳道B为RG288粗纯样品，泳道C为RG341粗纯样品。从电泳结果可以清晰的看到Gag(约55kDa)和Gag-GFP(约80kDa)的目标条带，证明上清经30％蔗糖垫密度超速离心后可以收获大量的VLP。为了去除残留的核酸，将R1242、RG288和RG341粗纯样品分别在室温下用酶浓度为200U/mL的Benzonase(ACROBiosystems)处理1小时。核酸去除后的样品分别用
Figure PCTCN2022107197-appb-000001
ExplorerTM 100低压液相色谱***(GE Healthcare)进行SEC实验，将Sepharose 6FF树脂装入XK 16/70色谱柱(GE Healthcare)，最终床体积为130mL。使用Blue Dextran 2000(HMW校准试剂盒，GE Healthcare)测定色谱柱的空体积。在eVLP分离之前，用3个柱体积的脱气Milli-Q超纯水清洗柱子，用1个柱体积的PBS，pH 7.4以2mL/min(60cm/h)的速度进行柱平衡。 The supernatants of the harvested R1242, RG288 and RG341 cells were centrifuged at 2000rpm at 4°C for 20min to remove cell debris and filtered with a 0.22μm filter membrane, then subjected to sucrose density gradient centrifugation, and the supernatant was subjected to 30% sucrose density ultracentrifugation (2,6000rpm /min, 1.5h at 4°C), and the precipitate was resuspended in PBS to obtain crude pure samples of R1242, RG288 and RG341eVLP respectively. Samples were taken for SDS-PAGE detection, and the results are shown in Figure 2, where lane A is the crude pure sample of R1242, lane B is the crude pure sample of RG288, and lane C is the crude pure sample of RG341. The target bands of Gag (about 55kDa) and Gag-GFP (about 80kDa) can be clearly seen from the electrophoresis results, which proves that a large number of VLPs can be harvested after the supernatant is ultracentrifuged at a density of 30% sucrose cushion. In order to remove residual nucleic acid, the crude pure samples of R1242, RG288 and RG341 were respectively treated with Benzonase (ACROBiosystems) at an enzyme concentration of 200 U/mL for 1 hour at room temperature. The samples after nucleic acid removal were used separately
Figure PCTCN2022107197-appb-000001
The ExplorerTM 100 low-pressure liquid chromatography system (GE Healthcare) was used for SEC experiments, and Sepharose 6FF resin was loaded into an XK 16/70 chromatographic column (GE Healthcare) with a final bed volume of 130 mL. The void volume of the column was determined using Blue Dextran 2000 (HMW calibration kit, GE Healthcare). Before eVLP separation, the column was washed with 3 column volumes of degassed Milli-Q ultrapure water, and the column was equilibrated with 1 column volume of PBS, pH 7.4 at a rate of 2 mL/min (60 cm/h).
将上述得到的R1242、RG288和RG341粗纯样品注入上样环中，使用PBS(pH 7.4)等速洗脱，流速为2mL/min(60cm/h)。在280nm和260nm处在线测定吸光度。在整个色谱过程中，使用Frac 950样品收集器(GE Healthcare)收集样品，并取样进行SDS-PAGE电泳分析，结果如图3所示，其中图A为R1242，图B为RG288，图C为RG341。根据电泳结果可以看出经粗纯样品SEC分离后，均可以获得高纯度样品(箭头所指)。根据电泳结果回收高纯度的样品，即为eVLP，样品ID分别记为R1242-210521F1、RG288-210503F1和RG341-210521F1。The crude pure samples of R1242, RG288 and RG341 obtained above were injected into the sample loop and eluted at a constant velocity with PBS (pH 7.4) at a flow rate of 2 mL/min (60 cm/h). Absorbance was measured online at 280 nm and 260 nm. During the entire chromatographic process, samples were collected using a Frac 950 sample collector (GE Healthcare), and samples were taken for SDS-PAGE electrophoresis analysis. The results are shown in Figure 3, where Figure A is R1242, Figure B is RG288, and Figure C is RG341 . According to the results of electrophoresis, it can be seen that after SEC separation of crude pure samples, high-purity samples (arrows) can be obtained. According to the results of electrophoresis, high-purity samples were recovered, namely eVLP, and the sample IDs were respectively recorded as R1242-210521F1, RG288-210503F1 and RG341-210521F1.
实施例5病毒样颗粒的形态学检测Morphological detection of embodiment 5 virus-like particles
为了进一步确认实施例4的方法能成功制备eVLP，对R1242-210521F1进行透射电镜形态学检测(委托北京中科百测技术服务有限公司)，结果见图4。可以看出R1242-210521F1在电镜下呈现为直径约150nm的空心球体。电镜照片结果从宏观上证明了实施例4的方法可以成功制备以HIV-1 Gag为骨架蛋白的eVLP。In order to further confirm that the method in Example 4 can successfully prepare eVLP, R1242-210521F1 was subjected to a transmission electron microscope morphology test (entrusted to Beijing Zhongke Baice Technology Service Co., Ltd.), and the results are shown in Figure 4. It can be seen that R1242-210521F1 appears as a hollow sphere with a diameter of about 150 nm under the electron microscope. The results of electron micrographs have proved macroscopically that the method of Example 4 can successfully prepare eVLP with HIV-1 Gag as the backbone protein.
实施例6 Claudin 18.2-eVLP与抗体IMAB362的活性检测Example 6 Activity detection of Claudin 18.2-eVLP and antibody IMAB362
本实施例中采用ELISA(酶联免疫吸附测定)方法检测本公开Claudin 18.2-eVLP与其特异抗体IMAB362的结合活性，以此证明Claudin 18.2四次跨膜蛋白以正确的构象成功展示在eVLP上。具体步骤如下：In this example, ELISA (enzyme-linked immunosorbent assay) method was used to detect the binding activity of Claudin 18.2-eVLP of the present disclosure and its specific antibody IMAB362, so as to prove that Claudin 18.2 four transmembrane proteins were successfully displayed on eVLP in the correct conformation. Specific steps are as follows:
1、包被：用0.5μg/孔(5μg/ml，100μl/孔)的RG288-210503F1或RG341-210521F1包被96孔板(Corning公司，货号：42592)，在4℃包被过夜(或16h)。RG288-210503F1或RG341-210521F1稀释所用包被缓冲液为15mM Na 2CO 3，35mM NaHCO 3，7.7mM NaN 3，pH9.6。 1. Coating: use 0.5 μg/well (5 μg/ml, 100 μl/well) of RG288-210503F1 or RG341-210521F1 to coat a 96-well plate (Corning Company, Cat. ). The coating buffer used for dilution of RG288-210503F1 or RG341-210521F1 is 15mM Na 2 CO 3 , 35mM NaHCO 3 , 7.7mM NaN 3 , pH 9.6.
2、洗涤：用每孔300μl的洗涤缓冲液(TBS，0.05％Tween-20，pH7.4)洗涤孔4次(注：彻底清除洗涤缓冲液很关键)。洗完之后通过抽吸除去残留的溶液，并确保其完全干燥。2. Washing: Wash the wells 4 times with 300 μl of washing buffer (TBS, 0.05% Tween-20, pH 7.4) per well (note: it is critical to completely remove the washing buffer). Remove residual solution by suction after washing and make sure it is completely dry.
3、封闭：在37℃条件下，每孔用300μl封闭缓冲液(TBS，2％BSA，pH7.4)封闭1.5h。3. Blocking: each well was blocked with 300 μl blocking buffer (TBS, 2% BSA, pH 7.4) for 1.5 h at 37° C.
4、洗涤：重复步骤2。4. Washing: Repeat step 2.
5、添加样品：每孔加入100μl 0.390625-50ng/mL IMAB362抗体，37℃孵育1h。样品提前用稀释缓冲液(含有0.5％BSA的TBS缓冲液，pH7.4)进行稀释。5. Add samples: add 100 μl 0.390625-50ng/mL IMAB362 antibody to each well, and incubate at 37°C for 1 hour. Samples were diluted in advance with dilution buffer (TBS buffer containing 0.5% BSA, pH 7.4).
6、洗涤：重复步骤2。6. Washing: Repeat step 2.
7、添加检测抗体：向每个孔中加入100μl anti-human IgG抗体(Jackson，货号：109-035-098)，37℃孵育1h。抗体提前用稀释缓冲液(含有0.5％BSA的TBS缓冲液，pH7.4)以1:20000比例进行稀释。7. Add detection antibody: add 100 μl anti-human IgG antibody (Jackson, catalog number: 109-035-098) to each well, and incubate at 37°C for 1 hour. The antibody was diluted with dilution buffer (TBS buffer containing 0.5% BSA, pH 7.4) at a ratio of 1:20000 in advance.
8、洗涤：重复步骤2。8. Washing: Repeat step 2.
9、添加底物：向各孔中加入200μl底物溶液，37℃孵育20min。避光。底物溶液配置：在10ml底物溶液(50mM Na 2HPO 4·12H 2O，25mM柠檬酸，pH5.5)中加入8μl 3％H 2O 2和100μl 10mg/ml TMB(BBI Life sciences，货号：A600954)。 9. Add substrate: add 200 μl substrate solution to each well, and incubate at 37° C. for 20 minutes. Avoid light. Substrate solution configuration: Add 8 μl 3% H 2 O 2 and 100 μl 10 mg/ml TMB (BBI Life sciences, Cat. No. : A600954).
10、终止反应：向各孔中加入1M硫酸50μl。10. Stop the reaction: add 50 μl of 1M sulfuric acid to each well.
11、读取OD值：在450nm处读取OD值，然后OD450-ODBlank为最终的OD值。其中ODBlank对应的孔为步骤5中不添加样品仅添加等体积稀释缓冲液的测定结果，作为空白对照。11. Read the OD value: read the OD value at 450nm, and then OD450-ODBlank is the final OD value. The well corresponding to ODBlank is the measurement result in step 5 where no sample is added but only an equal volume of dilution buffer is added, which is used as a blank control.
ELISA检测结果如图5所示，其中图A为RG288-210503F1和IMAB362结合的ELISA检测结果；图B为RG341-210521F1和IMAB362结合的ELISA检测结果。本公开Claudin 18.2-eVLP RG288-210503F1和RG341-210521F1与IMAB362结合的EC50值分别为1.64ng/mL和1.94ng/mL，表明本公开的Claudin 18.2-eVLP与IMAB362具有很好的结合活性，从而证明Claudin 18.2四次跨膜蛋白以正确的构象成功展示在eVLP上。The ELISA test results are shown in Figure 5, where Figure A is the ELISA test result for the combination of RG288-210503F1 and IMAB362; Figure B is the ELISA test result for the combination of RG341-210521F1 and IMAB362. The EC50 values of the combination of Claudin 18.2-eVLP RG288-210503F1 and RG341-210521F1 of the present disclosure with IMAB362 are 1.64ng/mL and 1.94ng/mL respectively, indicating that the Claudin 18.2-eVLP of the present disclosure has good binding activity to IMAB362, thus proving that Claudin 18.2 tetraspanin was successfully displayed on eVLP in the correct conformation.
实施例7 IMAB362 CAR-like(HEK293)细胞的准备Example 7 Preparation of IMAB362 CAR-like (HEK293) cells
本实施例中，模仿CAR-T细胞，制备IMAB362 CAR-like(HEK293)细胞，来验证Claudin 18.2-eVLP在评估CAR阳性表达率中的应用。In this example, imitating CAR-T cells, IMAB362 CAR-like (HEK293) cells were prepared to verify the application of Claudin 18.2-eVLP in evaluating the positive expression rate of CAR.
7.1IMAB362 CAR-like重组质粒的制备7.1 Preparation of IMAB362 CAR-like recombinant plasmid
IMAB362 CAR结构如图6所示。其对应的氨基酸序列为6，多核苷酸序列为SEQ ID No.11。质粒构建方法参考实施例1。The IMAB362 CAR structure is shown in Figure 6. The corresponding amino acid sequence is 6, and the polynucleotide sequence is SEQ ID No.11. Refer to Example 1 for the plasmid construction method.
7.2IMAB362 CAR-like(HEK293)细胞的制备7.2 Preparation of IMAB362 CAR-like (HEK293) cells
IMAB362 CAR重组质粒转染HEK293细胞的工艺参考实施例3。不同的是本实施例中需要通过G418抗性筛选出IMAB362 CAR-like(HEK293)单克隆细胞，标记ID为C633，用于CAR阳性表达率评估实验。Refer to Example 3 for the process of transfecting HEK293 cells with the IMAB362 CAR recombinant plasmid. The difference is that in this example, IMAB362 CAR-like (HEK293) monoclonal cells need to be screened out through G418 resistance, and the marker ID is C633, which is used in the CAR positive expression rate evaluation experiment.
实施例8 Claudin-18.2-eVLP用于CAR阳性表达率评估Example 8 Claudin-18.2-eVLP is used to evaluate the positive expression rate of CAR
本实施例中分别使用抗Fab抗体FITC-anti mFab Ab(Thermo Scientific,Cat.No.31543))、FITC-Protein L(ACROBiosystems,Cat.No.RPL-PF141)、Claudin 18.2胞外loop区蛋白：Claudin-18.2 Protein,His Tag TM(ACROBiosystems,Cat.No.CL2-H51H6)以及本公开的R1242-210521F1、RG288-210503F1和RG341-210521F1评估IMAB362 CAR表达情况。具体步骤如下： In this example, anti-Fab antibodies FITC-anti mFab Ab (Thermo Scientific, Cat. No. 31543)), FITC-Protein L (ACROBiosystems, Cat. No. RPL-PF141), and Claudin 18.2 extracellular loop region protein were used respectively: Claudin-18.2 Protein, His Tag TM (ACROBiosystems, Cat.No.CL2-H51H6) and R1242-210521F1, RG288-210503F1 and RG341-210521F1 of the present disclosure were used to evaluate the expression of IMAB362 CAR. Specific steps are as follows:
1、在含10％胎牛血清的DMEM培养基中培养C633细胞，置于CO 2培养箱中(37℃，5％CO 2)。 1. C633 cells were cultured in DMEM medium containing 10% fetal bovine serum, and placed in a CO 2 incubator (37° C., 5% CO 2 ).
2、收集细胞，用FACS缓冲液(2％BSA)清洗细胞一次。2. Collect the cells and wash the cells once with FACS buffer (2% BSA).
3、计数细胞数量和活率，将2×10^6活细胞放入每管中。3. Count the number of cells and viability, and put 2×10^6 viable cells into each tube.
4、将FITC-anti mFab Ab、FITC-Protein L、Claudin-18.2 Protein,His Tag TM、R1242-210521F1、RG288-210503F1和RG341-210521F1分别用FACS缓冲液(2％BSA)进行梯度稀释，然后将稀释后的样品溶液分别加入到含有细胞的管中。混合均匀，4℃孵育60min。(针对Claudin-18.2 Protein,His Tag TM，孵育后用FACS缓冲液(2％BSA)洗涤细胞1次，加入PE-anti His antibody(Biolegend,Cat.No.362603)，4℃孵育避光60min。) 4. FITC-anti mFab Ab, FITC-Protein L, Claudin-18.2 Protein, His Tag TM , R1242-210521F1, RG288-210503F1 and RG341-210521F1 were diluted with FACS buffer (2% BSA) respectively, and then The diluted sample solution was added separately to the tubes containing the cells. Mix well and incubate at 4°C for 60 min. (For Claudin-18.2 Protein, His Tag TM , wash the cells once with FACS buffer (2% BSA) after incubation, add PE-anti His antibody (Biolegend, Cat. No. 362603), and incubate at 4°C for 60 min in the dark. )
5、用FACS缓冲液(2％BSA)洗涤细胞3次，最后细胞样品用0.2mLPBS重悬。5. Wash the cells three times with FACS buffer (2% BSA), and finally resuspend the cell sample with 0.2 mL PBS.
6、将细胞悬液转入流管，并用流式细胞仪进行检测(激发波长488nm，发射波长530nm)。6. Transfer the cell suspension into a flow tube, and detect with a flow cytometer (excitation wavelength 488nm, emission wavelength 530nm).
7、使用FCS Express 6Plus和GraphPad Prism 5软件对结果数据进行分析。7. Use FCS Express 6Plus and GraphPad Prism 5 software to analyze the result data.
结果如图7所示，其中图A为当FITC-anti mFab Ab稀释200倍时，其评估IMAB362 CAR-like表达情况(阳性率为95.93％)；图B为当FITC-Protein L使用浓度为10μg/mL时，其评估IMAB362 CAR-like表达情况(阳性率为64.70％)；图C为当Claudin-18.2 Protein,His Tag TM使用浓度为10μg/mL时，其评估IMAB362 CAR-like表达情况(阳性率为1.17％)，说明仅表达Claudin 18.2的胞外loop区，无法识别IMAB362 CAR-like结构；图D为当RG288-210503F1使用浓度为10μg/mL时，其评估IMAB362 CAR-like表达情况(阳性率为99.52％)；图E为当RG341-210521F1使用浓度为10μg/mL时，其评估IMAB362 CAR-like表达情况(阳性率为99.60％)(R1242-210521F1为空白对照eVLP)。 The results are shown in Figure 7, where Figure A shows the evaluation of IMAB362 CAR-like expression when FITC-anti mFab Ab is diluted 200 times (positive rate is 95.93%); Figure B shows when FITC-Protein L is used at a concentration of 10 μg /mL, it evaluates the expression of IMAB362 CAR-like (positive rate is 64.70%); Figure C is when Claudin-18.2 Protein, His Tag TM is used at a concentration of 10 μg/mL, it evaluates the expression of IMAB362 CAR-like (positive rate is 64.70%); The ratio was 1.17%), indicating that only the extracellular loop region of Claudin 18.2 was expressed, and the IMAB362 CAR-like structure could not be recognized; Figure D shows the evaluation of IMAB362 CAR-like expression (positive The rate is 99.52%); Figure E shows the evaluation of IMAB362 CAR-like expression when RG341-210521F1 is used at a concentration of 10 μg/mL (positive rate is 99.60%) (R1242-210521F1 is the blank control eVLP).
从结果中可以看出，本公开Claudin18.2-eVLP上展示的Claudin18.2靶抗原构象正确，可以特异性的结合IMAB362 CAR-like，在通过FACS检测GFP的方法检测CAR阳性表达率情况时，表现出最佳效果。结果表明，本公开的含有绿色荧光蛋白GFP的Claudin 18.2-eVLP非常适用于评估CAR阳性表达率。It can be seen from the results that the conformation of the Claudin18.2 target antigen displayed on the Claudin18.2-eVLP of the present disclosure is correct and can specifically bind to IMAB362 CAR-like. When detecting the positive expression rate of CAR by FACS detection of GFP, show the best results. The results show that the Claudin 18.2-eVLP containing green fluorescent protein GFP of the present disclosure is very suitable for evaluating the positive expression rate of CAR.
实施例9 CD20-eVLP用于CAR阳性表达率评估Example 9 CD20-eVLP is used to evaluate the positive expression rate of CAR
为了验证本公开的技术方案可以应用于其他多次跨膜蛋白的制备，进而应用于其他靶向多次跨膜蛋白的CAR阳性率表达评估，本实施例针对另一种多次跨膜蛋白CD20，采用本公开的纳米颗粒，进行了CAR阳性表达率评估。具体步骤如下：In order to verify that the technical solution of the present disclosure can be applied to the preparation of other multi-spanning proteins, and then applied to the expression evaluation of CAR positive rate of other multi-spanning proteins, this example focuses on another multi-spanning protein CD20 , using the nanoparticles of the present disclosure, the positive expression rate of CAR was evaluated. Specific steps are as follows:
1、人CD20-eVLP的制备：根据实施例2的方法构建含有CD20编码基因的重组质粒，其中，CD20的氨基酸序列如SEQ ID No.15所示，其编码序列如SEQ ID No.16所示；根据实施例3的方法，将含有CD20编码基因的重组质粒与R1424质粒2：1混合转染HEK293细胞并收集培养上清；根据实施例4的方法分离纯化，制备得到的CD20-eVLP样品ID为RG344-210628F1；1. Preparation of human CD20-eVLP: according to the method of Example 2, a recombinant plasmid containing a gene encoding CD20 was constructed, wherein the amino acid sequence of CD20 is shown in SEQ ID No.15, and its coding sequence is shown in SEQ ID No.16 ;According to the method of Example 3, the recombinant plasmid containing the CD20 coding gene and the R1424 plasmid were mixed at 2:1 to transfect HEK293 cells and the culture supernatant was collected; the CD20-eVLP sample ID was prepared according to the method of Example 4 for separation and purification For RG344-210628F1;
2、Ofatumumab CAR-like(HEK293)细胞的准备，制备方法参考实施例7；其中，Ofatumumab CAR的氨基酸序列为SEQ ID No.17，多核苷酸序列为SEQ ID No.18；制备得到的Ofatumumab CAR-like(HEK293)单克隆细胞，标记ID为RC539b，用于CAR阳性表达率评估实验；2. Preparation of Ofatumumab CAR-like (HEK293) cells, the preparation method refers to Example 7; wherein, the amino acid sequence of Ofatumumab CAR is SEQ ID No.17, and the polynucleotide sequence is SEQ ID No.18; the prepared Ofatumumab CAR -like (HEK293) monoclonal cells, labeled ID is RC539b, used for CAR positive expression rate evaluation experiment;
3、CD20-eVLP用于CAR阳性表达率评估验证，具体方法参考实施例8。3. CD20-eVLP is used for evaluating and verifying the positive expression rate of CAR. For specific methods, refer to Example 8.
结果如图8所示，当RG344-210628F1使用浓度为20μg/mL时，其评估Ofatumumab CAR-like(HEK293)表达情况(阳性率为99.48％)(R1242-210521F1为空白对照eVLP)。The results are shown in Figure 8. When RG344-210628F1 was used at a concentration of 20 μg/mL, it evaluated the expression of Ofatumumab CAR-like (HEK293) (99.48% positive rate) (R1242-210521F1 was the blank control eVLP).
从结果中可以看出，本公开CD20-eVLP上展示的CD20靶抗原构象正确，可以特异性的结合Ofatumumab CAR-like细胞克隆，在通过FACS检测GFP的方法检测CAR阳性表达率情况时，表现出>99％的阳性率效果。该结果进一步表明本公开的技术手段可以广泛的适用于多次跨膜蛋白-eVLP的制备，进而应用于CAR阳性率表达评估。It can be seen from the results that the conformation of the CD20 target antigen displayed on the CD20-eVLP of the present disclosure is correct, and can specifically bind to Ofatumumab CAR-like cell clones. When the positive expression rate of CAR is detected by FACS detection of GFP, it shows >99% positive rate effect. This result further indicates that the technical means of the present disclosure can be widely applied to the preparation of multiple transmembrane protein-eVLP, and then applied to the evaluation of CAR positive rate expression.

Claims (10)

  1. 一种纳米颗粒，其包含：A nanoparticle comprising:
    (i)包膜病毒样颗粒(eVLP)骨架蛋白和/或其功能片段；和(i) enveloped virus-like particle (eVLP) backbone proteins and/or functional fragments thereof; and
    (ii)靶点蛋白和/或其功能片段，(ii) target protein and/or functional fragment thereof,
    其中，所述包膜病毒样颗粒骨架蛋白包含病毒核心蛋白或其功能片段，所述包膜病毒样颗粒骨架蛋白或其功能片段组装形成包膜病毒样颗粒，所述靶点蛋白和/或其功能片段展示在包膜病毒样颗粒上，形成靶点蛋白-eVLP纳米颗粒；Wherein, the enveloped virus-like particle skeleton protein comprises a viral core protein or a functional fragment thereof, and the enveloped virus-like particle skeleton protein or a functional fragment thereof assembles to form an enveloped virus-like particle, and the target protein and/or its Functional fragments are displayed on enveloped virus-like particles to form target protein-eVLP nanoparticles;
    优选地，所述靶点蛋白是CAR靶点蛋白；所述CAR靶点蛋白和/或其功能片段展示在包膜病毒样颗粒上，形成CAR靶点蛋白-eVLP纳米颗粒。Preferably, the target protein is a CAR target protein; the CAR target protein and/or its functional fragments are displayed on enveloped virus-like particles to form CAR target protein-eVLP nanoparticles.
  2. 权利要求1的纳米颗粒，其中，所述的病毒核心蛋白选自：逆转录病毒Gag蛋白、杆状病毒基质蛋白M蛋白、线状病毒核心蛋白、冠状病毒M、E和NP蛋白、流感病毒M1蛋白、副粘病毒M蛋白、呼吸道合胞病毒(RSV)M蛋白、沙粒病毒Z蛋白、新城疫病毒M蛋白、副流感病毒M蛋白、布尼亚病毒N蛋白、丙型肝炎病毒核心蛋白C、乙型肝炎病毒核心蛋白C和其组合物；The nanoparticle of claim 1, wherein said virus core protein is selected from: retrovirus Gag protein, baculovirus matrix protein M protein, filovirus core protein, coronavirus M, E and NP proteins, influenza virus M1 protein, paramyxovirus M protein, respiratory syncytial virus (RSV) M protein, arenavirus Z protein, Newcastle disease virus M protein, parainfluenza virus M protein, bunya virus N protein, hepatitis C virus core protein C , hepatitis B virus core protein C and compositions thereof;
    优选地，所述逆转录病毒Gag蛋白选自：人类免疫缺陷病毒Gag蛋白、猴免疫缺陷病毒Gag蛋白、小鼠白血病病毒Gag蛋白和牛白血病病毒Gag蛋白；更优选地，所述人类免疫缺陷病毒Gag蛋白包含与SEQ ID No.1具有至少80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列：更优选地，所述人类免疫缺陷病毒Gag蛋白的氨基酸序如SEQ ID No.1所示；Preferably, the retrovirus Gag protein is selected from: human immunodeficiency virus Gag protein, simian immunodeficiency virus Gag protein, mouse leukemia virus Gag protein and bovine leukemia virus Gag protein; more preferably, the human immunodeficiency virus Gag protein The protein comprises an amino acid sequence with at least 80% or more identity to SEQ ID No.1, preferably an amino acid sequence with more than 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, more An amino acid sequence preferably having an identity of 98% or more than 99%: more preferably, the amino acid sequence of the human immunodeficiency virus Gag protein is shown in SEQ ID No.1;
    优选地，所述杆状病毒基质蛋白M蛋白为水疱性口炎病毒M病毒核心蛋白；Preferably, the baculovirus matrix protein M protein is vesicular stomatitis virus M virus core protein;
    优选地，所述线状病毒核心蛋白为埃博拉病毒VP40病毒核心蛋白；Preferably, the filovirus core protein is Ebola virus VP40 virus core protein;
    优选地，所述冠状病毒核心蛋白选自新型冠状肺炎病毒(SARS-CoV-2)、SARS-CoV、MERS-Cov、229E、NL63、OC43和HKU1的核心蛋白。Preferably, the coronavirus core protein is selected from core proteins of novel coronavirus pneumonia virus (SARS-CoV-2), SARS-CoV, MERS-Cov, 229E, NL63, OC43 and HKU1.
  3. 权利要求1或2的纳米颗粒，其中，所述CAR靶点蛋白为膜蛋白，The nanoparticle of claim 1 or 2, wherein the CAR target protein is a membrane protein,
    优选地，所述膜蛋白包括跨膜蛋白；更优选地，所述跨膜蛋白包括多次跨膜蛋白；Preferably, the membrane protein comprises a transmembrane protein; more preferably, the transmembrane protein comprises a multispanning protein;
    优选地，所述CAR靶点蛋白选自CD20、Claudin18.1、Claudin18.2、CD133、GPRC5D、CCR5、CCR8、BCMA、GPCR、CD147、CD19、CD123、CD138、CD22、CD30、CD33、CD38、CD70、CAIX、EGFR、EGFRVIII、FOLR1、GPC3、HER2、HGFR、Anti-FMC63 Ab、CLL-1、SLAMF7、CD4、CD5、CD8A&CD8B、FAP、IL13RA2、GPC3、GUCY2C、Her3、PSMA、ROR1、SLAMF7、B7-H3、CD147、CEA、MUC16、Nectin-4、VEGFR2、Anti-RTX Ab、B7-H3、CAIX、CD7、CEA、MUC1、NKG2D、PSCA、uPAR、GD2、FR、PMEL、CA9、CD171/L1-CAM、IL-13Rα2、MART-1、ERBB2、ERBB3、ERBB4、NY-ESO-1、MAGE家族蛋白、BAGE家族蛋白、GAGE家族蛋白、AFP、CD44v7/8、IL-11Rα、EGP-2、EGP-40、FBP、GD3、FSA、PSA、HMGA2、胎儿型乙酰胆碱受体、LeY、EpCAM、间皮素、IGFR1、CA125、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、SCC、AFU、EBV-VCA、POA、β2-MG、PROGRP或MSLN中任一种；Preferably, the CAR target protein is selected from CD20, Claudin18.1, Claudin18.2, CD133, GPRC5D, CCR5, CCR8, BCMA, GPCR, CD147, CD19, CD123, CD138, CD22, CD30, CD33, CD38, CD70 , CAIX, EGFR, EGFRVIII, FOLR1, GPC3, HER2, HGFR, Anti-FMC63 Ab, CLL-1, SLAMF7, CD4, CD5, CD8A&CD8B, FAP, IL13RA2, GPC3, GUCY2C, Her3, PSMA, ROR1, SLAMF7, B7- H3, CD147, CEA, MUC16, Nectin-4, VEGFR2, Anti-RTX Ab, B7-H3, CAIX, CD7, CEA, MUC1, NKG2D, PSCA, uPAR, GD2, FR, PMEL, CA9, CD171/L1-CAM , IL-13Rα2, MART-1, ERBB2, ERBB3, ERBB4, NY-ESO-1, MAGE family protein, BAGE family protein, GAGE family protein, AFP, CD44v7/8, IL-11Rα, EGP-2, EGP-40 , FBP, GD3, FSA, PSA, HMGA2, fetal acetylcholine receptor, LeY, EpCAM, mesothelin, IGFR1, CA125, CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC , AFU, EBV-VCA, POA, β2-MG, PROGRP or MSLN;
    优选地，所述CAR靶点蛋白选自CD20、Claudin18.2、CD133、GPRC5D、CCR5、CCR8、CD19、BCMA、GPC3、CD30、CD22、EGFR、EGFRVIII、HER2或GPCR中任一种；Preferably, the CAR target protein is selected from any one of CD20, Claudin18.2, CD133, GPRC5D, CCR5, CCR8, CD19, BCMA, GPC3, CD30, CD22, EGFR, EGFRVIII, HER2 or GPCR;
    更优先地，所述CAR靶点蛋白为Claudin18.2；优选地，所述Claudin18.2的氨基酸序列包含与SEQ ID No.4具有至少80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列：更优选地，所述Claudin18.2的氨基酸序列如SEQ ID No.4所示；More preferably, the CAR target protein is Claudin18.2; preferably, the amino acid sequence of the Claudin18.2 comprises an amino acid sequence having at least 80% or more identity with SEQ ID No.4, preferably having 85%, Amino acid sequences with 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably amino acid sequences with 98% or more 99% identity: more preferably, the amino acid sequence of Claudin18.2 The sequence is shown in SEQ ID No.4;
    更优先地，所述CAR靶点蛋白为CD20；优选地，所述CD20的氨基酸序列包含与SEQ ID No.15具有至少80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列：更优选地，所述Claudin18.2的氨基酸序列如SEQ ID No.15所示。More preferably, the CAR target protein is CD20; preferably, the amino acid sequence of the CD20 comprises an amino acid sequence having at least 80% or more identity with SEQ ID No.15, preferably having 85%, 90%, 95% %, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences: more preferably, the amino acid sequence of Claudin18.2 is as SEQ ID Shown in No.15.
  4. 权利要求1-3任一项所述的纳米颗粒，其中，所述CAR靶点蛋白和/或其功能片段和/或所述病毒核心蛋白和/或其功能片段与标记物相连接；The nanoparticle according to any one of claims 1-3, wherein the CAR target protein and/or its functional fragment and/or the viral core protein and/or its functional fragment are linked to a marker;
    优选地，所述CAR靶点蛋白和/或其功能片段的N末端和/或C末端和/或病毒核心蛋白和/或其功能片段的N末端和/或C末端与标记物相连接；更优选地，在Claudin 18.2的C末端或Gag的C末端用标记物进行标记；更优选地，在CD20的C末端或Gag的C末端用标记物进行标记；Preferably, the N-terminal and/or C-terminal of the CAR target protein and/or its functional fragment and/or the N-terminal and/or C-terminal of the viral core protein and/or its functional fragment are connected to a label; more Preferably, the C-terminal of Claudin 18.2 or the C-terminal of Gag is labeled with a label; more preferably, the C-terminal of CD20 or the C-terminal of Gag is labeled with a label;
    优选地，所述标记物选自可检测标记物、纯化标签、报告标签和其组合物；Preferably, said label is selected from detectable labels, purification tags, reporter tags and combinations thereof;
    优选地，所述可检测标记物选自荧光基团、化学发光标记物、电化学发光标记物和其组合物；Preferably, the detectable label is selected from the group consisting of fluorescent groups, chemiluminescent labels, electrochemiluminescent labels and combinations thereof;
    优选地，所述荧光基团选自FITC、GFP、RFP、YFP、TRITC、PE、FAM、RRX、TR、Cy2、Cy3、Cy5、ECD、PC5.5、PC7、APC、APC-A70、APC-A75、Pac-Blue、Alexa488、mBBr、5-IAF、E-118、DTAF、罗丹明绿和KrO中的任一种；Preferably, the fluorescent group is selected from FITC, GFP, RFP, YFP, TRITC, PE, FAM, RRX, TR, Cy2, Cy3, Cy5, ECD, PC5.5, PC7, APC, APC-A70, APC- Any of A75, Pac-Blue, Alexa488, mBBr, 5-IAF, E-118, DTAF, rhodamine green and KrO;
    优选地，所述化学发光标记物为吖啶酯、异鲁米诺、辣根过氧化物酶或碱性磷酸酶；Preferably, the chemiluminescence marker is acridinium ester, isoluminol, horseradish peroxidase or alkaline phosphatase;
    优选地，所述电化学发光标记物为三联吡啶钌或其衍生N-羟基琥珀酰胺酯；Preferably, the electrochemiluminescent marker is ruthenium terpyridyl or its derivative N-hydroxysuccinamide ester;
    优选地，所述纯化标签选自HIS-Tag、GST-Tag、MBP-Tag、NusA-Tag、FLAG-Tag、SUMO、Avi-Tag、Halo-Tag和SNAP-Tag中的任一种；Preferably, the purification tag is selected from any one of HIS-Tag, GST-Tag, MBP-Tag, NusA-Tag, FLAG-Tag, SUMO, Avi-Tag, Halo-Tag and SNAP-Tag;
    优选地，所述纯化标签通过二抗检测；Preferably, the purification tag is detected by a secondary antibody;
    优选地，所述报告标签选自c-Myc、HA或荧光素酶中的任一种。Preferably, the reporter tag is selected from any one of c-Myc, HA or luciferase.
  5. 权利要求1-4任一项所述的纳米颗粒，其中，所述CAR靶点蛋白-eVLP纳米颗粒选自Claudin 18.2-eVLP纳米颗粒或CD20-eVLP纳米颗粒；The nanoparticle according to any one of claims 1-4, wherein the CAR target protein-eVLP nanoparticle is selected from Claudin 18.2-eVLP nanoparticles or CD20-eVLP nanoparticles;
    优选地，所述Claudin 18.2和/或其功能片段和/或所述病毒核心蛋白和/或其功能片段与标记物相连接；Preferably, the Claudin 18.2 and/or its functional fragments and/or the viral core protein and/or its functional fragments are linked to a marker;
    优选地，所述CD20和/或其功能片段和/或所述病毒核心蛋白和/或其功能片段与标记物相连接；Preferably, the CD20 and/or its functional fragments and/or the viral core protein and/or its functional fragments are linked to a marker;
    优选地，所述Claudin 18.2和/或其功能片段的N末端和/或C末端和/或病毒核心蛋白和/或其功能片段的N末端和/或C末端与标记物相连接；Preferably, the N-terminal and/or C-terminal of the Claudin 18.2 and/or its functional fragments and/or the N-terminal and/or C-terminal of the viral core protein and/or its functional fragments are linked to a marker;
    优选地，所述CD20和/或其功能片段的N末端和/或C末端和/或病毒核心蛋白和/或其功能片段的N末端和/或C末端与标记物相连接；Preferably, the N-terminal and/or C-terminal of the CD20 and/or its functional fragments and/or the N-terminal and/or C-terminal of the viral core protein and/or its functional fragments are connected to a marker;
    优选地，所述包膜病毒样颗粒骨架蛋白为Gag蛋白；Preferably, the enveloped virus-like particle skeleton protein is a Gag protein;
    优选地，所述Claudin 18.2的C末端或所述Gag蛋白的C末端与标记物相连接；Preferably, the C-terminal of the Claudin 18.2 or the C-terminal of the Gag protein is connected to a marker;
    优选地，所述CD20的C末端或所述Gag蛋白的C末端与标记物相连接；Preferably, the C-terminal of the CD20 or the C-terminal of the Gag protein is connected to a label;
    优选地，所述标记物为GPF；Preferably, the marker is GPF;
    优选地，所述GFP的氨基酸序列包含与SEQ ID No.2具有至少80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列：更优选地，所述GFP的氨基酸序列如SEQ ID No.2所示；Preferably, the amino acid sequence of the GFP comprises an amino acid sequence with at least 80% or more identity to SEQ ID No.2, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% An amino acid sequence with the above identity, more preferably an amino acid sequence with an identity of 98% or more than 99%: more preferably, the amino acid sequence of the GFP is shown in SEQ ID No.2;
    优选地，所述Gag蛋白的C末端与标记物GFP相连(Gag-GFP)，其氨基酸序列包含与SEQ ID No.3具有至少80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列：更优选地，所述Gag-GFP的氨基酸序列如SEQ ID No.3所示；Preferably, the C-terminus of the Gag protein is connected to the marker GFP (Gag-GFP), and its amino acid sequence comprises an amino acid sequence having at least 80% or more identity with SEQ ID No.3, preferably 85%, 90% , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences: more preferably, the amino acid sequence of Gag-GFP is as follows Shown in SEQ ID No.3;
    优选地，所述Claudin 18.2的C末端与标记物GFP相连(Claudin 18.2-GFP)，其氨基酸序列与SEQ ID No.5具有至少80％或以上同一性的氨基酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的氨基酸序列，更优选具有98％或99％以上同一性的氨基酸序列：更优选地，所述Claudin 18.2-GFP的氨基酸序列如SEQ ID No.5所示。Preferably, the C-terminus of the Claudin 18.2 is connected to the marker GFP (Claudin 18.2-GFP), and its amino acid sequence has at least 80% or more of the same amino acid sequence as SEQ ID No.5, preferably 85%, 90% , 95%, 96%, 97%, 98%, 99% or more identical amino acid sequences, more preferably 98% or 99% or more identical amino acid sequences: more preferably, the amino acid sequence of the Claudin 18.2-GFP As shown in SEQ ID No.5.
  6. 权利要求1-5任一项所述的纳米颗粒的制备方法，其包括：The preparation method of the nanoparticle described in any one of claim 1-5, it comprises:
    (1)重组质粒的构建：(1) Construction of recombinant plasmids:
    分别构建包含包膜病毒样颗粒(eVLP)骨架蛋白和/或其功能片段基因的重组质粒和靶点蛋白和/或其功能片段基因的重组质粒；或，构建同时包含包膜病毒样颗粒(eVLP)骨架蛋白和/或其功能片段基因和靶点蛋白和/或其功能片段基因的重组质粒；和Respectively constructing a recombinant plasmid comprising enveloped virus-like particle (eVLP) skeleton protein and/or its functional fragment gene and a recombinant plasmid of target protein and/or its functional fragment gene; ) a recombinant plasmid of the backbone protein and/or its functional fragment gene and the target protein and/or its functional fragment gene; and
    (2)细胞转染、蛋白表达和颗粒组装：(2) Cell transfection, protein expression and particle assembly:
    将步骤(1)构建的包膜病毒样颗粒(eVLP)骨架蛋白和/或其功能片段重组质粒和靶点蛋白和/或其功能片段重组质粒转染细胞，表达包膜病毒样颗粒骨架蛋白和/或其功能片段和靶点蛋白和/或其功能片段；所述包膜病毒样颗粒骨架蛋白或其功能片段组装形成包膜病毒样颗粒，所述靶点蛋白和/或其功能片段展示在包膜病毒样颗粒上，形成靶点蛋白-eVLP纳米颗粒；以及，任选地，The enveloped virus-like particle (eVLP) skeleton protein and/or its functional fragment recombinant plasmid and target protein and/or its functional fragment recombinant plasmid transfection cell with the enveloped virus-like particle (eVLP) skeleton protein and/or its functional fragment constructed in step (1), express enveloped virus-like particle skeleton protein and / or functional fragments thereof and target protein and/or functional fragments thereof; the enveloped virus-like particle skeleton protein or functional fragments thereof are assembled to form enveloped virus-like particles, and the target protein and/or functional fragments thereof are displayed on On the enveloped virus-like particle, forming target protein-eVLP nanoparticles; and, optionally,
    (3)纯化靶点蛋白-eVLP纳米颗粒；(3) purifying target protein-eVLP nanoparticles;
    优选地，所述GAG的编码核酸序列包含与SEQ ID No.7具有至少80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的核苷酸序列：更优选地，所述GFP的氨基酸序列如SEQ ID No.7所示；Preferably, the coding nucleic acid sequence of the GAG comprises a nucleotide sequence with at least 80% or more identity to SEQ ID No.7, preferably 85%, 90%, 95%, 96%, 97%, 98% 1. A nucleotide sequence with more than 99% identity, more preferably a nucleotide sequence with 98% or more identity: more preferably, the amino acid sequence of the GFP is shown in SEQ ID No.7;
    优选地，所述Gag-GFP编码核酸序列包含与SEQ ID No.8具有至少80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的核苷酸序列：更优选地，所述GFP的氨基酸序列如SEQ ID No.8所示；Preferably, the Gag-GFP encoding nucleic acid sequence comprises a nucleotide sequence with at least 80% or more identity to SEQ ID No.8, preferably 85%, 90%, 95%, 96%, 97%, 98% %, more than 99% identical nucleotide sequence, more preferably 98% or above 99% identical nucleotide sequence: more preferably, the amino acid sequence of the GFP is shown in SEQ ID No.8;
    优选地，所述Claudin 18.2的编码核酸序列包含与SEQ ID No.9具有至少80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的核苷酸序列：更优选地，所述GFP的氨基酸序列如SEQ ID No.9所示；Preferably, the coding nucleic acid sequence of Claudin 18.2 comprises a nucleotide sequence with at least 80% or more identity to SEQ ID No.9, preferably 85%, 90%, 95%, 96%, 97%, 98% %, more than 99% identical nucleotide sequence, more preferably 98% or more than 99% identical nucleotide sequence: more preferably, the amino acid sequence of the GFP is shown in SEQ ID No.9;
    优选地，所述Claudin 18.2-GFP的编码核酸序列包含与SEQ ID No.10具有至少80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的核苷酸序列：更优选地，所述GFP的氨基酸序列如SEQ ID No.10所示；Preferably, the nucleic acid sequence encoding Claudin 18.2-GFP comprises a nucleotide sequence with at least 80% or more identity to SEQ ID No.10, preferably 85%, 90%, 95%, 96%, 97% , 98%, 99% or more identical nucleotide sequences, more preferably 98% or 99% or more identical nucleotide sequences: more preferably, the amino acid sequence of the GFP is shown in SEQ ID No.10 ;
    优选地，所述CD20的编码核酸序列包含与SEQ ID No.16具有至少80％或以上同一性的核苷酸序列，优选具有85％、90％、95％、96％、97％、98％、99％以上同一性的核苷酸序列，更优选具有98％或99％以上同一性的核苷酸序列：更优选地，所述GFP的氨基酸序列如SEQ ID No.16所示。Preferably, the nucleic acid sequence encoding CD20 comprises a nucleotide sequence with at least 80% or more identity to SEQ ID No.16, preferably 85%, 90%, 95%, 96%, 97%, 98% , A nucleotide sequence with more than 99% identity, more preferably a nucleotide sequence with 98% or more identity: more preferably, the amino acid sequence of the GFP is shown in SEQ ID No.16.
  7. 权利要求6的制备方法，所述的细胞选自原核宿主细胞或真核宿主细胞；The preparation method according to claim 6, wherein said cells are selected from prokaryotic host cells or eukaryotic host cells;
    优选地，所述原核宿主细胞为细菌细胞、枯草芽孢杆菌和分枝杆菌中的任一种；优选地，所述细菌细胞为大肠杆菌；Preferably, the prokaryotic host cell is any one of bacterial cells, Bacillus subtilis and mycobacteria; preferably, the bacterial cells are Escherichia coli;
    优选地，所述真核宿主细胞选自动物细胞、植物细胞或真菌中的任一种；Preferably, the eukaryotic host cell is selected from any one of animal cells, plant cells or fungi;
    优选地，所述真核宿主细胞选自酵母、昆虫、禽类、植物、秀丽隐杆线虫和哺乳动物宿主细胞中的任一种；Preferably, the eukaryotic host cell is selected from any one of yeast, insect, avian, plant, Caenorhabditis elegans and mammalian host cells;
    优选地，所述昆虫细胞选自草地夜蛾细胞(例如Sf9、Sf21)、粉纹夜蛾细胞(例如High Five细胞)和果蝇S2细胞中的任一种；Preferably, the insect cell is selected from any one of Spodoptera frugiperda cells (such as Sf9, Sf21), Trichoplusia cells (such as High Five cells) and Drosophila S2 cells;
    优选地，所述真菌选自酿酒酵母、乳克鲁维酵母、假丝酵母属物种(例如白色假丝酵母或光滑假丝酵母)、构巢曲霉、粟酒裂殖酵母、巴斯德毕赤酵母、和解脂耶罗酵母中的任一种；Preferably, the fungus is selected from Saccharomyces cerevisiae, Kluyveromyces lactis, Candida species (such as Candida albicans or Candida glabrata), Aspergillus nidulans, Schizosaccharomyces pombe, Pichia pastoris Any of yeast, and Yarrowia lipolytica;
    优选地，所述哺乳动物细胞选自COS细胞、小鼠L细胞、LNCaP细胞、中国仓鼠卵巢(CHO)细胞、人胚肾(HEK)细胞(例如HEK293细胞)、633细胞、Vero、BHK细胞、非洲绿猴细胞、CV1细胞、HeLa细胞、MDCK细胞和Hep-2细胞中的任一种。Preferably, the mammalian cells are selected from COS cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells (such as HEK293 cells), 633 cells, Vero, BHK cells, Any of Vero cells, CV1 cells, HeLa cells, MDCK cells, and Hep-2 cells.
  8. 一种检测CAR阳性表达率的方法，其包括：将权利要求1-5任一项所述的纳米颗粒或权利要求6或7所述方法制备的纳米颗粒与CAR修饰的细胞孵育，然后进行检测；优选地，在细胞孵育后、进行检测前洗涤细胞；A method for detecting the positive expression rate of CAR, comprising: incubating the nanoparticle according to any one of claims 1-5 or the nanoparticle prepared by the method according to claim 6 or 7 with CAR-modified cells, and then detecting ; preferably, washing the cells after incubation of the cells and prior to detection;
    优选地，所述CAR修饰的细胞选自CAR-like(HEK293)细胞、CAR-T细胞、CAR-NK细胞、CAR-M细胞、CAR-NKT细胞、CAR-Treg细胞及CAR-γδT细胞中的任一种；Preferably, the CAR-modified cells are selected from CAR-like (HEK293) cells, CAR-T cells, CAR-NK cells, CAR-M cells, CAR-NKT cells, CAR-Treg cells, and CAR-γδT cells. any;
    优选地，所述孵育的温度为2℃-40℃，优选37℃，和/或，所述孵育的时间为15min-2小时，优选1小时；和/或，所述孵育的CO 2的浓度为2％-8％，优选5％； Preferably, the incubation temperature is 2°C-40°C, preferably 37°C, and/or, the incubation time is 15min-2 hours, preferably 1 hour; and/or, the incubation CO 2 concentration 2%-8%, preferably 5%;
    优选地，所述洗涤的次数为1-6次，优选3次；Preferably, the number of washings is 1-6 times, preferably 3 times;
    优选地，所述洗涤缓冲液为0.5％-5％BSA，优选2％BSA；Preferably, the washing buffer is 0.5%-5% BSA, preferably 2% BSA;
    优选地，所述检测选自流式细胞术检测、免疫检测、ELISA、SPR、BLI中的任一种。Preferably, the detection is selected from any one of flow cytometry detection, immunoassay, ELISA, SPR, BLI.
  9. 权利要求8所述的方法，其包括将Claudin 18.2-eVLP纳米颗粒或CD20-eVLP纳米颗粒与CAR修饰的细胞孵育，然后进行检测；The method of claim 8, comprising incubating Claudin 18.2-eVLP nanoparticles or CD20-eVLP nanoparticles with CAR-modified cells, and then detecting;
    优选地，所述包膜病毒样颗粒骨架蛋白为Gag蛋白；Preferably, the enveloped virus-like particle skeleton protein is a Gag protein;
    优选地，所述Claudin 18.2的C末端或所述Gag蛋白的C末端与标记物相连接；Preferably, the C-terminal of the Claudin 18.2 or the C-terminal of the Gag protein is connected to a marker;
    优选地，所述CD20的C末端或所述Gag蛋白的C末端与标记物相连接。Preferably, the C-terminal of the CD20 or the C-terminal of the Gag protein is connected with a label.
  10. 一种试剂盒，其包含权利要求1-5任一项所述的纳米颗粒或权利要求6或7所述方法制备的纳米颗粒；A kit comprising the nanoparticle according to any one of claims 1-5 or the nanoparticle prepared by the method according to claim 6 or 7;
    优选地，所述纳米颗粒是CAR靶点蛋白-eVLP纳米颗粒；Preferably, the nanoparticles are CAR target protein-eVLP nanoparticles;
    优选地，所述试剂盒包含带标记物的CAR靶点蛋白-eVLP纳米颗粒；Preferably, the kit comprises labeled CAR target protein-eVLP nanoparticles;
    优选地，所述试剂盒包含带标记物的Claudin 18.2-eVLP纳米颗粒；Preferably, the kit comprises labeled Claudin 18.2-eVLP nanoparticles;
    优选地，所述试剂盒包含带标记物的CD20-eVLP纳米颗粒；Preferably, the kit comprises labeled CD20-eVLP nanoparticles;
    优选地，所述试剂盒为ELISA试剂盒、SPR试剂盒或BLI试剂盒；Preferably, the kit is an ELISA kit, an SPR kit or a BLI kit;
    优选地，所述ELISA试剂盒包含所述CAR靶点蛋白-eVLP、酶标板、封闭液、样品稀释液、酶结合物、浓缩洗涤液、酶底物溶液和终止液；Preferably, the ELISA kit comprises the CAR target protein-eVLP, microtiter plate, blocking solution, sample diluent, enzyme conjugate, concentrated washing solution, enzyme substrate solution and stop solution;
    优选地，所述SPR试剂盒包含所述CAR靶点蛋白-eVLP纳米颗粒；Preferably, the SPR kit comprises the CAR target protein-eVLP nanoparticles;
    优选地，所述BLI试剂盒包含所述CAR靶点蛋白-eVLP纳米颗粒。Preferably, the BLI kit comprises the CAR target protein-eVLP nanoparticles.
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