WO2023001256A1 - Nanoparticule et utilisation correspondante dans la détection du taux d'expression car-positif - Google Patents

Nanoparticule et utilisation correspondante dans la détection du taux d'expression car-positif 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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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/zh
Publication of WO2023001256A1 publication Critical patent/WO2023001256A1/fr

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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

L'invention concerne une nanoparticule et l'utilisation correspondante dans la détection d'un taux d'expression CAR-positif. La nanoparticule comprend : (i) une protéine de squelette de particule enveloppée de type virus (eVLP) et/ou un fragment fonctionnel correspondant ; et (ii) une protéine cible et/ou un fragment fonctionnel correspondant. La nanoparticule peut être utilisée pour détecter un taux d'expression CAR-positif et peut également être utilisée pour prouver qu'un CAR peut se lier spécifiquement à une protéine cible.
PCT/CN2022/107197 2021-07-22 2022-07-21 Nanoparticule et utilisation correspondante dans la détection du taux d'expression car-positif WO2023001256A1 (fr)

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