CN116981684A

CN116981684A - Nanoparticle and application thereof in detection of CAR positive expression rate

Info

Publication number: CN116981684A
Application number: CN202280020744.2A
Authority: CN
Inventors: 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
Original assignee: Beijing Baipusai Biotechnology Co ltd
Current assignee: Beijing Baipusai Biotechnology Co ltd
Priority date: 2021-07-22
Filing date: 2022-07-21
Publication date: 2023-10-31
Also published as: WO2023001256A1

Abstract

A nanoparticle comprising (i) an enveloped virus-like particle (ekp) scaffold protein and/or a functional fragment thereof, and the use thereof to detect CAR positive expression rate; and (ii) a target protein and/or a functional fragment thereof. The nanoparticle can be used to detect the positive expression rate of the CAR, and can also be used to demonstrate that the CAR can specifically bind to a target protein.

Description

Nanoparticle and application thereof in detection of CAR positive expression rate

Technical Field

The disclosure relates to the field of biotechnology, in particular to a nanoparticle and application thereof in detecting positive expression rate of CAR.

Background

In the immune system, T cells need to kill abnormal cells by means of Antigen Presenting Cells (APCs). The APC surface has a number of MHC (major histocompatibility complex) molecules that recognize cell surface antigens and, after the APC recognizes abnormal cells, the MHC binds to the T Cell Receptor (TCR) and signals T cells. With the help of molecules such as CD3, CD4 and CD8, T cells can eventually recognize and kill abnormal cells. However, tumor cells destroy their own "fingerprints" and thus cannot be recognized by APC cells.

Chimeric antigen receptor (Chimeric Antigen Receptor, CAR) is an artificial receptor molecule made by genetic engineering techniques that confers specificity to immune effector cells (e.g., T lymphocytes) against a target epitope, thereby enhancing the T lymphocyte recognition antigen signal and activation function (Sadelain, m., r.brentjens, and i.rivere.the promise and potential pitfalls of chimeric antigen receptors.curr Opin Immunol,2009.21 (2): p.215-23.). CAR-T cells recognize specific antigens of interest in an MHC non-limiting manner and continue to activate expanded T cells. CAR-T cell therapy is essentially an Adoptive Cell Transfer (ACT), which was originally proposed as a process in which donor lymphocytes are infused into recipients during organ transplantation, which antigen mediates the development of graft rejection, and later gradually evolves into therapy for the treatment of 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.).

For CAR-T cells, the active ingredient that exerts a tumor killing effect is T cells with a positive expression rate of CAR. The packaging specification and clinical dosage of the CAR-T cell product are expressed by the number of CAR-T positive cells, and the detection of the CAR positive expression rate is an important quality control step in the CAR-T cell production process.

In the prior art, the positive expression rate of the CAR is detected mainly by Anti-Fab antibody or Protein L Protein of a light chain or a hinge region, but the method has the defect of non-specific binding, and cannot prove whether the CAR can bind to a target antigen. Therefore, the target protein has the advantage of strong specificity for detecting the positive expression rate of the CAR and is favored by the industry. However, most target proteins are difficult to prepare, and the resulting target proteins may also adversely affect the detection of the positive expression rate of the CAR. For example, due to the hydrophobic nature of the transmembrane region of the target protein, a detergent must be introduced during purification to solubilize the target protein, resulting in detergent content in the prepared target protein, and the presence of detergent can result in membrane lysis of T cells, which makes the target protein in a detergent system unusable for detection of CAR positive rate. In addition, the extracellular loop region of the target protein is used for detecting the positive expression rate of the CAR, and although the preparation difficulty of the target protein is low, the conformation of the target protein cannot be ensured to be consistent with the natural conformation due to the fact that the result of the transmembrane region is not limited, so that the defect that the CAR cannot be identified correctly exists.

To address these issues, there is a need to develop a CAR target protein that both retains the intact native conformation and is capable of specifically binding to the CAR.

Virus-like particles (VLPs), also known as Core-like particles (CLPs), are spherical or tubular protein nanostructures formed by self-assembly of viral Capsid Proteins (CPs), having a structure similar to that of a natural viral capsid, free of genome, and non-infectious. VLPs are largely classified into eblp and non-enveloped VLPs. In the case of VLPs from enveloped viruses, this process is by budding from the host cell, which results in encapsulation of the VLPs by lipid bilayer (chinese patent application CN101146522 a). U.S. patent application US20110189159A1 is based on the finding that a protein of interest can be delivered to a cell as a fused or unfused protein; in particular, VLPs made with two Gag fusions (Gag-protein of interest and Gag-protease) can deliver the protein of interest as an unfused protein to cells for treatment. The vaccine disclosed in PCT patent application WO2021022008A1 comprises VLPs, pharmaceutically acceptable excipients, carriers and/or adjuvants; wherein the VLP comprises (a) a synthetic or natural lipid bilayer, (b) an anchor molecule embedded in the lipid bilayer, and (c) an antigen bound to the anchor molecule. In recent years, eVLP has shown unique research and application value in numerous fields such as vaccine development, drug targeted delivery, biomedical imaging and sensing, tissue engineering and the like. However, studies of the rate of expression of eVLPs in CAR positives are rarely reported.

Disclosure of Invention

In order to solve the problems in the prior art, the present disclosure provides a nanoparticle and its application in detecting the positive expression rate of CAR.

Specifically, the present disclosure solves the technical problems to be solved by the present disclosure through the following technical solutions.

In one aspect of the present disclosure, there is provided a nanoparticle comprising:

(i) An enveloped virus-like particle (eVLP) scaffold protein and/or a functional fragment thereof; and

(ii) A target protein and/or a functional fragment thereof,

wherein the enveloped virus-like particle framework protein comprises a virus core protein or a functional fragment thereof, the enveloped virus-like particle framework protein or the functional fragment thereof is assembled to form enveloped virus-like particles, and the target protein and/or the functional fragment thereof is displayed on the enveloped virus-like particles to form target protein-eVLP nano particles.

In one or more embodiments, the target protein is a CAR target protein; the CAR target protein and/or functional fragment thereof is displayed on an enveloped virus-like particle to form a CAR target protein-ehlp nanoparticle.

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, parainfluenza virus, bunyavirus, hepatitis c virus, hepatitis b virus.

In one or more embodiments, the retrovirus is selected from the group consisting of human immunodeficiency virus, monkey immunodeficiency virus, mouse leukemia virus, and bovine leukemia virus.

In one or more embodiments, the baculovirus is vesicular stomatitis virus.

In one or more embodiments, the filovirus is ebola virus.

In one or more embodiments, the coronavirus is selected from the group consisting of novel coronapneumoviruses (SARS-CoV-2), SARS-CoV, MERS-CoV, 229E, NL, OC43 and HKU1.

In one or more embodiments, the viral core protein is selected from the group consisting of: 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, bunyavirus N protein, hepatitis C virus core protein C, hepatitis b virus core protein C, and combinations thereof.

In one or more embodiments, the retroviral Gag protein is selected from the group consisting of: human immunodeficiency virus Gag protein, monkey immunodeficiency virus Gag protein, mouse leukemia virus Gag protein and bovine leukemia virus Gag protein. In one or more preferred embodiments, the retroviral Gag protein is the human immunodeficiency virus Gag protein. In one or more preferred embodiments, the human immunodeficiency virus Gag protein comprises an amino acid sequence having at least 80% or more identity to SEQ ID No.1, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity. In one or more preferred embodiments, the amino acid sequence of the human immunodeficiency virus Gag protein is shown as SEQ ID No. 1.

In one or more embodiments, the baculovirus matrix protein M protein is a vesicular stomatitis virus M virus core protein.

In one or more embodiments, the filovirus core protein is an ebola virus VP40 virus core protein.

In one or more embodiments, the core protein of the coronavirus is selected from the group consisting of novel coronapneumovirus (SARS-CoV-2), SARS-CoV, MERS-CoV, 229E, NL, OC43 and HKU 1.

In one or more embodiments, the virus is a virus that obtains an envelope when budding from the cell membrane of a host cell.

In one or more embodiments, the CAR target protein comprises a membrane protein.

In one or more embodiments, the membrane proteins include transmembrane proteins.

In one or more embodiments, the transmembrane protein comprises a plurality of transmembrane proteins.

In one or more embodiments of the present invention, the CAR target protein is selected from the group consisting of CD20, claudin18.1, claudin18.2, CD133, GPRC5D, CCR, 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 any of 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.

In one or more embodiments, the CAR target protein comprises a multi-transmembrane protein.

In one or more embodiments, the CAR target protein is selected from any one of CD20, claudin18.2, CD133, GPRC5D, CCR, CCR8, CD19, BCMA, GPC3, CD30, CD22, EGFR, EGFRVIII, HER2, or GPCR.

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 having at least 80% or more identity to SEQ ID No.4, preferably an amino acid sequence having more than 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, more preferably an amino acid sequence having more than 98% or 99% identity. In one or more preferred embodiments, the amino acid sequence of Claudin18.2 is shown as SEQ ID No. 4.

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 having at least 80% or more identity to SEQ ID No.15, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity: more preferably, the amino acid sequence of CD20 is shown in SEQ ID No. 15.

In one or more embodiments, the CAR target protein and/or functional fragment thereof and/or the viral core protein and/or functional fragment thereof is linked to a marker.

In one or more embodiments, the N-terminus and/or C-terminus of the CAR target protein and/or functional fragment thereof and/or the N-terminus and/or C-terminus of the viral core protein and/or functional fragment thereof are linked to a tag. 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 preferred embodiment or embodiments, the marker is at the C-terminus of CD20 or the C-terminus of Gag.

In one or more embodiments, the label is selected from the group consisting of a detectable label, a purification label, a reporter label, and combinations thereof.

In one or more embodiments, the detectable label is selected from the group consisting of a fluorescent group, a chemiluminescent label, an electrochemiluminescent label, and combinations thereof.

In one or more embodiments, the fluorophore is selected from any one of FITC, GFP, RFP, YFP, TRITC, PE, FAM, RRX, TR, cy, cy3, cy5, ECD, PC5.5, PC7, APC-A70, APC-A75, pac-Blue, alexa488, mBBr, 5-IAF, E-118, DTAF, rhodamine Green, and KrO.

In one or more embodiments, the chemiluminescent label is an acridinium ester, isoluminol, horseradish peroxidase, or alkaline phosphatase.

In one or more embodiments, the electrochemiluminescent label is ruthenium terpyridyl or a derivative N-hydroxysuccinimide ester thereof.

In one or more embodiments, the purification Tag is selected from any of HIS-Tag, GST-Tag, MBP-Tag, nusA-Tag, FLAG-Tag, SUMO, avi-Tag, halo-Tag, and SNAP-Tag.

In one or more embodiments, the purification tag is detected by a secondary antibody.

In one or more embodiments, the reporter tag is selected from any of c-Myc, HA, or luciferase.

In one or more embodiments, the CAR target protein is displayed on the ekp, while being labeled with a marker at the C-terminus of the CAR target protein or the C-terminus of Gag.

In another aspect of the disclosure, a labeled Claudin 18.2-eVLP is provided, wherein the Claudin 18.2-eVLP is linked to a label.

In one or more embodiments, the CAR target protein-ehlp nanoparticle is a Claudin 18.2-ehlp nanoparticle, the Claudin18.2 and/or functional fragment thereof and/or the viral core protein and/or functional fragment thereof being linked to a label.

In one or more embodiments, the N-terminus and/or C-terminus of the Claudin 18.2 and/or functional fragment thereof and/or the N-terminus and/or C-terminus of the viral core protein and/or functional fragment thereof are linked to a tag.

In one or more embodiments, the enveloped virus-like particle scaffold protein is Gag protein.

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 tag.

In another aspect of the disclosure, a labeled CD20-eVLP is provided, the CD20-eVLP and the label being linked.

In one or more embodiments, the CAR target protein-ehlp nanoparticle is a CD 20-ehlp nanoparticle, and the CD20 and/or functional fragment thereof and/or the viral core protein and/or functional fragment thereof is linked to a label.

In one or more embodiments, the CD20 and/or functional fragment thereof is linked to a tag at the N-terminus and/or C-terminus and/or viral core protein and/or functional fragment thereof.

In one or more embodiments, the C-terminus of the CD20 or the C-terminus of the Gag protein is linked to a label.

In one or more embodiments, the marker is GPF.

In one or more embodiments, the amino acid sequence of GFP comprises an amino acid sequence having at least 80% or more identity to SEQ ID No.2, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity. In one or more preferred embodiments, the amino acid sequence of GFP is shown as SEQ ID No. 2.

In one or more embodiments, the C-terminus of the Gag protein is linked to a marker GFP, denoted Gag-GFP, and the amino acid sequence of the Gag-GFP comprises an amino acid sequence having at least 80% or more identity to SEQ ID No.3, preferably an amino acid sequence having more than 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, more preferably an amino acid sequence having more than 98% or 99% identity. In one or more preferred embodiments, the Gag-GFP has the amino acid sequence shown in SEQ ID No. 3.

In one or more embodiments, the C-terminus of Claudin 18.2 is linked to a marker GFP, denoted Claudin 18.2-GFP, and the amino acid sequence of Claudin 18.2-GFP has an amino acid sequence with at least 80% or more identity to SEQ ID No.5, 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 99% or more identity. In one or more preferred embodiments, the amino acid sequence of Claudin 18.2-GFP is shown as SEQ ID No. 5.

In another aspect of the present disclosure, a method of preparing nanoparticles includes:

(1) Construction of recombinant plasmids:

respectively constructing a recombinant plasmid containing envelope virus-like particle (eVLP) skeleton proteins and/or functional fragment genes thereof and a recombinant plasmid containing target proteins and/or functional fragment genes thereof; or constructing a recombinant plasmid simultaneously comprising an envelope virus-like particle (eVLP) framework protein and/or a functional fragment gene thereof and a target protein and/or a functional fragment gene thereof; and

(2) Host cell transfection, protein expression and particle assembly:

transfecting the recombinant plasmid of the skeleton protein and/or the functional fragment thereof of the enveloped virus-like particle (eVLP) constructed in the step (1) and the recombinant plasmid of the target protein and/or the functional fragment thereof into a host cell, and expressing the skeleton protein and/or the functional fragment thereof of the enveloped virus-like particle and the target protein and/or the functional fragment thereof; the coated virus-like particle skeleton proteins or functional fragments thereof are assembled to form coated virus-like particles, and the target proteins and/or the functional fragments thereof are displayed on the coated virus-like particles to form target protein-eVLP nanoparticles; and, optionally,

(3) Purifying the target protein-eVLP nanoparticles.

In one or more embodiments, the coding nucleic acid sequence of the GAG comprises a nucleotide sequence having at least 80% or more identity to SEQ ID No.7, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity: more preferably, the amino acid sequence of GFP is shown as SEQ ID No. 7.

In one or more embodiments, the Gag-GFP encoding nucleic acid sequence comprises a nucleotide sequence having at least 80% or more identity to SEQ ID No.8, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity: more preferably, the amino acid sequence of GFP is shown as SEQ ID No. 8.

In one or more embodiments, the Claudin 18.2 encoding nucleic acid sequence comprises a nucleotide sequence having at least 80% or more identity to SEQ ID No.9, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity: more preferably, the amino acid sequence of GFP is shown as SEQ ID No. 9.

In one or more embodiments, the Claudin 18.2-GFP encoding nucleic acid sequence comprises a nucleotide sequence having at least 80% or more identity to SEQ ID No.10, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity: more preferably, the amino acid sequence of GFP is shown as SEQ ID No. 10.

In one or more embodiments, the coding nucleic acid sequence of CD20 comprises a nucleotide sequence having at least 80% or more identity to SEQ ID No.16, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity: more preferably, the amino acid sequence of GFP is shown as SEQ ID No. 16.

In one or more embodiments, the host cell is a prokaryotic host cell or a eukaryotic host cell.

In one or more embodiments, the prokaryotic host cell is selected from bacterial cells, such as e.coli, bacillus subtilis, and mycobacterium.

In one or more embodiments, the eukaryotic host cell is selected from any one of an animal cell, a plant cell, or a fungus.

In one or more embodiments, the eukaryotic host cell is selected from any one of a yeast, insect, avian, plant, caenorhabditis elegans, and mammalian host cell.

Non-limiting examples of such insect cells are: spodoptera frugiperda (Spodoptera frugiperda) (Sf) cells, e.g., sf9, sf21, trichoplusia ni (Trichoplusia ni) cells, e.g., high Five cells, and Drosophila S2 cells.

Examples of such fungal (including yeast) host cells are Saccharomyces cerevisiae (S.cerevisiae), kluyveromyces lactis (Kluy veromyces lactis; K.lactis), candida species including Candida albicans (C.albicans) and Candida glabrata (C.glabra), aspergillus nidulans (Aspergillus nidulans), schizosaccharomyces pombe (Schizosaccharomyces pombe; S.pombe), pichia pastoris (Pichia pastoris), and yarrowia lipolytica (Yarrowia lipolytica).

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.

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.

In another aspect of the disclosure, a method of detecting a positive expression rate of a CAR comprises incubating the nanoparticle or nanoparticle prepared by the method with a CAR-modified cell, and then detecting.

In one or more embodiments, the cells are washed after incubation of the cells prior to detection.

In one or more preferred embodiments, the CAR-modified cell is selected from any one of a CAR-like (HEK 293) cell, a CAR-T cell, a CAR-NK cell, a CAR-M cell, a CAR-NKT cell, a CAR-Treg cell, and a CAR- γδ T cell.

In one or more embodiments, a method of detecting a positive expression rate of a CAR comprises incubating, washing, and then detecting the labeled Claudin 18.2-ekp described above with CAR-modified cells.

Preferably, the method for detecting the positive expression rate of the CAR comprises incubating the Claudin 18.2-eVLP with the marker and the CAR-T cells, and washing for detection.

In one or more embodiments, the Claudin 18.2-eklp ensures a natural intact conformation of the CAR target protein, increasing the success rate of isolating antibodies that recognize the native membrane protein structure.

In one or more embodiments, a method of detecting a positive expression rate of a CAR comprises incubating, washing, and then detecting the aforementioned labeled CD 20-ehlp with a CAR-modified cell.

Preferably, the method for detecting the positive expression rate of the CAR comprises incubating the CD20-eVLP with the marker and the CAR-T cells, and washing for detection.

In one or more embodiments, the CD 20-ehlp ensures a natural intact conformation of the CAR target protein, increasing the success rate of isolating antibodies that recognize the native membrane protein structure.

In one or more embodiments, the incubation is at a temperature of 2 ℃ to 40 ℃, preferably 37 ℃, and/or the incubation is for a time of 15min to 2 hours, preferably 1 hour; and/or, the incubated CO ₂ The concentration of (2) to 8%, preferably 5%.

In one or more embodiments, the number of washes is 1-6, preferably 3.

In one or more embodiments, the wash buffer is 0.5% -5% bsa, preferably 2% bsa.

In one or more embodiments, the assay is any one of flow cytometry assay, immunodetection, ELISA, SPR, BLI.

In one or more embodiments, the enveloped virus-like particle ehlp framework protein is a Gag protein.

In one or more embodiments, the CAR target protein is displayed on the membrane surface (fig. 1) using the ability of HIV-1 Gag to self-assemble into an ehlp. Preferably, the four transmembrane protein Claudin 18.2 is displayed on the HIV-1 Gag envelope VLP, with a marker at the C-terminus of Claudin 18.2 or the C-terminus of Gag. The rate of CAR positive expression was assessed using a Claudin 18.2-eVLP using flow cytometry detection. All Claudin 18.2-eVLPs produced by the present disclosure can be evaluated for expression of target antigen binding CARs.

In another aspect of the disclosure, a kit is provided that contains the aforementioned nanoparticle.

In one or more embodiments, the nanoparticle is a CAR target protein-ehlp nanoparticle.

In one or more embodiments, the kit contains the aforementioned labeled CAR target protein-ehlp.

In one or more embodiments, the kit contains the aforementioned labeled Claudin 18.2-eVLP.

In one or more embodiments, the kit contains the aforementioned labeled CD20-eVLP.

In one or more embodiments, the kit is an ELISA kit, SPR kit, or BLI kit.

In one or more embodiments, the ELISA kit contains the aforementioned CAR target protein-ekp, an ELISA plate, a blocking solution, a sample diluent, an enzyme conjugate, a concentrated wash solution, an enzyme substrate solution, and a stop solution.

In one or more embodiments, the SPR kit comprises the aforementioned CAR target protein-ehlp nanoparticle.

In one or more embodiments, the BLI kit comprises the aforementioned CAR target protein-ehlp nanoparticle.

The kit disclosed by the disclosure can be used for detecting the positive expression rate of the CAR, and can also prove that the CAR can specifically bind to a target protein.

In a non-limiting example, reagents for preparing and/or administering CAR target protein-ehlp can be included in the kit. The kit may further comprise reagents for assessing CAR target protein-ehlp activity in vitro and in vivo. In certain aspects, the kit may include reagents and/or devices for administration, such as an inhaler or nebulizer, and the kit may further include one or more buffers, and the like.

When the components of the kit are provided in the form of one and/or more liquid solutions, the liquid solutions are aqueous solutions, with sterile aqueous solutions being particularly preferred. Furthermore, the components of the kit may also be provided in the form of a dry powder. When the reagents and/or components are provided in dry powder form, the liquid solution may be reconstituted by the addition of a suitable solvent.

It is to be understood that within the scope of the present disclosure, the above-described technical features of the present disclosure and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 is a schematic diagram of the principle of HIV-Gag coated VLPs displaying multiple transmembrane target antigens.

FIG. 2 shows the result of electrophoresis of crude pure samples after sucrose density gradient centrifugation, wherein lane A is R1242 crude pure sample, lane B is RG288 crude pure sample, and lane C is RG341 crude pure sample.

Fig. 3 shows the result of electrophoresis detection of samples after Sepharose 6FF separation, wherein fig. a is R1242, fig. B is RG288, and fig. C is RG341.

FIG. 4 shows the results of R1242-210521F1 transmission electron microscopy.

FIG. 5 shows ELISA assay for binding activity of Claudin18.2-eVLP to its specific antibody IMAB362, wherein FIG. A is the ELISA assay for binding of RG288-210503F1 to IMAB 362; panel B shows ELISA detection results of RG341-210521F1 and IMAB362 binding.

Fig. 6 is a schematic diagram of an IMAB361 CAR-like structure.

FIG. 7 shows that Claudin18.2-eVLP of the present disclosure was used to evaluate IMAB362 CAR-like expression: wherein panel a is the evaluation of IMAB362 CAR-like expression when FITC-anti mFab was diluted 200-fold; panel B is an evaluation of IMAB362 CAR-like expression when FITC-Protein L is used at a concentration of 10 μg/mL; panel C is when Claudin-18.2 Protein,His Tag ^TM At a concentration of 10 μg/mL, it was evaluated for IMAB362 CAR-like expression; panel D is an evaluation of IMAB362 CAR-like expression when RG288-210503F1 was used at a concentration of 10 μg/mL; panel E is an evaluation of IMAB362 CAR-like expression when RG341-210521F1 was used at a concentration of 10. Mu.g/mL.

FIG. 8 shows that CD20-eVLP of the present disclosure was used to evaluate Ofatumumab CAR-like expression: RG344-210628F1 was evaluated for Ofatumumab CAR-like expression when used at a concentration of 20. Mu.g/mL.

Detailed Description

The present disclosure discloses an eVLP and an application thereof in detecting a CAR positive expression rate. Those skilled in the art can refer to the disclosure herein to obtain the eVLP, to realize its applications, and it is specifically noted that all similar substitutions and modifications will be apparent to those skilled in the art, and are considered to be included in the present disclosure. While the methods and applications of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the disclosed techniques can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the present disclosure.

Definition of the definition

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.

The term "virus-like particle" (VLP) refers to a structure that resembles a virus in at least one attribute 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 generally be produced in large quantities by heterologous expression and can be easily purified.

The term "enveloped VLP" or alternatively "eblp" refers to an enveloped virus-like particle formed by encapsulation of a virus-like particle by a lipid envelope derived from a host cell.

The term "CAR target protein" refers to a protein that specifically binds to a CAR.

The term "vlp scaffold protein" may be understood in the present disclosure as a protein forming the scaffold structure of an vlp, which may consist essentially of or include viral core proteins, as well as other proteins.

The term "viral core protein" refers to an envelope protein that in some cases is also capable of driving budding and release of particles from a host cell.

The term "functional fragment" corresponds to a fragment of a full-length protein having a truncated structure, but still retaining all or part of the function of the full-length protein; for example, for a core protein, its corresponding functional fragment may be understood as a fragment of a truncated core protein which still has the ability to form at least part of the capsid of the virus or drive budding and release of the particle from the host cell; for a target protein, its corresponding functional fragment can be understood as a fragment of a truncated target protein, which still has the ability to specifically bind to the CAR.

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 cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulatory molecule.

The term "CAR-like (HEK 293)" refers to Chimeric Antigen Receptor (CAR) -modified HEK293 cells, i.e., HEK293 is edited with CAR means, displaying the CAR on the surface of HEK 293.

The term "CAR-NK" refers to Chimeric Antigen Receptor (CAR) modified NK cells, where NK cells are natural killer cells (natural killer cell), which are important immune cells of the body, involved not only in anti-tumor, anti-viral infection and immune modulation, but also in the development of hypersensitivity and autoimmune diseases in some cases, capable of recognizing target cells, killing mediators. NK cells are considered as effector cells which have the same potential to enhance the antitumor ability by CAR modification because of the advantages of specific target cell recognition mechanism, short physiological cycle, wide tumor killing ability and the like.

The term "CAR-M" refers to Chimeric Antigen Receptor (CAR) -modified macrophages (CAR macroges), i.e. human macrophages are edited by CAR means so that they can directly phagocytose a tumor.

The term "CAR-NKT" refers to Chimeric Antigen Receptor (CAR) modified NKT cells (Natural killer T cell), wherein NKT cells are a specific T cell subset with both T cell receptor TCRs and NK cell receptors on the cell surface. The CAR-NKT cells combine the original advantages of the NKT cells and the specificity of the CAR therapy, so that the tumor killing effect is better realized.

The term "CAR-Treg" refers to Chimeric Antigen Receptor (CAR) modified regulatory T cells (Tregs), where Tregs are a subset of T cells that control autoimmune reactivity in vivo, also referred to as suppressor T cells (suppressor T cells) in the early stage. Regulatory T cells can be classified 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 and Tr1, and CD8 Treg and NKT cells, etc., and are closely related to autoimmune diseases, and abnormal expression thereof may lead to autoimmune diseases.

The term "CAR- γδ T" refers to Chimeric Antigen Receptor (CAR) modified γδ T cells, wherein γδ T cells are a class of T cells between adaptive immunity and innate immunity, accounting for 1% -5% of peripheral blood T lymphocytes, distributed primarily in mucosal and epithelial tissues. The γδ T cells recognize antigen without MHC restriction, not only can kill tumor cells in various ways, but also exert antigen-presenting effects as antigen-presenting cells (APC). The gamma delta T cells are changed into CAR-gamma delta T cells, so that specific antigens can be accurately identified, tumor cells can be killed efficiently, and the treatment of solid tumors can be challenged by utilizing the characteristics of the gamma delta T cells.

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 chain and heavy chain variable regions are linked consecutively by a short flexible polypeptide linker and are capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

The term "antigen" or "Ag" refers to a molecule that causes an immune response. The immune response may involve antibody production or activation of specific immunocompetent cells or both. The skilled artisan will appreciate that virtually any macromolecule, including all proteins or peptides, can act as an antigen. Furthermore, the antigen may be derived from recombinant or genomic DNA. The skilled artisan will appreciate that any DNA comprising a nucleotide sequence or portion of a nucleotide sequence encoding a protein that elicits an immune response, thus encodes an "antigen".

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single or double stranded form, as well as polymers thereof.

The term "ELISA" is short for Enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbnent Assay). The method comprises the steps of combining known antigen or antibody on the surface of a solid phase carrier, then incubating the antigen or antibody marked (coupled) by enzyme, and developing by a chromogenic substance, wherein the color development depth is in direct proportion to the content of a substance to be detected, and the substance can be observed by naked eyes. In ELISA experiments, there are three necessary reagents: known antigens or antibodies (for binding to a solid support); enzyme-labeled antibodies or antigens (markers); color developer (for color development). Four types of ELISA experiments are common: direct ELISA, indirect ELISA, sandwich ELISA, and competition ELISA.

The term "SPR" is a short term for surface plasmon resonance (Surface plasmon resonance), which uses the SPR principle to detect the interaction condition between a ligand and an analyte on a biosensing chip (biosensing chip), and is widely used as a universal detection platform in the fields of drug screening, scientific research and the like for analysis of affinity, binding specificity, concentration quantification and the like between biomolecules.

The term "BLI" is an abbreviation for biological film interference technique (Bio-Layer Interferometry) and is an experimental method for detecting interactions between biomolecules based on shift changes in interference patterns.

The term FACS is short for flow cytometry fluorescence sorting technology (Fluorescence activated Cell Sorting), and the flow cytometer operates on the principle that cells to be measured are stained with a specific fluorescent dye and then placed into a sample tube, and enter a flow chamber filled with sheath fluid under the pressure of gas. Cells are arranged in a single row under the restriction of sheath liquid and are ejected by a nozzle of a flow chamber to form a cell column which is perpendicularly intersected with an incident laser beam, and cells in the liquid column are excited by the laser to generate fluorescence. A series of optical systems (lenses, diaphragms, filters, detectors, etc.) in the instrument collect signals such as fluorescence, light scattering, light absorption or cell impedance, and a computer system collects, stores, displays and analyzes the various signals to be measured and performs statistical analysis on various indexes.

The term "HIS-Tag" consists of 6-10 histidine residues with a molecular weight of less than 0.84KD, typically inserted at the C-or N-terminus of the protein of interest. HIS-Tag is the most commonly used Tag for prokaryotic expression at present, and the Tag can be removed after the protein is purified, so that the function of the protein is not influenced. Meanwhile, the protein purification steps are simple and convenient, the purification conditions are mild, and the protein is not greatly influenced.

The term "GST-Tag" is a glutathione thiol transferase Tag, which has a relatively large molecular mass of about 26KD, and is inserted into the C-terminal or N-terminal of a target protein, commonly used in E.coli. GST (glutathione-sulfhydryl transferase) protein itself is a transferase that plays an important role in detoxification processes. The GST tag is generally selected for two purposes, namely, to improve the solubility of the protein expression and to improve the protein expression level. After the purification of the protein expression is finished, whether the tag is cut off or not is determined according to different protein applications, the tag is large, and whether the tag is cut off or not is considered according to downstream applications. If the GST fusion moiety is to be removed, it can be excised with a site-specific protease.

The term "MBP-Tag" is a maltose binding protein Tag, has the amino acid residue number of 346 and the molecular weight of 42.5kDa, is encoded by the malE gene of Escherichia coli K12, and can be placed at the N-terminal during construction to improve the solubility (especially eukaryotic proteins). Folding of MBP requires the assistance of two chaperone systems, dnaK-DnaJ-GrpE and GroEL-GeoES, which can bring these chaperones into the vicinity of the protein of interest to assist in its correct folding. In addition, the maltose binding protein in the form of the tag protein can reduce the degradation of the target protein, improve the water solubility of the expression product and provide a basis for the purification of the target protein in the future. The maltose binding protein can be adsorbed by the polysaccharide resin, so that the fusion protein can be separated from other protein components when passing through the column.

The term "NusA-Tag" is a transcription termination/anti-termination protein Tag, nusA is a protein of E.coli itself, namely transcription anti-termination factor, amino acid residue number 495, molecular weight: 54.87kDa, from 4000 E.coli protein libraries, davia, 1999. NusA does not have a separate purification tag function and is therefore used in combination with other tags (e.g. His tag).

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 makes the expression of the FLAG-bearing fusion protein more efficient in eukaryotic expression systems.

The term "SUMO" is SUMO tag protein, is a small molecule ubiquitin related modified protein, and is a type of large protein which exists in eukaryotes and is highly conserved and involved in protein ubiquitination related modification. Compared with GST, MBP or NusA, SUMO not only can be used as a fusion tag of recombinant protein expression, but also has the function of molecular chaperones, can promote correct folding of the protein, has tolerance to heat and protease, and is more beneficial to maintaining the stability of target protein.

The term "Avi-Tag" is a 15 amino acid short peptide with a single biotinylated lysine site, which, in complete contrast to known natural biotinylated sequences, can be added at the N-and C-terminus of the target protein. After fusion expression, it can be biotinylated by biotin ligase, and low-affinity monomeric avidin or avidin derivatives are selected for purification of the recombinant protein.

The term "Halo-Tag" is a genetically modified derivative of a dehalogenase that is effectively covalently bound to a variety of synthetic HaloTag ligands. This monomeric protein with a molecular weight of 33kDa can be fused to the N-or C-terminus of the recombinant protein and expressed in both prokaryotic and eukaryotic systems. HaloTag ligands are small molecule chemicals capable of covalently binding to HaloTag proteins in vitro or in vivo.

The term "SNAP-Tag" is obtained from the methyl transfer of human O6-methylguanine-DNA (O6-alkylguanine-DNA-alkyltransferase). SNAP-Tag can be covalently bound to a substrate with high specificity, either in vivo or in vitro, to label proteins with biotin or fluorescent groups (e.g., fluorescein and rhodamine). The active sulfhydryl site carried by SNAP accepts a side chain benzyl group carried by benzyl guanine, releasing guanine. This novel thioether bond covalently binds to the target protein carried by SNAP carrying the tag carried by the benzyl group. Benzyl guanine is stable under biochemical conditions and no other proteins will interact with this class of substances, so SNAP tag reactions are highly specific.

The term "c-Myc" tag protein is a small tag of 10 amino acids (EQKLISEEDL, SEQ ID No. 13) that can still recognize its corresponding antibody as an epitope expressed in a different protein framework. C-Myc tag has been successfully used in Western-blot hybridization techniques, immunoprecipitation and flow cytometry, and can be used to detect expression of recombinant proteins in target cells. Common vectors are pCMV-MYC, pcDNA3.1 (+) -MYC-hisA, pCMV-RFP-C-Myc, pCMV-Myc, etc.

The term "HA" tag protein, tag sequence YPYDVPDYA (SEQ ID No. 14), is derived from hemagglutinin surface antigen determinant of influenza virus, 9 amino acids, HAs little influence on the spatial structure of exogenous target protein, and is easy to construct into a tag protein fused to N-terminal or C-terminal. Anti-HA antibody detection and ELISA detection are commonly used.

The term "luciferase" is derived from luciferin in an organism, and is commonly known as firefly luciferase, renilla luciferase and Guassia luciferase.

Examples

EXAMPLE 1 construction of eVLP backbone plasmid

The eVLP backbone used in this example was HIV-1 _SF2 p ⁵⁵ Gag (GenBank accession No. K02007) protein (SEQ ID No. 1), the Gag protein alone has the ability to self-assemble into coated VLP particles.

First, recombinant plasmids R1221 and R1242 containing Gag-GFP encoding genes were constructed, respectively: the polynucleotides SEQ ID No.7 and 8 which code for the amino acid sequences shown in SEQ ID No.1 and 3 are synthesized by Shanghai bioengineering Co., ltd, and the restriction enzyme BamHI cleavage site and the Kozak sequence are added to the 5' end; a stop codon TAA and a restriction enzyme XhoI cleavage site are added to the 3' -end. The synthesized product was digested with BamHI and XhoI restriction enzymes, and then subjected to agarose gel electrophoresis, and the desired fragment was recovered by gel cutting. The target fragment was ligated with pcNDA3.1 (+) which was also digested with the restriction enzymes BamHI and XhoI, respectively, to construct recombinant eukaryotic expression vectors, i.e., recombinant plasmids R1221 and R1242. Then, respectively converting recombinant plasmids R1221 and R1242 into E.coli DH5 alpha, culturing at 37 ℃ for 16 hours, picking single colonies, extracting plasmids, carrying out agarose gel electrophoresis identification on the extracted plasmids after double enzyme digestion of restriction enzymes BamHI and XhoI, sequencing positive clones by Shanghai bioengineering limited company, selecting clones with correct sequencing, amplifying and extracting plasmids, carrying out aseptic filtration on the extracted plasmids, and preserving at-20 ℃ for later use.

EXAMPLE 2 construction of Claudin 18.2 recombinant expression plasmid

First, recombinant plasmids R1353 and R1303 containing Claudin 18.2-GFP encoding genes were constructed, respectively: the polynucleotides SEQ ID No.9 and 10 encoding the amino acid sequences shown in SEQ ID No.4 and 5 are synthesized by Shanghai bioengineering Co-Ltd, and a restriction enzyme BamHI cleavage site and a Kozak sequence are added to the 5' end; a stop codon TAA and a restriction enzyme XhoI cleavage site are added to the 3' -end. The synthesized product was digested with BamHI and XhoI restriction enzymes, and then subjected to agarose gel electrophoresis, and the desired fragment was recovered by gel cutting. The target fragment was ligated with pcNDA3.1 (+) which was also digested with restriction enzymes BamHI and XhoI, respectively, to construct recombinant eukaryotic expression vectors, i.e., recombinant plasmids R1353 and R1303. Then, respectively converting recombinant plasmids R1353 and R1303 into E.coli DH5 alpha, culturing at 37 ℃ for 16 hours, picking single colonies, extracting plasmids, carrying out agarose gel electrophoresis identification on the extracted plasmids after double enzyme digestion of restriction enzymes BamHI and XhoI, sequencing positive clones by Shanghai bioengineering limited company, selecting clones with correct sequencing, amplifying and extracting plasmids, carrying out aseptic filtration on the extracted plasmids, and preserving at-20 ℃ for later use.

EXAMPLE 3 blank HIV-1 Gag-eVLP and Claudin 18.2-eVLP expression in HEK293 cells

3.1 expression of blank HIV-1 Gag-eVLP in HEK293 cells

The constructed recombinant plasmid R1242 is transfected into HEK293 cells (corresponding to the cell ID of R1242) by using PEI transfection reagent, and blank control eVLP expression and assembly are carried out. HEK293 cells were passaged 1X 10≡6 cells/mL the day before transfection and cultured at 37 ℃. The number is counted on the day of transfection, the cell density is adjusted to be 2X 10-6 cells/mL, and the activity rate is more than 95%.

The amounts of each component in the transfection complex were calculated from the cell density: the corresponding relation between the plasmid dose and the number of cells is that 1X 10≡6 cells correspond to 0.6 mug plasmid, and the corresponding relation between the PEI dose and the plasmid dose is that: the mass of PEI is 3 times the mass of DNA.

The transfection complexes were configured according to the amounts of the components calculated above: 1.2mg of recombinant plasmid with the concentration of 200 mug/mL is added into CD 293TGE culture medium (Acrobiosystems; product number CM-1156-11) to 50mL (solution A), 3.6mg of PEI transfection reagent with the concentration of 1mg/mL is added into CD 293TGE culture medium to 50mL (solution B), after the mixture is uniformly mixed, solution B is slowly added into solution A, the mixture is uniformly mixed, and after standing for 10-15min at room temperature, the transfection mixture is slowly dripped into 1000mL HEK293 cells. 37 ℃,5% CO ₂ After 48 hours of culture at 135rpm, the culture supernatant of R1242 was collected.

3.2 Expression of Claudin 18.2-eVLP in HEK293 cells

HEK293 cells (corresponding cell IDs RG288 (transfected with R1303 and R1221) and RG341 (transfected with R1353 and R1242)) are obtained by respectively transfecting the constructed recombinant plasmids with the PEI transfection reagent according to the ratio of R1303:R1221=2:1 and R1353:2, and the expression and the assembly of eVLPs are carried out. HEK293 cells were passaged 1X 10≡6 cells/mL the day before transfection and cultured at 37 ℃. The number is counted on the day of transfection, the cell density is adjusted to be 2X 10-6 cells/mL, and the activity rate is more than 95%.

The transfection complexes were configured according to the amounts of the components calculated above: adding 1.2mg of recombinant plasmid with the concentration of 200 mug/mL into CD 293TGE culture medium to 50mL (solution A), adding 3.6mg of PEI transfection reagent with the concentration of 1mg/mL into CD 293TGE culture medium to 50mL (solution B), respectively uniformly mixing, slowly adding the solution B into the solution A, uniformly mixing, and standing at room temperature After 10-15min, the transfection mixture was slowly added dropwise to 1000mL HEK293 cells. 37 ℃,5% CO ₂ Culture supernatants of RG288 and RG341 were collected after 48 hours of culture at 135rpm, respectively.

EXAMPLE 4 isolation and purification of blank eVLP and Claudin 18.2-eVLP

The supernatant of the harvested R1242, RG288 and RG341 cells was centrifuged at 2000rpm at 4℃for 20min to remove cell debris and filtered with a 0.22 μm filter, and the supernatant was subjected to sucrose density gradient centrifugation, and the supernatant was subjected to 30% sucrose density ultracentrifugation (2,6000 rpm/min at 4℃for 1.5 h) and the pellet was resuspended in PBS to give crude pure samples of R1242, RG288 and RG341eVLP, respectively. SDS-PAGE was performed with separate samples, and the results are shown in FIG. 2, wherein lane A is R1242 as a crude pure sample, lane B is RG288 as a crude pure sample, and lane C is RG341 as a crude pure sample. The target bands for Gag (about 55 kDa) and Gag-GFP (about 80 kDa) were clearly seen from the electrophoresis results, demonstrating that the supernatant could be ultracentrifuged with a 30% sucrose pad density to yield a large amount of VLPs. To remove residual nucleic acid, crude and pure samples of R1242, RG288 and RG341 were treated with Benzonase (ACROBiosystems) having an enzyme concentration of 200U/mL at room temperature for 1 hour, respectively. The samples after nucleic acid removal were used separatelyExplorer (TM) 100 low pressure liquid chromatography System (GE Healthcare) SEC experiments were performed with Sepharose 6FF resin loaded into XK 16/70 chromatography columns (GE Healthcare) with a final bed volume of 130mL. The void volume of the column was determined using Blue Dextran 2000 (HMW calibration kit, GE Healthcare). Prior to eVLP separation, the column was washed with 3 column volumes of degassed Milli-Q ultrapure water, column equilibrated with 1 column volume of PBS, pH 7.4 at a rate of 2mL/min (60 cm/h).

Crude pure samples of R1242, RG288 and RG341 obtained above were injected into the loading ring and eluted at constant speed using PBS (pH 7.4) at a flow rate of 2mL/min (60 cm/h). Absorbance was measured at 280nm and 260nm on line. During the whole chromatography, samples were collected using a Frac 950 sample collector (GE Healthcare) and sampled for SDS-PAGE electrophoresis analysis, as shown in fig. 3, where plot a is R1242, plot B is RG288, and plot C is RG341. From the electrophoresis results, it can be seen that after separation by the crude pure sample SEC, high purity samples (indicated by arrows) can be obtained. And recovering high-purity samples according to electrophoresis results, namely eVLP, wherein the sample IDs are respectively marked as R1242-210521F1, RG288-210503F1 and RG341-210521F1.

EXAMPLE 5 morphological detection of Virus-like particles

To further confirm that the method of example 4 successfully produced eVLPs, transmission electron microscopy morphological examination (assigned to Peking, boseki technologies Co., ltd.) was performed on R1242-210521F1, and the results are shown in FIG. 4. It can be seen that R1242-210521F1 appears under electron microscopy as a hollow sphere with a diameter of about 150 nm. The electron microscopy results macroscopically demonstrate that the method of example 4 can successfully produce eVLPs with HIV-1 Gag as the scaffold protein.

Example 6 Activity detection of Claudin 18.2-eVLP with antibody IMAB362

In this example, ELISA (enzyme-linked immunosorbent assay) was used to detect the binding activity of Claudin 18.2-eVLP of the present disclosure to its specific antibody IMAB362, thereby demonstrating that Claudin 18.2 four transmembrane proteins were successfully displayed on eVLP in the correct conformation. The method comprises the following specific steps:

1. coating: 96-well plates (Corning Corp., cat# 42592) were coated with either RG288-210503F1 or RG341-210521F1 at 0.5 μg/well (5 μg/ml,100 μl/well) overnight (or 16 h) at 4deg.C. The coating buffer used for diluting RG288-210503F1 or RG341-210521F1 was 15mM Na ₂ CO ₃ ，35mM NaHCO ₃ ，7.7mM NaN ₃ ，pH9.6。

2. Washing: wells were washed 4 times with 300. Mu.l of wash buffer (TBS, 0.05% Tween-20, pH 7.4) per well (note: thorough removal of wash buffer is critical). After washing, the residual solution was removed by suction and was ensured to be completely dried.

3. Closing: each well was blocked with 300. Mu.l of blocking buffer (TBS, 2%BSA,pH7.4) for 1.5h at 37 ℃.

4. Washing: and (5) repeating the step 2.

5. Adding a sample: mu.l 0.390625-50ng/mL IMAB362 antibody was added to each well and incubated for 1h at 37 ℃. Samples were diluted in advance with dilution buffer (TBS buffer, pH7.4, containing 0.5% BSA).

6. Washing: and (5) repeating the step 2.

7. Adding a detection antibody: to each well 100. Mu.l of anti-human IgG antibody (Jackson, cat. No. 109-035-098) was added and incubated at 37℃for 1h. The antibodies were diluted in advance with dilution buffer (TBS buffer containing 0.5% BSA, pH 7.4) at a ratio of 1:20000.

8. Washing: and (5) repeating the step 2.

9. Adding a substrate: 200 μl of substrate solution was added to each well and incubated at 37deg.C for 20min. And (5) light shielding. Substrate solution preparation: after 10ml of substrate solution (50 mM Na ₂ HPO ₄ ·12H ₂ O,25mM citric acid, pH 5.5) was added 8. Mu.l 3%H ₂ O ₂ And 100 μl of 10mg/ml TMB (BBI Life sciences, cat# A600954).

10. Terminating the reaction: to each well was added 50 μl of 1M sulfuric acid.

11. Reading OD value: OD values were read at 450nm, then OD450-ODBlank was the final OD value. Wherein the ODBlank corresponding wells were the results of the determination in step 5 without addition of sample and with addition of only an equal volume of dilution buffer, as a blank.

ELISA detection results are shown in FIG. 5, wherein FIG. A shows ELISA detection results of RG288-210503F1 and IMAB362 binding; panel B shows ELISA detection results of RG341-210521F1 and IMAB362 binding. The EC50 values of Claudin 18.2-eVLP RG288-210503F1 and RG341-210521F1 binding to IMAB362 of the present disclosure were 1.64ng/mL and 1.94ng/mL, respectively, indicating that Claudin 18.2-eVLP of the present disclosure has good binding activity to IMAB362, thereby proving that Claudin 18.2 tetraspanins were successfully displayed on eVLP in the correct conformation.

Example 7 preparation of IMAB362 CAR-like (HEK 293) cells

In this example, IMAB362 CAR-like (HEK 293) cells were prepared to mimic CAR-T cells to verify the use of Claudin 18.2-eVLP in assessing CAR positive expression rates.

7.1 preparation of IMAB362 CAR-like recombinant plasmid

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. Plasmid construction methods refer to example 1.

7.2 preparation of IMAB362 CAR-like (HEK 293) cells

Procedure of IMAB362 CAR recombinant plasmid transfection HEK293 cells is referred to in example 3. Except that in this example IMAB362 CAR-like (HEK 293) monoclonal cells, labeled with ID C633, were required to be screened for resistance to G418 for CAR positive expression rate assessment experiments.

Example 8 Claudin-18.2-eVLP for CAR positive expression Rate assessment

In this example, anti-Fab antibodies FITC-anti-mFab Ab (Thermo Scientific, cat. No. 31543)), FITC-Protein L (ACROBiosystems, cat.No.RPL-PF 141), claudin 18.2 extracellular loop domain Protein were used, respectively: claudin-18.2 Protein,His Tag ^TM (ACROBiosystems, cat.No.CL2-H51H 6) R1242-210521F1, RG288-210503F1 and RG341-210521F1 of the present disclosure evaluate IMAB362 CAR expression. The method comprises the following specific steps:

1. Culturing C633 cells in DMEM medium containing 10% foetal calf serum, and placing in CO ₂ In an incubator (37 ℃,5% CO) ₂ )。

2. Cells were collected and washed once with FACS buffer (2% bsa).

3. The number and viability of cells were counted and 2X 10-6 viable cells were placed into each tube.

4. FITC-anti-mFab Ab, FITC-Protein L, claudin-18.2 Protein,His Tag ^TM The samples R1242-210521F1, RG288-210503F1 and RG341-210521F1 were each serially diluted with FACS buffer (2% BSA) and the diluted sample solutions were then added to the tubes containing the cells, respectively. Mixing well, and incubating at 4 ℃ for 60min. (for Claudin-18.2 Protein,His Tag ^TM After incubation, cells were washed 1 time with FACS buffer (2% BSA), PE-anti His antibody (Biolegend, cat. No. 362603) was added and incubated at 4℃for 60min in the dark. )

5. Cells were washed 3 times with FACS buffer (2% bsa) and finally cell samples were resuspended with 0.2ml pbs.

6. The cell suspension was transferred to a flow tube and detected by flow cytometry (excitation wavelength 488nm, emission wavelength 530 nm).

7. The resulting data was analyzed using FCS Express 6Plus and GraphPad Prism 5 software.

The results are shown in figure 7, where panel a is the assessment of IMAB362 CAR-like expression (positive rate 95.93%) when FITC-anti mFab was diluted 200-fold; panel B is an evaluation of IMAB362 CAR-like expression (positive rate 64.70%) when FITC-Protein L was used at a concentration of 10 μg/mL; panel C is when Claudin-18.2 Protein,His Tag ^TM When the concentration is 10 mug/mL, the expression condition of the IMAB362 CAR-like is evaluated (the positive rate is 1.17%), which indicates that only the extracellular loop region of Claudin18.2 is expressed and the IMAB362 CAR-like structure cannot be identified; panel D is an evaluation of IMAB362 CAR-like expression (99.52% positive rate) when RG288-210503F1 was used at a concentration of 10 μg/mL; panel E is an evaluation of IMAB362 CAR-like expression (positive rate 99.60%) when RG341-210521F1 was used at a concentration of 10. Mu.g/mL (R1242-210521F 1 is blank eVLP).

From the results, it can be seen that claudin18.2 target antigen displayed on claudin 18.2-eklp of the present disclosure is in correct conformation, can specifically bind to IMAB362 CAR-like, and shows the best effect when the positive expression rate of CAR is detected by FACS detection of GFP. The results indicate that Claudin 18.2-eklp containing green fluorescent protein GFP of the present disclosure is well suited for assessing CAR positive expression rates.

Example 9 CD20-eVLP for CAR positive expression rate assessment

In order to verify that the technical scheme of the disclosure can be applied to preparation of other multi-transmembrane proteins, and further applied to expression evaluation of CAR positive rates of other targeted multi-transmembrane proteins, the embodiment adopts the nano-particles of the disclosure to evaluate the CAR positive expression rates aiming at another multi-transmembrane protein CD 20. The method comprises the following specific steps:

1. Preparation of human CD 20-eVLP: constructing a recombinant plasmid containing a CD20 coding gene according to the method of the embodiment 2, wherein the amino acid sequence of the CD20 is shown as SEQ ID No.15, and the coding sequence of the CD20 is shown as SEQ ID No. 16; according to the method of example 3, a recombinant plasmid containing the CD20 encoding gene was combined with R1424 plasmid 2:1, mixing transfected HEK293 cells and collecting culture supernatant; the CD20-eVLP sample ID obtained by separation and purification according to the method of example 4 is RG344-210628F1;

2. preparation of Ofatumumab CAR-like (HEK 293) cells, preparation method was as described in example 7; wherein the amino acid sequence of the Ofatumumab CAR is SEQ ID No.17, and the polynucleotide sequence is SEQ ID No.18; the prepared Ofatumumab CAR-like (HEK 293) monoclonal cell is marked with an ID of RC539b and is used for a CAR positive expression rate evaluation experiment;

3. CD20-eVLP was used for CAR positive expression rate evaluation verification, specific methods refer to example 8.

The results are shown in FIG. 8, which evaluates the Ofatumumab CAR-like (HEK 293) expression (positive rate 99.48%) when RG344-210628F1 is used at a concentration of 20. Mu.g/mL (R1242-210521F 1 is blank eVLP).

From the results, it can be seen that the CD20 target antigen displayed on CD 20-eklp of the present disclosure is conformationally correct, can specifically bind to Ofatumumab CAR-like cell clones, and exhibits a positive rate effect of >99% when the positive expression rate of CAR is detected by FACS detection of GFP. The results further indicate that the technical means of the present disclosure can be widely applied to the preparation of multiple transmembrane protein-eVLPs, and further applied to the expression evaluation of CAR positive rates.

Claims

A nanoparticle, comprising:

(i) An enveloped virus-like particle (eVLP) scaffold protein and/or a functional fragment thereof; and

(ii) A target protein and/or a functional fragment thereof,

wherein the enveloped virus-like particle framework protein comprises a virus core protein or a functional fragment thereof, the enveloped virus-like particle framework protein or the functional fragment thereof is assembled to form enveloped virus-like particles, and the target protein and/or the functional fragment thereof is displayed on the enveloped virus-like particles to form target protein-eVLP nanoparticles;

preferably, the target protein is a CAR target protein; the CAR target protein and/or functional fragment thereof is displayed on an enveloped virus-like particle to form a CAR target protein-ehlp nanoparticle.
The nanoparticle of claim 1, wherein the viral core protein is selected from the group consisting of: 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, bunyavirus N protein, hepatitis C virus core protein C, hepatitis b virus core protein C and combinations thereof;

Preferably, the retroviral Gag protein is selected from: human immunodeficiency virus Gag protein, monkey immunodeficiency virus Gag protein, mouse leukemia virus Gag protein and bovine leukemia virus Gag protein; more preferably, the human immunodeficiency virus Gag protein comprises an amino acid sequence having at least 80% or more identity to SEQ ID No.1, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity: more preferably, the amino acid sequence of the human immunodeficiency virus Gag protein is shown as SEQ ID No. 1;

preferably, the baculovirus matrix protein M protein is vesicular stomatitis virus M virus core protein;

preferably, the filovirus core protein is ebola virus VP40 virus core protein;

preferably, the coronavirus core protein is selected from the group consisting of the core proteins of novel coronapneumoviruses (SARS-CoV-2), SARS-CoV, MERS-CoV, 229E, NL63, OC43 and HKU 1.
The nanoparticle of claim 1 or 2, wherein the CAR target protein is a membrane protein,

preferably, the membrane protein comprises a transmembrane protein; more preferably, the transmembrane protein comprises a plurality of transmembrane proteins;

Preferably, the method comprises the steps of, the CAR target protein is selected from the group consisting of CD20, claudin18.1, claudin18.2, CD133, GPRC5D, CCR, 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 any of 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;

preferably, the CAR target protein is selected from any one of CD20, claudin18.2, CD133, GPRC5D, CCR, CCR8, CD19, BCMA, GPC3, CD30, CD22, EGFR, EGFRVIII, HER2, or GPCR;

More preferably, the CAR target protein is claudin18.2; preferably, the amino acid sequence of claudin18.2 comprises an amino acid sequence having at least 80% or more identity to SEQ ID No.4, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity: more preferably, the amino acid sequence of Claudin18.2 is shown as SEQ ID No. 4;

more preferably, the CAR target protein is CD20; preferably, the amino acid sequence of CD20 comprises an amino acid sequence having at least 80% or more identity to SEQ ID No.15, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity to SEQ ID No. 15: more preferably, the amino acid sequence of Claudin18.2 is shown as SEQ ID No. 15.
A nanoparticle according to any one of claims 1 to 3, wherein the CAR target protein and/or functional fragment thereof and/or the viral core protein and/or functional fragment thereof is linked to a label;

preferably, the N-terminus and/or C-terminus of the CAR target protein and/or functional fragment thereof and/or the N-terminus and/or C-terminus of the viral core protein and/or functional fragment thereof are linked to a tag; more preferably, the C-terminus of Claudin18.2 or the C-terminus of Gag is labeled with a label; more preferably, the C-terminal end of CD20 or the C-terminal end of Gag is labeled with a label;

Preferably, the label is selected from the group consisting of a detectable label, a purification label, a reporter label, and combinations thereof;

preferably, the detectable label is selected from the group consisting of a fluorescent group, a chemiluminescent label, an electrochemiluminescent label, and combinations thereof;

preferably, the fluorophore is selected from any one of FITC, GFP, RFP, YFP, TRITC, PE, FAM, RRX, TR, cy, cy3, cy5, ECD, PC5.5, PC7, APC-A70, APC-A75, pac-Blue, alexa488, mBBr, 5-IAF, E-118, DTAF, rhodamine Green, and KrO;

preferably, the chemiluminescent label is acridinium ester, isoluminol, horseradish peroxidase or alkaline phosphatase;

preferably, the electrochemiluminescence label is ruthenium terpyridyl or a derivative N-hydroxysuccinimide ester thereof;

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;

preferably, the purification tag is detected by a secondary antibody;

preferably, the reporter tag is selected from any one of c-Myc, HA or luciferase.
The nanoparticle of any one of claims 1-4, wherein the CAR target protein-vlp nanoparticle is selected from a Claudin 18.2-vlp nanoparticle or a CD 20-vlp nanoparticle;

Preferably, said Claudin 18.2 and/or a functional fragment thereof and/or said viral core protein and/or a functional fragment thereof is linked to a marker;

preferably, said CD20 and/or a functional fragment thereof and/or said viral core protein and/or a functional fragment thereof is linked to a label;

preferably, the N-terminus and/or C-terminus of the Claudin 18.2 and/or functional fragment thereof and/or the N-terminus and/or C-terminus of the viral core protein and/or functional fragment thereof are linked to a tag;

preferably, the N-terminus and/or C-terminus of the CD20 and/or functional fragment thereof and/or the N-terminus and/or C-terminus of the viral core protein and/or functional fragment thereof are linked to a label;

preferably, the enveloped virus-like particle scaffold protein is Gag protein;

preferably, the C-terminus of said Claudin 18.2 or the C-terminus of said Gag protein is linked to a marker;

preferably, the C-terminus of the CD20 or the C-terminus of the Gag protein is linked to a label;

preferably, the marker is GPF;

preferably, the amino acid sequence of GFP comprises an amino acid sequence having at least 80% or more identity to SEQ ID No.2, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity: more preferably, the amino acid sequence of GFP is shown as SEQ ID No. 2;

Preferably, the C-terminal end of the Gag protein is linked to a marker GFP (Gag-GFP) whose amino acid sequence comprises an amino acid sequence having at least 80% or more identity to SEQ ID No.3, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or 99% or more identity: more preferably, the amino acid sequence of Gag-GFP is shown as SEQ ID No. 3;

preferably, the C-terminal end of said Claudin 18.2 is linked to a marker GFP (Claudin 18.2-GFP) having an amino acid sequence with at least 80% or more identity to SEQ ID No.5, preferably an amino acid sequence with more than 85%, 90%, 95%, 96%, 97%, 98%, 99% identity, more preferably an amino acid sequence with more than 98% or 99% identity: more preferably, the amino acid sequence of Claudin 18.2-GFP is shown in SEQ ID No. 5.
A method of preparing the nanoparticle of any one of claims 1-5, comprising:

(1) Construction of recombinant plasmids:

respectively constructing a recombinant plasmid containing envelope virus-like particle (eVLP) skeleton proteins and/or functional fragment genes thereof and a recombinant plasmid containing target proteins and/or functional fragment genes thereof; or constructing a recombinant plasmid simultaneously comprising an envelope virus-like particle (eVLP) framework protein and/or a functional fragment gene thereof and a target protein and/or a functional fragment gene thereof; and

(2) Cell transfection, protein expression and particle assembly:

transfecting the recombinant plasmid of the skeleton protein and/or the functional fragment thereof of the enveloped virus-like particle (eVLP) constructed in the step (1) and the recombinant plasmid of the target protein and/or the functional fragment thereof into cells, and expressing the skeleton protein and/or the functional fragment thereof of the enveloped virus-like particle and the target protein and/or the functional fragment thereof; the coated virus-like particle skeleton proteins or functional fragments thereof are assembled to form coated virus-like particles, and the target proteins and/or the functional fragments thereof are displayed on the coated virus-like particles to form target protein-eVLP nanoparticles; and, optionally,

(3) Purifying target protein-eVLP nanoparticles;

preferably, the coding nucleic acid sequence of the GAG comprises a nucleotide sequence having at least 80% or more identity to SEQ ID No.7, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or more identity to SEQ ID No. 7: more preferably, the amino acid sequence of GFP is shown as SEQ ID No. 7;

preferably, the Gag-GFP encoding nucleic acid sequence comprises a nucleotide sequence having at least 80% or more identity to SEQ ID No.8, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or more identity to SEQ ID No. 8: more preferably, the amino acid sequence of GFP is shown as SEQ ID No. 8;

Preferably, the coding nucleic acid sequence of Claudin 18.2 comprises a nucleotide sequence having at least 80% or more identity to SEQ ID No.9, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity: more preferably, the amino acid sequence of GFP is shown as SEQ ID No. 9;

preferably, the Claudin 18.2-GFP encoding nucleic acid sequence comprises a nucleotide sequence having at least 80% or more identity to SEQ ID No.10, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity: more preferably, the amino acid sequence of GFP is shown as SEQ ID No. 10;

preferably, the coding nucleic acid sequence of CD20 comprises a nucleotide sequence having at least 80% or more identity to SEQ ID No.16, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or more identity to SEQ ID No. 16: more preferably, the amino acid sequence of GFP is shown as SEQ ID No. 16.
The method of claim 6, wherein the cell is selected from the group consisting of a prokaryotic host cell and a eukaryotic host cell;

preferably, the prokaryotic host cell is any one of a bacterial cell, bacillus subtilis, and mycobacterium; preferably, the bacterial cell is escherichia coli;

preferably, the eukaryotic host cell is selected from any one of an animal cell, a plant cell or a fungus;

preferably, the eukaryotic host cell is selected from any one of yeast, insect, avian, plant, caenorhabditis elegans and mammalian host cells;

preferably, the insect cell is selected from any one of Spodoptera frugiperda cells (e.g., sf9, sf 21), spodoptera frugiperda cells (e.g., high Five cells), and Drosophila S2 cells;

preferably, the fungus is selected from any one of Saccharomyces cerevisiae, kluyveromyces lactis, candida species (e.g., candida albicans or candida glabrata), aspergillus nidulans, schizosaccharomyces pombe, pichia pastoris, and yarrowia lipolytica;

preferably, the mammalian cells are selected from any one of 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.
A method of detecting a CAR positive expression rate, comprising: incubating the nanoparticle of any one of claims 1-5 or the nanoparticle prepared by the method of claim 6 or 7 with a CAR-modified cell, and then detecting; preferably, the cells are washed after incubation of the cells, prior to detection;

preferably, the CAR-modified cell is selected from any one of a CAR-like (HEK 293) cell, a CAR-T cell, a CAR-NK cell, a CAR-M cell, a CAR-NKT cell, a CAR-Treg cell, and a CAR- γδ T cell;

preferably, the temperature of the incubation is between 2 ℃ and 40 ℃, preferably 37 ℃, and/or the time of the incubation is between 15min and 2 hours, preferably 1 hour; and/or, the incubated CO ₂ The concentration of (2) to 8%, preferably 5%;

preferably, the number of washes is 1-6, preferably 3;

preferably, the wash buffer is 0.5% -5% bsa, preferably 2% bsa;

preferably, the detection is selected from any one of flow cytometry detection, immunodetection, ELISA, SPR, BLI.
The method of claim 8, comprising incubating the Claudin 18.2-ehvlp nanoparticle or CD 20-ehvlp nanoparticle with a CAR-modified cell and then detecting;

preferably, the enveloped virus-like particle scaffold protein is Gag protein;

Preferably, the C-terminus of said Claudin 18.2 or the C-terminus of said Gag protein is linked to a marker;

preferably, the C-terminus of the CD20 or the C-terminus of the Gag protein is linked to a label.
A kit comprising the nanoparticle of any one of claims 1-5 or the nanoparticle prepared by the method of claim 6 or 7;

preferably, the nanoparticle is a CAR target protein-ehlp nanoparticle;

preferably, the kit comprises a labeled CAR target protein-ehlp nanoparticle;

preferably, the kit comprises a labeled Claudin 18.2-ehlp nanoparticle;

preferably, the kit comprises a labeled CD 20-ehlp nanoparticle;

preferably, the kit is an ELISA kit, SPR kit or BLI kit;

preferably, the ELISA kit comprises the CAR target protein-ekp, an ELISA plate, a blocking solution, a sample diluent, an enzyme conjugate, a concentrated wash solution, an enzyme substrate solution, and a stop solution;

preferably, the SPR kit comprises the CAR target protein-ehlp nanoparticle;

preferably, the BLI kit comprises the CAR target protein-ehlp nanoparticle.