Chimeric protein and use thereof

Abstract

Provided are a chimeric protein and a use thereof. The chimeric protein comprises: a transmembrane transport region, a target protein display region, a membrane anchoring region, and an intracellular signal transduction region which are sequentially connected, wherein the transmembrane transport region comprises a transmembrane transport peptide segment; the target protein display region comprises a target protein peptide segment; the membrane anchoring region comprises a membrane anchoring peptide segment; and the intracellular signal transduction region comprises a budding aid peptide segment.

Description

Chimeric protein and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of biology. Specifically, the present application relates to a chimeric protein and application thereof. BACKGROUND

[0002] Chimeric protein display (CPD) technology is a protein modification strategy. The modified protein can be expressed and located on the membrane structure surface of a carrier, and can realize remodeling of protein structure, change of protein function and protein subcellular localization, etc., and has wide application prospects in the fields of vaccine development, biopharmaceuticals and bioengineering, etc.

[0003] However, the current chimeric protein display technology still needs to be studied.

[0004] SUMMARY

[0005] The present application aims to at least partially solve the technical problems existing in the prior art. To this end, the present application provides a chimeric protein. The target protein modified by the chimeric protein can be displayed on the membrane structure surface of a carrier, which is conducive to promoting signal transmission between cells, can be used as a target for immune recognition and drug action, can enhance immune response, can realize specific targeting, and can be used for drug screening, and can realize remodeling of protein structure, change of protein function and protein subcellular localization, etc., and has important application prospects in the fields of vaccine development, antibody engineering (such as antibody preparation or modification, etc.), gene therapy, etc.

[0006] In one aspect of the present application, the present application provides a chimeric protein. According to an embodiment of the present application, the chimeric protein comprises, in sequence: a transmembrane transport region, a target protein display region, a membrane anchoring region and an intracellular signal transduction region; the transmembrane transport region comprises a transmembrane transport peptide segment; the target protein display region comprises a target protein peptide segment; the membrane anchoring region comprises a membrane anchoring peptide segment; and the intracellular signal transduction region comprises a positioning auxiliary peptide segment.

[0007] The present application modifies the transmembrane transport region, the membrane anchoring region and the intracellular signal transduction region on the target protein display region, so that the target protein can be displayed on the membrane structure surface of a carrier, which is conducive to promoting signal transmission between cells, can be used as a target for immune recognition and drug action, can enhance immune response, can realize specific targeting, and can be used for drug screening, and can realize remodeling of protein structure, change of protein function and protein subcellular localization, etc., and has important application prospects in the fields of vaccine development, antibody engineering (such as antibody preparation or modification, etc.), gene therapy, etc.

[0008] In another aspect of the present application, a chimeric protein construct is provided. According to embodiments of the present application, the chimeric protein construct comprises: the chimeric protein as described above; and a carrier, wherein the chimeric protein is loaded on a surface of a membrane structure of the carrier.

[0009] In yet another aspect of the present application, a nucleic acid molecule is provided. According to embodiments of the present application, the nucleic acid molecule encodes the chimeric protein as described above.

[0010] In yet another aspect of the present application, a recombinant expression vector is provided. According to embodiments of the present application, the recombinant expression vector comprises: the nucleic acid molecule as described above.

[0011] In yet another aspect of the present application, a recombinant cell is provided. According to embodiments of the present application, the recombinant cell comprises: the recombinant expression vector as described above.

[0012] In yet another aspect of the present application, a medicament is provided. According to embodiments of the present application, the medicament comprises: the chimeric protein as described above, the chimeric protein construct as described above, or the recombinant cell as described above.

[0013] In yet another aspect of the present application, use of the chimeric protein as described above, the chimeric protein construct as described above, or the recombinant cell as described above in the manufacture of a medicament is provided. According to embodiments of the present application, the medicament is for preventing or treating a disease.

[0014] In yet another aspect of the present application, a method for preventing or treating a disease is provided. According to embodiments of the present application, the method comprises: administering the chimeric protein as described above, the chimeric protein construct as described above, or the recombinant cell as described above to a subject.

[0015] In yet another aspect of the present application, use of the chimeric protein as described above, the chimeric protein construct as described above, or the recombinant cell as described above in screening a medicament is provided.

[0016] In yet another aspect of the present application, a method for screening a medicament is provided. According to embodiments of the present application, the method comprises: co-culturing the chimeric protein as described above, the chimeric protein construct as described above, or the recombinant cell as described above with a candidate medicament; detecting a detection signal associated with the chimeric protein before and after the co-culturing; and when the detection signal associated with the chimeric protein changes before and after the co-culturing, the candidate medicament is a target medicament that can react with the target protein.

[0017] Additional aspects and advantages of the present application will be made apparent from the following description. BRIEF DESCRIPTION OF DRAWINGS

[0018] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the references to the figures, in which:

[0019] FIG. 1 shows a schematic diagram of a chimeric protein structure according to an embodiment of the present application;

[0020] FIG. 2 shows a schematic diagram of CAD drugs using VSV virus as a carrier according to an embodiment of the present application;

[0021] FIG. 3 shows a cell membrane localization electron microscope image of a chimeric antigen according to an embodiment of the present application;

[0022] FIG. 4 shows an EGF protein expression level analysis diagram of a secretory form EGF antigen vaccine (SP-EGF) and a chimeric antigen EGF vaccine (CAD-EGF) according to an embodiment of the present application;

[0023] FIG. 5 shows a chimeric antigen particle localization electron microscope image according to an embodiment of the present application;

[0024] FIG. 6 shows a schematic diagram of a secretory antigen and a CAD designed antigen structure according to an embodiment of the present application;

[0025] FIG. 7 shows a comparison diagram of EGF antigen immunization results according to an embodiment of the present application;

[0026] FIG. 8 shows a comparison diagram of mTNFα antigen immunization results according to an embodiment of the present application;

[0027] FIG. 9 shows a diagram of immunization effects of a CAD designed and DR modified vaccine of an SVN antigen according to an embodiment of the present application;

[0028] FIG. 10 shows a diagram of immunization effects of a CAD designed and DR modified vaccine of a HER2 antigen according to an embodiment of the present application;

[0029] FIG. 11 shows a diagram of FCGR and cetuximab binding analysis according to an embodiment of the present application;

[0030] FIG. 12 shows a diagram of CPD cetuximab SCFV particle targeting EGFR positive cell analysis according to an embodiment of the present application;

[0031] FIG. 13 shows a diagram of CPD-gp160 assisted targeting T cell analysis according to an embodiment of the present application. DETAILED DESCRIPTION

[0032] Embodiments of the present application are described in detail below. The embodiments described below are examples for explaining the present application and should not be understood as limiting the present application.

[0033] It should be noted that the terms "first", "second" are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined as "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise stated, the meaning of "a plurality" is two or more.

[0034] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges or values should be construed to be roughly about the ranges or values. For ranges, the endpoints are included as well as intervening points to the same extent as points explicitly listed. For values, the same applies.

[0035] In the present document, the term "comprising" or "including" is an open term, i.e. including the contents indicated by the present application, but not excluding other contents.

[0036] Chimeric protein

[0037] In one aspect of the present application, a chimeric protein is provided. According to an embodiment of the present application, referring to FIG. 1, the chimeric protein comprises, in sequence: a transmembrane transport region, a target protein display region, a membrane anchoring region, and an intracellular signal transduction region; the transmembrane transport region comprises a transmembrane transport peptide segment; the target protein display region comprises a target protein peptide segment; the membrane anchoring region comprises a membrane anchoring peptide segment; and the intracellular signal transduction region comprises a targeting peptide segment.

[0038] In the chimeric protein according to an embodiment of the present application, by connecting the target protein display region to the transmembrane transport region (TTS), the target protein can be transported to the membrane structure through the secretion pathway of the cell and possibly embedded in the membrane structure.

[0039] By connecting the target protein to the membrane anchoring region (AR), which usually contains one or more transmembrane helices, the target protein can be fixed at a specific location of the membrane by penetrating the cell membrane. This anchoring effect helps to stabilize the expression of the protein on the membrane and can affect its function or interaction with other molecules.

[0040] By connecting the membrane anchoring region to the intracellular signal transduction region (TR), it can interact with intracellular signaling molecules to achieve intracellular signal transduction.

[0041] By the above-mentioned chimeric protein display method, the localization, stability and function of the target protein in the cell can be accurately controlled, which has important application prospects in the fields of vaccine development, antibody engineering, gene therapy, etc.

[0042] According to embodiments of the present application, the target protein comprises an antigen protein. The localization of the antigen protein to the membrane structure is beneficial to enhance the immune response reaction, for example, beneficial to the recognition of the BCR to the antigen protein, thereby activating the B cell and inducing the humoral immunity. According to embodiments of the present application, the antigen protein comprises a self-antigen and / or a heterologous antigen. When the target protein comprises an antigen protein, the constructed chimeric protein can be referred to as CAD.

[0043] According to embodiments of the present application, the self-antigen comprises at least one of HER2, PSA, TRP-2, VEGF, EpCAM, GPC3, MSLN, TNFα, PCSK9, EGFR, EGF, Survivin, ANGPTL3 and IgE.

[0044] According to embodiments of the present application, the heterologous antigen comprises a pathogen protein.

[0045] According to embodiments of the present application, the pathogen protein comprises at least one of Zika virus protein, HIV protein, herpes virus protein, cytomegalovirus protein, novel coronavirus protein, and coxsackievirus protein.

[0046] According to embodiments of the present application, the target protein comprises at least one of a receptor ligand, a single-chain variable fragment of an antibody, and a virus protein. In this way, the chimeric protein can target and locate the drug molecules to specific tissues or cells.

[0047] In the present application, the term “receptor ligand” refers to a molecule capable of specifically binding to a receptor on the cell surface or in the cell, which can include proteins, polypeptides, lipids, hormones, neurotransmitters, cytokines, etc. After the ligand binds to the receptor, the conformation of the receptor can be changed, leading to the activation of the receptor, and then triggering the downstream signal cascade reaction.

[0048] The single-chain variable fragment (scFv) of an antibody is formed by connecting the variable regions of the light chain and the heavy chain of the antibody, which can provide highly specific and high-affinity binding to the antigen.

[0049] Different virus proteins can recognize different receptors in the body, and this property can be used to achieve the targeting of different chimeric proteins. Exemplarily, the virus protein can be a glycoprotein.

[0050] According to an embodiment of the present application, the transmembrane transport region has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereto. The transmembrane transport region with the above sequence can guide the transport of the protein from the inside of the cell to the outside of the cell, which is the first step to ensure that the target protein can be displayed on the membrane.

[0051] According to an embodiment of the present application, the membrane anchoring region has an amino acid sequence as shown in any one of SEQ ID NO: 2, 3, 14, 15 or an amino acid sequence with at least 80% homology thereto. The membrane anchoring region with the above sequence can change the protein localization, such as anchoring the protein in a secretory form on the membrane.

[0052] According to an embodiment of the present application, the intracellular signal transduction region has an amino acid sequence as shown in any one of SEQ ID NO: 4, 5, 28 or an amino acid sequence with at least 80% homology thereto. The intracellular signal transduction region with the above sequence is the region of signal interaction between the protein inside and outside the membrane, and the binding carrier realizes the accurate positioning of the carrier particle membrane.

[0053] According to an embodiment of the present application, a helping region (HR) is further included between the target protein display region and the membrane anchoring region. In this way, the protein positioned on the membrane structure surface of the carrier can be better displayed, and the immune response reaction is further enhanced, and the targeting is improved.

[0054] According to an embodiment of the present application, the helping region has an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence with at least 80% homology thereto. The helping region with the above sequence can further better display the protein positioned on the membrane structure surface of the carrier, enhance the immune response reaction, and improve the targeting.

[0055] Chimeric protein construct

[0056] In another aspect of the present application, a chimeric protein construct is provided. According to an embodiment of the present application, the chimeric protein construct comprises: the chimeric protein described above; and a carrier, wherein the chimeric protein is loaded on the membrane structure surface of the carrier. In the chimeric protein construct of the present application, the target protein can be displayed on the membrane structure surface of the carrier, which is conducive to promoting the signal transmission between cells, can be used as a target for immune recognition and drug action, enhances the immune response reaction, realizes specific targeting, and can also be used for drug screening, and has important application prospects in the fields of vaccine development, antibody engineering, gene therapy, etc.

[0057] According to embodiments of the present application, the vector comprises at least one of a viral vector, a virus-like particle, an exosome, and a cell.

[0058] According to embodiments of the present application, the virus comprises a modified virus or a defective virus. The “modified virus” refers to a virus that is improved or changed in certain properties by genetic engineering or mutation. The “defective virus” refers to a virus that cannot independently complete the life cycle due to genome deletion or mutation.

[0059] According to embodiments of the present application, the virus comprises a DNA virus or an RNA virus.

[0060] According to embodiments of the present application, the virus comprises at least one of a lentivirus, an adenovirus, an adeno-associated virus, a poxvirus, a herpes simplex virus, a vesicular stomatitis virus, a lymphocytic choriomeningitis virus, a measles virus, a coronavirus, and a Newcastle disease virus.

[0061] The chimeric protein is co-displayed with the envelope protein VSV-G protein on the surface of the virus particle using the vesicular stomatitis virus (VSV) as the vector, and the nucleic acid sequence of the chimeric antigen can also be carried on the virus genome, which can be expressed in the body after immunization to play the related functions of the protein (FIG. 2).

[0062] Nucleic acid molecules, recombinant expression constructs, and recombinant cells

[0063] In yet another aspect of the present application, a nucleic acid molecule is provided. According to embodiments of the present application, the nucleic acid molecule encodes the aforementioned chimeric protein. Thus, the nucleic acid molecule of the present application can express the aforementioned chimeric protein, so that the target protein is displayed on the membrane structure surface of the vector, which is beneficial to promote the signal transmission between cells, can be used as a target for immune recognition and drug action, enhances the immune response, realizes specific targeting, and can also be used for drug screening, and has important application prospects in the fields of vaccine development, antibody engineering, gene therapy, etc.

[0064] According to embodiments of the present application, the nucleic acid molecule comprises a first nucleic acid molecule, a second nucleic acid molecule, a third nucleic acid molecule, and a fourth nucleic acid molecule connected in sequence;

[0065] The first nucleic acid molecule encodes the transmembrane transport region;

[0066] The second nucleic acid molecule encodes the target protein display region;

[0067] The third nucleic acid molecule encodes the membrane anchoring region;

[0068] The fourth nucleic acid molecule encodes the intracellular signal transduction region;

[0069] The first nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 7 or a nucleotide sequence with at least 80% homology thereto, whereby the amino acid sequence shown in SEQ ID NO: 1 can be encoded.

[0070] The third nucleic acid molecule has a nucleotide sequence shown in any one of SEQ ID NO: 8, 9, 21, 22 or a nucleotide sequence with at least 80% homology thereto, whereby the amino acid sequence shown in SEQ ID NO: 2, 3, 14, 15 can be encoded respectively.

[0071] The fourth nucleic acid molecule has a nucleotide sequence shown in any one of SEQ ID NO: 10, 11, 35 or a nucleotide sequence with at least 80% homology thereto, whereby the amino acid sequence shown in SEQ ID NO: 4, 5, 28 can be encoded respectively.

[0072] In the present application, the term "homology" refers to the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences. The term "at least 80% homology" refers to at least 80% similarity to each reference sequence, which can be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%.

[0073] According to an embodiment of the present application, the second nucleic acid molecule and the third nucleic acid molecule further comprise:

[0074] A fifth nucleic acid molecule, the fifth nucleic acid molecule encoding the helper display region;

[0075] The fifth nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO: 12 or a nucleotide sequence with at least 80% homology thereto. By using the sequence, the helper display region encoded by the sequence can modify the target protein, and the obtained chimeric protein can be better positioned to the membrane structure surface of the carrier, achieving specific targeting.

[0076] In yet another aspect of the present application, a recombinant expression construct is provided. According to embodiments of the present application, the recombinant expression construct comprises the nucleic acid molecule described above. The recombinant expression construct of the present application can express the chimeric protein described above in a cell. When the nucleic acid molecule described above is linked to an expression construct, the nucleic acid molecule can be linked to control elements on the expression construct directly or indirectly, as long as the control elements can control the translation and expression of the nucleic acid molecule. Of course, the control elements can be directly from the expression construct itself or exogenous, i.e., not from the expression construct itself. Of course, the nucleic acid molecule can be operably linked to the control elements.

[0077] As used herein, "operably linked" means that an exogenous gene is linked to a recombinant expression construct, so that the control elements in the recombinant expression construct, such as transcription control sequences and translation control sequences, etc., can perform their intended function of regulating the transcription and translation of the exogenous gene.

[0078] According to embodiments of the present application, the recombinant expression vector comprises at least one of a plasmid, a viral vector with a membrane structure, a virus-like particle with a membrane structure, an exosome, and a cell. Illustratively, the virus comprises at least one of a lentivirus, an adenovirus, an adeno-associated virus, a poxvirus, a herpes simplex virus, a vesicular stomatitis virus, a lymphocytic choriomeningitis virus, a measles virus, a coronavirus, and a Newcastle disease virus. After the recombinant expression vector according to some specific embodiments of the present application is introduced into a suitable recipient cell, the expression of the chimeric protein described above can be effectively realized under the mediation of a regulatory system, and then a large amount can be obtained in vitro.

[0079] In yet another aspect of the present application, a recombinant cell is provided. According to embodiments of the present application, the recombinant cell comprises the recombinant expression vector described above. Thus, the recombinant expression vector can express the chimeric protein described above in a cell, so that the target protein can be displayed on the surface of the membrane structure of the vector, which is beneficial for promoting signal transmission between cells, serving as a target for immune recognition and drug action, enhancing immune response, achieving specific targeting, and being used for drug screening, and having important application prospects in the fields of vaccine development, antibody engineering, gene therapy, etc.

[0080] It should be noted that the recombinant cell of the present application is not an animal fertilized egg cell or a plant cell, and can comprise a HEK 293T cell, a HeLa cell, a COS cell, a Vero cell, an MDCK cell, a CHO cell, a BHK-21 cell, etc.

[0081] It should be noted that the features and advantages described above for the chimeric protein also apply to the nucleic acid molecule, the recombinant expression construct, and the recombinant cell, which will not be described here again.

[0082] Pharmaceutical, uses and methods

[0083] In yet another aspect of the present application, a pharmaceutical is provided. According to embodiments of the present application, the pharmaceutical comprises the aforementioned chimeric protein, the aforementioned chimeric protein construct, or the aforementioned recombinant cell. In the pharmaceutical of the present application, the target protein can be displayed on the surface of the membrane structure of the carrier, which is conducive to promoting the signal transmission between cells, can be used as a target for immune recognition and drug action, enhances the immune response, achieves specific targeting, and thus enhances the drug efficacy.

[0084] According to embodiments of the present application, the pharmaceutical comprises a vaccine.

[0085] According to embodiments of the present application, the pharmaceutical further comprises a pharmaceutically acceptable excipient.

[0086] In the present application, the term "pharmaceutically acceptable" means that the substance or chimeric protein construct must be chemically and / or toxicologically compatible with other ingredients contained in the formulation and / or the mammal being treated therewith. Preferably, the "pharmaceutically acceptable" of the present application means that it is approved by the national government or listed in the Chinese Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, especially in humans.

[0087] In the present application, the term "pharmaceutically acceptable excipient" is a pharmaceutically or food acceptable carrier, solvent, suspending agent or excipient for delivering the active component (e.g. self-replicating RNA molecule) in the pharmaceutical of the present application to animals or humans. Exemplary excipients can be liquid or solid, including but not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ion strength enhancers, preservatives, carriers, flow aids, sweeteners, dyes / colorants, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, emulsifiers, sprays, compressed air or other suitable gases, or other suitable non-active ingredients used with the pharmaceutical compounds. Examples of excipients include various lactose, mannitol, oils such as corn oil, buffers such as PBS, saline, polyethylene glycol, glycerol, polypropylene glycol, dimethyl sulfoxide, amides such as dimethylacetamide, proteins such as albumin, monosaccharides and oligosaccharides such as glucose, lactose, cyclodextrin and starch.

[0088] In yet another aspect of the present application, the aforementioned chimeric protein, the aforementioned chimeric protein construct, or the aforementioned recombinant cell is used for the preparation of a pharmaceutical. According to embodiments of the present application, the pharmaceutical is used for preventing or treating diseases. In the pharmaceutical of the present application, the target protein can be displayed on the surface of the membrane structure of the carrier, which is conducive to promoting the signal transmission between cells, can be used as a target for immune recognition and drug action, enhances the immune response, achieves specific targeting, and thus enhances the drug efficacy, and can be used for preventing or treating diseases.

[0089] In yet another aspect of the present application, a method for preventing or treating a disease is provided. According to embodiments of the present application, the method comprises administering the aforementioned chimeric protein, the aforementioned chimeric protein construct, or the aforementioned recombinant cell to a subject. Thus, by using the method of the present application, the target protein administered to the subject can be displayed on the surface of the membrane structure of the carrier, which is beneficial for promoting intercellular signal transduction, enhancing immune response, achieving specific targeting, and thus achieving prevention or treatment of the disease.

[0090] According to embodiments of the present application, the disease comprises cancer, autoimmune disease, infectious disease, metabolic disease, or neurological disease.

[0091] According to embodiments of the present application, the cancer comprises non-small cell lung cancer, papillary thyroid cancer, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, cholangiocarcinoma or sarcoma, acute myeloid leukemia, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma, or gastric cancer;

[0092] The autoimmune disease comprises lupus erythematosus, rheumatoid arthritis, or psoriasis;

[0093] The metabolic disease comprises diabetes, hypertension, or hyperlipidemia;

[0094] The infectious disease comprises hepatitis B, AIDS, or hand-foot-mouth disease;

[0095] The neurological disease comprises Alzheimer's disease.

[0096] In yet another aspect of the present application, the aforementioned chimeric protein, the aforementioned chimeric protein construct, or the aforementioned recombinant cell is used for screening drugs. In the chimeric protein of the present application, the target protein is modified and can be displayed on the surface of the membrane structure of the carrier, which can be used as a target for drug action and applied to drug screening.

[0097] In yet another aspect of the present application, a method for screening drugs is provided. According to embodiments of the present application, the method comprises co-culturing the aforementioned chimeric protein, the aforementioned chimeric protein construct, or the aforementioned recombinant cell with a candidate drug; detecting a detection signal related to the chimeric protein before and after the co-culturing; and when the detection signal related to the chimeric protein changes before and after the co-culturing, the candidate drug is a target drug that can react with the target protein.

[0098] In the chimeric protein of the present application, the target protein is modified to be displayed on the surface of the membrane structure of the carrier. If it reacts with the target drug, a change in the detection signal associated with the chimeric protein will occur. Further, by detecting whether the detection signal associated with the chimeric protein changes before and after co-culture, it is confirmed whether the candidate drug is the target drug.

[0099] In some embodiments, the term "detection signal associated with the chimeric protein" can be a fluorescence signal, an electrical signal, a chemiluminescence signal, a cell activity signal, etc.

[0100] It should be noted that the features and advantages described above for the chimeric protein, the chimeric protein construct or the recombinant cell also apply to the drug, the use and the method, which will not be repeated here.

[0101] The scheme of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0102] Example 1

[0103] Membrane localization of chimeric protein

[0104] 1. Use the chimeric protein to design a chimeric-EGF antigen plasmid. Specifically, design a first nucleic acid molecule, a second nucleic acid molecule, a third nucleic acid molecule, and a fourth nucleic acid molecule, respectively. The first nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 7 and can encode a transmembrane transporter protein; the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 13 and can encode EGF; the third nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 8 and can encode a membrane anchor region; and the fourth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 10 and can encode an intracellular signal transduction region.

[0105] The first nucleic acid molecule, the second nucleic acid molecule, the third nucleic acid molecule, and the fourth nucleic acid molecule are sequentially connected from the 5' end to synthesize a new coding nucleotide sequence and a stop codon TGA is added at the 3' end. The entire synthesized sequence is inserted into a pcDNA3.1 expression vector.

[0106] 2. Transfect the plasmid into 293T cells, and after 24 hours, the cells are fixed with 4% paraformaldehyde, and then stained with WGA594, DAPI, and EGF antibody. Cells without transfection are used as the Ctrl group.

[0107] The results are shown in Figure 3. It can be seen that the chimeric-EGF has a clear cell membrane localization.

[0108] Example 2

[0109] Chimeric antigen drug particle membrane localization

[0110] The VSV viral vector and VSV viral defective vector (VSV-ΔG) were used to construct the secretory form of EGF antigen vaccine (SP-EGF) and chimeric antigen EGF vaccine (CAD-EGF), respectively. Specifically, the nucleotide sequence encoding the foreign gene was inserted before the L gene of the VSV genome, and a packaging plasmid was synthesized. The vaccine drug particles were purified by upstream production (using Vero cells as production cells) and downstream ultracentrifugation. The EGF content in the drug particle harvest and purified solution was determined by ELISA. The specific experimental procedure was as follows: BHK-21 cells were transfected with the packaging plasmid (containing the first nucleic acid molecule, the second nucleic acid molecule, the third nucleic acid molecule, and the fourth nucleic acid molecule in the chimeric-EGF antigen plasmid of Example 1) and the helper plasmid (pcDNA3.1-N, pcDNA3.1-P, pcDNA3.1-G, pcDNA3.1-L), and after 72 h, the seed was filtered and harvested, and the seed was added to Vero cells (VSV viral vector production) or VSV-G transfected vero cells (VSV-ΔG production). After 24 h, the supernatant was harvested, and the residual cell debris was removed to obtain the clarified supernatant. The clarified supernatant was digested with Benzonase to remove the Vero host cell DNA. The enzyme-digested solution was concentrated by hollow fiber column ultrafiltration, and then subjected to 30% and 55% sucrose density gradient centrifugation. The target layer was collected, and the buffer was replaced to remove the sugar to obtain the purified supernatant. The EGF protein content in the solution was determined by ELISA.

[0111] The results are shown in Figure 4. The EGF expressed in the secretory form (SP-EGF) had a higher content in the harvest, but almost no residual EGF protein could be detected after purification. In contrast, the chimeric antigen group (CAD-EGF) could detect EGF protein after purification, whether it was the VSV virus group or the defective virus (VSV-ΔG) group, indicating that the EGF antigen was located on the particle.

[0112] The VSV-ΔG-CAD-EGF particles were mixed with 4% paraformaldehyde in an equal volume, 20 μl of which was dropped on a plastic film, and the front of the electron microscope carbon grid was buckled on the droplet and placed for 20 min. After washing with PBS and neutralizing with glycine, 5% BSA was blocked for 10 min. 20 μl of diluted primary antibody was added and incubated for 3 h. The colloidal gold secondary antibody was diluted at a ratio of 1:100, and the carbon grid was placed on it and incubated for 1 h. After 1% glutaraldehyde fixation and washing, phosphotungstic acid was used for staining, and natural air drying or oven drying was followed by electron microscope observation.

[0113] The results are shown in Figure 5, and there is obvious enrichment of VSV-G and EGF on the VSV-ΔG-CAD-EGF particles.

[0114] Example 3

[0115] Screening of sequences of functional regions of CAD structure

[0116] The core functional regions of CAD structure are the membrane anchoring region (AR) and the intracellular signal transduction region (TR), which are derived from 5 human transmembrane proteins (CD4, Integrin, VAMP2, ICAM1, HLA-Dra1) and 4 viral glycoproteins (VSV-G, MARV-G (Marburg virus glycoprotein), ZEBOV-G (Zaire ebolavirus glycoprotein), LCMV-GP (Lymphocytic choriomeningitis virus glycoprotein).

[0117] Table 1 Sequence information of membrane anchoring region

[0118] Table 2 Sequence information of intracellular signal transduction region

[0119] The specific method for synthesizing plasmids is to replace the AR and TR regions of the CAD part in the packaging plasmid of the CAD-EGF vaccine constructed by the VSV-ΔG vector in Example 2 in whole or in part. The specific process is: synthesis of plasmids, packaging of particles, production and purification of particles. The purified particles are subjected to magnetic bead flow detection of the EGF protein content on the surface of the particles, and the specific method is: the magnetic beads are washed and resuspended in buffer, and an excess of VSV-G capture antibody is added and incubated with the magnetic beads. Use magnetic bead adsorption to wash away the unbound antibody. Resuspend the magnetic beads with diluted particle suspension and react fully on a rotary mixer. Wash away the unbound particles and resuspend in buffer. Add EGF detection antibody with a fluorescent group, and after incubation, washing and resuspension, use for flow detection.

[0120] The experimental results are shown in the following table. In addition to the sequence derived from VSV-G, the CAD particles derived from the HLA-Dra1 sequence also have a good particle membrane display positive rate (> 90%). And the AR region of VSV-G and the TR region of HLA-Dra1 are combined, and the particle membrane display positive rate is still > 90%, which is the best sequence combination. In addition, in addition to the sequence combination of ICAM1 and LCMV-G, other sequences also have different degrees of particle membrane display positive rate, which can be used as a target for regulating the positive rate of particle membrane display of the target protein.

[0121] Table 3 Particle membrane display positive rate of different membrane anchor regions and budding helper regions

[0122] Example 4

[0123] CAD antigen modified vaccine achieves strong immune response

[0124] The VSV-ΔG vector was used to design a soluble protein (SP) vaccine and a chimeric antigen display (CAD) vaccine to further verify the immune enhancement effect induced by the CAD technology. The specific operation is as follows: different modified antigen sequences are inserted into the VSV virus defective vector (VSV-ΔG) genome packaging plasmid, and the construction method of the CAD-EGF antigen plasmid is referred to Example 1. The difference between the construction method of the control CAD-EGF antigen plasmid and Example 1 is that it does not contain AR and TR, and the plasmid structure is shown in Figure 6. The vaccine particles are packaged, amplified and ultracentrifuged in vitro to obtain VSV-ΔG-SP-EGF (antigen soluble expression group) and VSV-ΔG-CAD-EGF (chimeric antigen (CAD) modified group). The purified vaccines of different groups are immunized in mice at the same dose. The specific experiment is as follows: 6-8 week old Balb / C female mice are used, and muscle immunization is used, and the time is 0 day and 14 days. The blood sampling time is 0 day, 7 days, 15 days, 21 days, 28 days and 35 days. The mouse serum titer is detected by indirect ELISA method. The antigen design is shown in Figure 6.

[0125] Verification of antigen selection:

[0126] 1) hEGF

[0127] As shown in Figure 7, the immune results show that 35 days after the primary immunization, the antigen soluble expression group cannot induce specific antibody production, and the chimeric antigen (CAD) modified group can reach a high level of antibody (antibody titer > 128000).

[0128] 2) mTNFα

[0129] As shown in Figure 8, the immune results show that 35 days after the first immunization, the antigen secretion expression group cannot induce specific antibody production, the chimeric antigen (CAD) modified group can reach a certain level of antibody.

[0130] Example 5

[0131] Enhancement of the help region (HR) in CPD modification on the immune effect of the vaccine

[0132] The help region (HR) (SEQ ID NO: 12) is introduced in the CAD sequence design for certain antigens to achieve better immune effect. The specific process is as follows: different modified antigen sequences are inserted into the VSV virus defective vector (VSV-ΔG) packaging plasmid, the AR nucleotide sequence in the CAD sequence is SEQ ID NO: 9, and the TR nucleotide sequence is SEQ ID NO: 10. The vaccine particles are packaged, amplified and ultracentrifuged in vitro. The purified vaccines of different groups are immunized in mice at the same dose. The specific experiment is as follows: 6-8 week old Balb / C female mice are used, and the muscle immunization method is used, and the time is 0 day and 14 days. The blood sampling time is 0 day, 7 days, 15 days, 21 days, 28 days and 35 days.

[0133] SVN sequence:

[0134] Verification of antigen selection:

[0135] 1) Survivin (SVN)

[0136] As shown in Figure 9, the immune results show that 35 days after the first immunization, compared with the secreted antigen, the chimeric antigen modified (CAD-SVN) vaccine can immunize a certain degree of specific antibody (titer > 10 3 ), and the antibody titer needs to be improved. After adding the help region (HR) on the basis of the chimeric antigen modification, the specific antibody titer is significantly improved, and can reach 10 5 .

[0137] 2) HER2

[0138] As shown in Figure 10, the immune results show that 35 days after the first immunization, the HER2 antigen in the secretion state can immunize a certain degree of antibody, and the CAD modification does not achieve significant improvement. After adding the help region (HR) on the basis of the chimeric antigen modification, the antibody level induced by the vaccine realizes a great breakthrough, and can reach 10 5 above.

[0139] Example 6

[0140] Specific targeting of drugs by CPD

[0141] Targeting proteins include, but are not limited to, ligands for specific receptors, antibody SCFV regions, viral proteins, etc.

[0142] Verification of target selection:

[0143] 1) Fc receptor FCGR

[0144] The Fc receptor FCGR designed by CPD can be displayed on the surface of the particle membrane to achieve binding with existing antibody drugs and change the targeting of the drugs. Taking the monoclonal antibody cetuximab of EGFR as an example, indirect ELISA detection is used to verify the binding of the antibody and CPD-FCGR.

[0145] The specific method is: 1. CPD-FCGR particle construction: referring to Example 2, the Fc receptor FCGR (AR: SEQ ID NO: 8, TR: SEQ ID NO: 11) designed by CPD is inserted into the genome of the VSV virus defective vector (VSV-ΔG), and the particle is packaged, amplified and ultracentrifuged. Purification. 2. Cetuximab and CPD-FCGR particles were mixed at 4°C for 2h for binding reaction. The mixture was added to the well plate coated with EGFR protein for adsorption, and the particles with cetuximab connected could bind to EGFR protein. After washing away the unbound components, the content of cetuximab-particle complex in the mixture was displayed using anti-VSV-G antibody and corresponding secondary antibody reaction.

[0146] HER2 sequence:

[0147] As shown in the results of Figure 11, the CPD-FCGR + cetuximab binding group showed a positive reaction, indicating that CPD-FCGR can produce a binding reaction with cetuximab. The CPD-FCGR particle complex combined with CPD-FCGR and cetuximab achieved EGFR-positive cell-specific targeting.

[0148] 2) Cetuximab SCFV

[0149] Using CPD technology, the antibody SCFV region can be located on the surface of the particle membrane to achieve specific targeting of tissues or cells. In this experiment, the SCFV of cetuximab was co-located with the VSV-G mutant (VSV-G mut) on the surface of the VSV virus defective vector (VSV-ΔG) particle. The VSV-G mutant can block the binding with LDLR while maintaining the lysosome escape function, so that the particle realizes the specific targeting of EGFR-positive cells through the cetuximab SCFV.

[0150] The particle construction method is: 1. Refer to Example 2, CPD design cetuximab SCFV, connect CPD sequence in turn at the C terminal of cetuximab SCFV, AR: SEQ ID NO: 3, TR: SEQ ID NO: 5, connect TSS: SEQ ID NO: 1 at the N terminal. Insert the nucleotide sequence corresponding to the amino acid sequence of this design into the VSV virus defective vector (VSV-ΔG) packaging plasmid, then perform in vitro packaging of the particle, and use VSV-G or VSV-G mut (K47Q, R354A) transfected vero cells for production, and then perform ultracentrifugation purification.

[0151] The in vitro verification experiment method is: digest and collect EGFR positive cells (A549) and EGFR negative cells (DMS53) after infection, add PE-coupled protein L and SCFV expressed on the cell surface, detect PE by flow cytometry, and the expression of SCFV on the cell surface reflects the targeting and intracellular function and level.

[0152] The results are shown in Figure 12: the infection level of CPD cetuximab SCFV particles produced using VSV-G as a helper protein is not different in the two cells. But the CPD cetuximab SCFV particles produced using VSV-G mut helper protein can significantly target EGFR positive cells.

[0153] 3) gp160

[0154] Different viral proteins are selected to change the targeting of the drug.

[0155] The mesothelin CAR sequence is inserted into the VSV defective vector (ΔG / ΔM) genome, and the VSV-ΔG-ΔM-MCAR particles packaged with the HIV virus coat protein gp160 plasmid modified by the CPD technology are used for activation experiments. The specific construction method is: refer to Example 2, connect CPD sequence in turn at the C terminal of gp160 protein, AR: SEQ ID NO: 2, TR: SEQ ID NO: 4, insert the corresponding nucleotide sequence into pcDNA3.1 vector, and synthesize plasmid pcDNA3.1-CPD-gp160. Co-transfect BHK-21 cells with the packaging plasmid of VSV-ΔG-ΔM-MCAR and the packaging helper plasmids pcDNA3.1-N, pcDNA3.1-P, pcDNA3.1-M, pcDNA3.1-L, pcDNA3.1-CPD-gp160, harvest the particles after 72 hours and purify them.

[0156] gp160 protein:

[0157] The experimental procedure is as follows: PanT cells are isolated according to the kit procedure, counted, and seeded into well plates. Cells are infected with MCAR (gp160) particles or control particles packaged with VSV-G at an MOI of 0.1. After 48 hours of incubation, the T cells are resuspended by trypsinization and counted. Cells are washed and incubated with PE-labeled protein L to bind to the surface CAR structure. After incubation and washing, the cells are analyzed by flow cytometry.

[0158] The results are shown in Figure 13, which shows that the gp160 particles can specifically target T cells and achieve intracellular expression of CAR.

[0159] Although the embodiments of the present application have been shown and described above, it should be understood by those skilled in the art that the above-mentioned embodiments are exemplary and cannot be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-mentioned embodiments within the scope of the present application.

Claims

1. A chimeric protein, characterized in that, The chimeric protein comprises: a transmembrane transport region, a target protein display region, a membrane anchoring region and an intracellular signal transduction region connected in sequence; the transmembrane transport region comprises a transmembrane transport peptide segment; the target protein display region comprises a target protein peptide segment; the membrane anchoring region comprises a membrane anchoring peptide segment; the intracellular signal transduction region comprises a positioning auxiliary peptide segment.

2. The chimeric protein of claim 1, wherein, The target protein comprises an antigen protein.

3. The chimeric protein of claim 2, wherein, The antigen protein comprises a self antigen and / or a heterologous antigen.

4. The chimeric protein of claim 3, wherein, The self antigen comprises at least one of HER2, PSA, TRP-2, VEGF, EpCAM, GPC3, MSLN, TNFα, PCSK9, EGFR, EGF, Survivin, ANGPTL3 and IgE.

5. The chimeric protein of claim 3, wherein, The heterologous antigen comprises a pathogen protein.

6. The chimeric protein of claim 5, wherein, The pathogen protein comprises at least one of Zika virus protein, HIV protein, herpes virus protein, cytomegalovirus protein, novel coronavirus protein, coxsackievirus protein.

7. The chimeric protein of claim 1, wherein, The target protein comprises at least one of a receptor ligand, a single-chain variable fragment of an antibody and a virus protein.

8. The chimeric protein of claim 1, wherein, The transmembrane transport region has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence with at least 80% homology thereto.

9. The chimeric protein of claim 1, wherein, The membrane anchoring region has an amino acid sequence as shown in any one of SEQ ID NO: 2, 3, 14, 15 or an amino acid sequence with at least 80% homology thereto.

10. The chimeric protein of claim 1, wherein, The intracellular signal transduction region has an amino acid sequence as shown in any one of SEQ ID NO: 4, 5, 28 or an amino acid sequence with at least 80% homology thereto.

11. The chimeric protein of claim 1, wherein, The target protein display region and the membrane anchoring region further comprise a helper display region.

12. The chimeric protein of claim 11, wherein, The helper display region has an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence with at least 80% homology thereto.

13. A chimeric protein construct, characterized in that, The chimeric protein comprises: The chimeric protein of any one of claims 1-12; The carrier, wherein the chimeric protein is loaded on the surface of the membrane structure of the carrier.

14. The chimeric protein construct of claim 13, wherein, The carrier comprises at least one of a viral vector, a virus-like particle, an exosome and a cell.

15. The chimeric protein construct of claim 14, wherein, The virus comprises a modified virus or a defective virus.

16. The chimeric protein construct of claim 14, wherein, The virus comprises a DNA virus or an RNA virus.

17. The chimeric protein construct of claim 14, wherein, The virus comprises at least one of a lentivirus, an adenovirus, an adeno-associated virus, a poxvirus, a herpes simplex virus, a vesicular stomatitis virus, a lymphocytic choriomeningitis virus, a measles virus, a coronavirus and a Newcastle disease virus.

18. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the chimeric protein of any one of claims 1-12.

19. The nucleic acid molecule of claim 18, wherein, The nucleic acid molecule comprises a first nucleic acid molecule, a second nucleic acid molecule, a third nucleic acid molecule and a fourth nucleic acid molecule connected in sequence; The first nucleic acid molecule encodes the transmembrane transport region; The second nucleic acid molecule encodes the target protein display region; The third nucleic acid molecule encodes the membrane anchoring region; The fourth nucleic acid molecule encodes the intracellular signal transduction region; The first nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 7 or a nucleotide sequence with at least 80% homology thereto; the third nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID NO: 8, 9, 21, 22 or a nucleotide sequence with at least 80% homology thereof; the fourth nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID NO: 10, 11, 35 or a nucleotide sequence with at least 80% homology thereof.

20. The nucleic acid molecule of claim 19, wherein, the second nucleic acid molecule and the third nucleic acid molecule further comprise: a fifth nucleic acid molecule, the fifth nucleic acid molecule encoding the helper display region; the fifth nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 12 or a nucleotide sequence with at least 80% homology thereof.

21. A recombinant expression vector, characterized in that, comprise: the nucleic acid molecule of any one of claims 18-21.

22. The recombinant expression vector of claim 21, wherein, the recombinant expression vector comprises at least one of a plasmid, a viral vector with a membrane structure, a virus-like particle with a membrane structure, an exosome and a cell.

23. The recombinant expression vector of claim 22, wherein, the virus comprises at least one of a lentivirus, an adenovirus, an adeno-associated virus, a poxvirus, a herpes simplex virus, a vesicular stomatitis virus, a lymphocytic choriomeningitis virus, a measles virus, a coronavirus and a Newcastle disease virus.

24. A recombinant cell, comprising: comprise: the recombinant expression vector of any one of claims 21-23.

25. A medicament, characterized by comprising: comprise: the chimeric protein of any one of claims 1-12, the chimeric protein construct of any one of claims 13-17 or the recombinant cell of claim 24.

26. The medicament according to claim 25, characterized in that, the drug comprises a vaccine.

27. Use of the chimeric protein according to any one of claims 1 to 12, the chimeric protein construct according to any one of claims 13 to 17 or the recombinant cell according to claim 24 for the manufacture of a medicament, characterized in that, the drug is used for preventing or treating a disease.

28. A method for preventing or treating a disease, characterized in that, comprise: administering the chimeric protein of any one of claims 1-12, the chimeric protein construct of any one of claims 13-17 or the recombinant cell of claim 24 to a subject.

29. Use according to claim 27 or method according to claim 28, characterized in that, the disease comprises a cancer, an autoimmune disease, an infectious disease, a metabolic disease or a neurological disease.

30. Use or method according to claim 29, characterized in that, the cancer comprises non-small cell lung cancer, papillary thyroid carcinoma, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, cholangiocarcinoma or sarcoma, acute myeloid leukemia, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma or gastric cancer; the autoimmune disease comprises lupus erythematosus, rheumatoid arthritis or psoriasis; the metabolic disease comprises diabetes, hypertension or hyperlipidemia; the infectious disease comprises hepatitis B, AIDS or hand-foot-mouth disease; the neurological disease comprises Alzheimer's disease.

31. Use of the chimeric protein of any one of claims 1-12, the chimeric protein construct of any one of claims 13-17 or the recombinant cell of claim 24 in screening a drug.

32. A method for screening drugs, characterized in that, comprise: co-culturing the chimeric protein of any one of claims 1-12, the chimeric protein construct of any one of claims 13-17 or the recombinant cell of claim 24 with a candidate drug; detecting a detection signal related to the chimeric protein before and after the co-culturing; When the detection signal associated with the chimeric protein changes before and after the co-culture, the candidate drug is a target drug that can react with the target protein.

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