Gas6 fusion protein, preparation method therefor and use thereof

Abstract

Provided are a GAS6 fusion protein, a preparation method therefor and the use thereof. The provided fusion protein contains an scFv capable of specifically recognizing tumor cell antigens, and can disguise tumor cells as apoptotic cells and enable phagocytosis of tumor cells by phagocytes by means of interaction with TAM RTK. The provided GAS6 fusion protein is applicable for phagocytosis of various tumor cells, showing strong broad-spectrum properties.

Description

GAS6 fusion protein and preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological immunity, and particularly relates to a GAS6 fusion protein and a preparation method and application thereof. BACKGROUND

[0002] The TYRO 3, AXL and MERTK (TAM) family of receptor tyrosine kinases (RTK) is abnormally expressed in various cancers, and has been identified as a promising therapeutic target based on its different functions in cancer cells and tumor-promoting immune cells.

[0003] TAM RTKs are expressed by epithelial cells, platelets and various immune cells, and play an important role in tissue homeostasis and immune regulation. The three TAM receptor tyrosine kinases (RTK), TYR03, AXL and MERTK (TAM), have a common structure, including two immunoglobulin-like (Ig-like) and two fibronectin III (FNIII) domains in their extracellular amino-terminal region, a single-pass transmembrane domain and a cytoplasmic carboxy-terminal region with a kinase domain. TAM RTKs induce their homodimerization, autophosphorylation and kinase activation by ligand binding, thereby altering the control of subsequent downstream signaling, participating in the development of cancer.

[0004] Currently, the two most studied TAM ligands are growth-arrest-specific protein 6 (GAS6) and PROS1 secreted protein, and TAM RTKs bridge phosphatidylserine (PtdSer) on the membrane of apoptotic cells with TAM receptors through soluble mediators GAS6 or PROS1. The unique mode of PtdSer-GAS6-TAM complex, the largest activation of the RTK family, has been shown to be crucial in the continuous clearance of apoptotic cells and debris produced by various tissues.

[0005] Endocytosis is the process by which phagocytes clear apoptotic cells, and the phagocyte TAM (TYRO3, AXL and MERTK) receptor tyrosine kinase family recognizes the “Eat-me” signal, and initiates endocytosis after mediating interaction with PtdSer through GAS6 or PROS1, achieving the effect of phagocytic clearance of apoptotic cells and debris.

[0006] In cancer cells, TAM RTK activation of signaling pathways promotes cancer cell survival, metastasis, and resistance to multiple chemotherapeutic agents and targeted therapies. Meanwhile, TAM RTK also plays a role in innate immune cells, and activation of TAM RTK promotes an immunosuppressive phenotype, produces anti-inflammatory cytokines and chemokines, suppresses pro-inflammatory mediators, and reduces antigen presentation function. In addition, TAM RTK can recruit myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME) with immunosuppressive effects and activate immune checkpoints.

[0007] Therefore, immunologically mediated therapies by TAM antagonists as inhibitors of a single target or in combination with other cancer therapies are of great potential. Many TYRO3, AXL and / or MERTK (TAM) targeting drugs are being developed as cancer therapies, some of which have progressed to clinical testing, including selective small molecule tyrosine kinase inhibitors (TKIs), decoy receptor fusion proteins, antagonistic monoclonal antibodies, antibody-drug conjugates, and AXL-directed chimeric antigen receptor (CAR) T cells. And some emerging preclinical drugs, including inactive ligands that prevent TAM receptor tyrosine kinase (RTK) binding to endogenous ligands and DNA aptamers, nanoparticles delivering small interfering RNA (siRNA) that silences TAM gene expression at the mRNA level, and heterobifunctional protein degraders that induce TAM RTK ubiquitination and subsequent proteasomal degradation.

[0008] The prior art closer to the GAS6-mediated phagocytosis is mainly the preparation and application of chimeric antigen receptor immune cells based on GAS6 construction. It provides a chimeric antigen receptor (CAR) based on GAS6 modification, and the extracellular binding domain of the CAR can specifically target GAS6 receptors. The purpose is to make the CAR immune cells have strong specificity and target affinity, so that the killing ability of the target cells is strong and the safety is high.

[0009] However, the conventional technology based on GAS6-mediated phagocytosis has the problems of single targeting and lack of broad spectrum.

[0010] SUMMARY

[0011] Therefore, according to various embodiments of the present application, a GAS6 fusion protein is provided, and the technical scheme is as follows:

[0012] In one aspect, the present application provides a GAS6 fusion protein, which comprises, from N-terminus to C-terminus, an antigen-specific binding protein, a linker and a GAS6c.

[0013] The amino acid sequence of the GAS6c is shown in SEQ ID NO. 1.

[0014] In one embodiment, the C-terminal of the GAS6 fusion protein further comprises a fragment encoded by an EPM gene as shown in SEQ ID NO. 2.

[0015] In one embodiment, the amino acid sequence of the scFv is as shown in SEQ ID NO. 3 or SEQ ID NO. 4.

[0016] In one embodiment, the N-terminal of the GAS6 fusion protein further comprises a signal peptide as shown in SEQ ID NO. 11.

[0017] The signal peptide comprises one or more of GAS6, CSF2RA and hCD8A.

[0018] In one embodiment, the linker comprises one or more of a flexible linker.

[0019] In one embodiment, the amino acid sequence of the linker is as shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0020] In one embodiment, the antigen-specific binding protein comprises one or more of a single-chain antibody, a ligand and an antibody-binding engineered protein.

[0021] In one embodiment, the amino acid sequence is as shown in SEQ ID NO. 7 or SEQ ID NO. 8.

[0022] In one embodiment, the amino acid sequence is as shown in SEQ ID NO. 9 or SEQ ID NO. 10.

[0023] In one embodiment, the GAS6 fusion protein is delivered by MSCs.

[0024] In one aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned GAS6 fusion protein.

[0025] In one aspect, the present application provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0026] The recombinant vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0027] In one aspect, the present application provides a recombinant cell expressing the above-mentioned GAS6 fusion protein, or comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector.

[0028] The recombinant cell is a eukaryotic cell or a prokaryotic cell.

[0029] Another aspect of the present application provides a method for preparing a GAS6 fusion protein, comprising:

[0030] culturing the recombinant cell, and isolating the GAS6 fusion protein from the resulting culture.

[0031] Another aspect of the present application provides a pharmaceutical composition comprising the GAS6 fusion protein as described above, and a pharmaceutically acceptable excipient.

[0032] In one embodiment, the pharmaceutical composition further comprises immune cells.

[0033] In one embodiment, the immune cells comprise one or more of monocytes or macrophages, dendritic cells, natural killer cells, or natural killer T cells.

[0034] In one embodiment, the macrophages comprise M1-type macrophages or M2-type macrophages.

[0035] Another aspect of the present application provides a method for treating a disease in a subject, comprising:

[0036] administering to the subject a therapeutically effective amount of the pharmaceutical composition.

[0037] The details of one or more embodiments of the application are set forth in the description below, and other features, objects, and advantages of the application will be apparent from the description and the claims. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the drawings needed to be used in the description of the embodiments will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0039] FIG. 1 is a schematic diagram of a GAS6 fusion protein modification;

[0040] FIG. 2 is a vector map of plasmid construction of pCDH-SFFV-copGFP-EF1a-myc-HER2 scFv-GAS6c;

[0041] FIG. 3 is a vector map of plasmid construction of pCDH-SFFV-copGFP-EF1a-myc-TRP1-TA99 scFv-GAS6c;

[0042] FIG. 4 is the result of HER2-GAS6c fusion protein-mediated phagocytosis of N87 by human primary macrophages in vitro;

[0043] Figure 5 is the result of RAW264.7 phagocytosis of N87-Fluc in vitro mediated by HER2-GAS6c fusion protein after infection with adenovirus;

[0044] Figure 6 is the expression of TRP1-TA99-scFv-GAS6c fusion protein in cells and supernatant after transfection;

[0045] Figure 7 is the result of RAW264.7 phagocytosis of B16 in vitro mediated by TRP1-TA99-GAS6c fusion protein. DETAILED DESCRIPTION

[0046] The present application will be further described below in conjunction with embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not intended to limit the scope of the present application, and the purpose of providing these embodiments and examples is to make the disclosure of the present application more thoroughly and comprehensively understood. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein, and those skilled in the art can make various modifications or changes without departing from the spirit of the present application, and the equivalent forms obtained thereby also fall within the scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0047] Unless otherwise defined, 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 application belongs.

[0048] Terms

[0049] All documents referred to in this application are incorporated by reference in this application as if each document were individually incorporated by reference. Unless otherwise stated, the documents referred to in this application are incorporated by reference in their entirety, for all purposes. When the present application refers to the definitions of relevant technical features, terms, names, phrases, etc. in the cited documents, the definitions in the cited documents are also incorporated by reference. When the present application refers to the cited documents, the examples and preferred modes of the relevant technical features incorporated by reference can also be incorporated by reference into the present application, but only to the extent that the present application can be implemented. It should be understood that when the cited content conflicts with the description in the present application, the present application is given priority or is modified according to the description in the present application.

[0050] Unless otherwise stated or contradictory, the terms or phrases used herein have the following meanings:

[0051] The selection scope of the terms "and / or", "or / and", "and / or" used in the present application includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, including any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0052] In the present application, when referring to a numerical interval (i.e. a numerical range), if no specific description is provided, the optional numerical distribution within the above numerical interval is considered to be continuous, and includes the two numerical end points (i.e. the minimum value and the maximum value) of the numerical range, and each numerical value between the two numerical end points. If no specific description is provided, when the numerical interval only refers to the integers within the numerical interval, including the two end point integers of the numerical range and each integer between the two end points, in the present application, each integer is directly listed, for example, t is an integer selected from 1-10, which means that t is any one integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in the present application should be understood to include any and all sub-ranges included therein.

[0053] In one aspect, the present application provides a GAS6 fusion protein, which comprises, from N-terminus to C-terminus, a signal peptide, an antigen-specific binding protein, a linker and a GAS6c, wherein the amino acid sequence of the GAS6c is shown as SEQ ID NO. 1.

[0054] The amino acid sequence of the human GAS6c is shown as SEQ ID NO. 1:

[0055] In one specific example, an EPM fragment shown as SEQ ID NO. 2 is further included.

[0056] The amino acid sequence of the EPM is shown in SEQ ID NO. 2:

[0057] ALPGNPDHREMGETLPEEVGEYRQPSGGSVPVSPGPPSGLEPTSSSPY

[0058] It can be understood that the scFv described in the present application can be one, two or more (for example, three or four) of any antibody or antigen-binding fragment.

[0059] Optionally, the sequence of the scFv is shown in SEQ ID NO. 3 or SEQ ID NO. 4.

[0060] The amino acid sequence of the human HER2-scFv is shown in SEQ ID NO. 3:

[0061] The amino acid sequence of the TRP1-TA99-scFv is shown in SEQ ID NO. 4:

[0062] In one specific example, the linker includes, but is not limited to, one or more of a flexible linker.

[0063] Optionally, the sequence of the linker is shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0064] The amino acid sequence of the G4S linker is shown in SEQ ID NO. 5: GGGGSGGGGSGGGGS

[0065] The amino acid sequence of the G3S linker is shown in SEQ ID NO. 6: GGGS

[0066] Optionally, the GAS6 fusion protein has an amino acid sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8.

[0067] The amino acid sequence of the HER2-scFv-G4S-GAS6c-EPM is shown in SEQ ID NO. 7:

[0068] The amino acid sequence of the HER2-scFv-G4S-GAS6c is shown in SEQ ID NO. 8:

[0069] Further optionally, the GAS6 fusion protein targeting TRP1 has an amino acid sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10.

[0070] wherein the TRP1-TA99-scFv-GAS6c-EPM amino acid sequence is shown as SEQ ID NO. 9:

[0071] wherein the TRP1-TA99-scFv-GAS6c amino acid sequence is shown as SEQ ID NO. 10

[0072] Optionally, the signal peptide amino acid sequence of GAS6 is shown as SEQ ID NO. 11:

[0073] MAPSLSPGPAALRRAPQLLLLLLAAECALA

[0074] In one specific example, the GAS6 fusion protein includes but is not limited to delivery by MSC.

[0075] In one aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned GAS6 fusion protein. The nucleotide sequence of the scFv capable of specifically recognizing the target cell antigen can be obtained by PCR amplification, recombination or artificial synthesis.

[0076] More specifically, the target cell includes but is not limited to tumor cells, apoptotic cells, senescent cells.

[0077] The present application also provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0078] Optionally, the recombinant vector is a eukaryotic expression vector. The present application does not make special limitations on the type of the eukaryotic expression vector, which can include but is not limited to HEK293FT cells.

[0079] In another aspect, the present application also provides a recombinant cell comprising the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0080] Optionally, the recombinant cell is a eukaryotic cell or a prokaryotic cell.

[0081] In another aspect, the present application provides a preparation method of the GAS6 fusion protein, comprising:

[0082] culturing the above-mentioned recombinant cell, and isolating the GAS6 fusion protein from the obtained culture.

[0083] In another aspect, the present application provides a pharmaceutical composition comprising the above-mentioned GAS6 fusion protein and a pharmaceutically acceptable adjuvant.

[0084] It can be understood that the pharmaceutical composition can include but is not limited to:

[0085] (1) GAS6 fusion protein + common excipients;

[0086] (2) GAS6 fusion protein + macrophages + common excipients;

[0087] (3) GAS6 fusion protein + macrophages + other immune cells + common excipients;

[0088] (4) GAS6 fusion protein + immune cells that phagocytose apoptotic cells + common excipients;

[0089] (5) GAS6 fusion protein + immune cells + common excipients.

[0090] The type of excipient is not particularly limited in the present application, and appropriate pharmaceutical excipients can be selected and the amount of excipients can be adjusted according to the required dosage form in clinic.

[0091] Further, the pharmaceutical composition disclosed in the present application can be used for treating tumors, i.e. indications include: esophageal tumors such as squamous cell carcinoma, adenocarcinoma, lymphoma, neuroendocrine tumor and sarcoma, etc. Gastric tumors such as common leiomyoma, hemangioma, lipoma, gastric stromal tumor, gastric sarcoma, gastric cancer, etc. Intestinal tumors such as common large intestinal polyps, colon cancer, rectal cancer, colon carcinoid, rectal cancer, etc. Liver tumors including focal nodular hyperplasia of the liver, liver hemangioma, liver cyst, liver adenoma, liver cirrhosis, liver cancer, etc. Skin tumors such as sebaceous cyst, sebaceous nevus, syringoma, keloid, fibrosarcoma, basal cell carcinoma, squamous cell carcinoma, eczema-like cancer, etc. In addition, there are hematological tumors, nervous system tumors, urinary system tumors, head and neck malignant tumors, etc.

[0092] Further, the pharmaceutical composition disclosed in the present application can also be used for treating chronic inflammatory diseases or inflammation-related disease types, i.e. indications further include: common chronic pharyngolaryngitis, chronic gastritis, chronic pancreatitis, etc.

[0093] Further, the inflammatory diseases include but are not limited to infectious and non-infectious inflammation, as well as degenerative, exudative, proliferative and specific inflammation.

[0094] Further, the inflammatory diseases include abnormal activation of the inflammatory pathway without antigen stimulation of autoimmunity, including Hashimoto's thyroiditis, systemic lupus erythematosus, rheumatoid arthritis, primary biliary cirrhosis, etc. and inflammatory diseases caused by bacterial, viral, fungal, mycoplasma, chlamydia, rickettsia, etc. infections.

[0095] In the present application, "pharmaceutically acceptable" means those ligands, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for administration to patients and are commensurate with a reasonable benefit / risk ratio.

[0096] In the present application, "pharmaceutically acceptable excipient" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.

[0097] As used herein, the phrase "pharmaceutically acceptable excipient" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0098] Suitable examples include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch, potato starch, and substituted or unsubstituted beta- cyclodextrin; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laureate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0099] In the present application, "excipients" include, but are not limited to, mannitol, sorbitol, sodium pyrosulfite, sodium bisulfite, sodium thiosulfite, cysteine hydrochloride, mercaptoacetic acid, methionine, vitamin C, disodium edetate (disodium EDTA), sodium calcium EDTA, carbonates of monovalent alkali metals, acetates of monovalent alkali metals, phosphates of monovalent alkali metals or aqueous solutions thereof, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, fructooligosaccharides, dextran, glycine, starch, sucrose, dextrin (e.g., maltodextrin), lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinylpyrrolidone, glycerol, Tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate.

[0100] The application also provides a method for phagocytosis mediated by TAM receptors by GAS6 fusion protein, comprising: obtaining the GAS6 fusion protein as described above, synthesizing a plasmid vector with the encoded GAS6 fusion protein, transfecting cells, separating a culture containing the GAS6 fusion protein, and co-culturing tumor cells and immune cells under the condition of culture, so that phagocytosis mediated by GAS6-TAM pathway can occur.

[0101] Optionally, the immune cells include, but are not limited to, one or more of monocytes or macrophages or dendritic cells or natural killer cells or natural killer T cells.

[0102] Immune cells (immune cells) are commonly known as white blood cells, including lymphocytes and various phagocytes, etc., and also specifically refer to lymphocytes and the like that can recognize antigens and produce specific immune responses. Lymphocytes are the basic components of the immune system and are widely distributed in the body. They are mainly T lymphocytes, B lymphocytes, which are activated by antigen stimulation, proliferate, and produce specific immune responses. In addition to T lymphocytes and B lymphocytes, there are K lymphocytes and NK lymphocytes, a total of four types. T lymphocytes are a multifunctional cell population. In addition to lymphocytes, cells involved in immune responses also include plasma cells, granulocytes, mast cells, antigen-presenting cells, and cells of the mononuclear phagocyte system.

[0103] Further optionally, the macrophages include M1 type macrophages or M2 type macrophages.

[0104] Another aspect of the application provides a method for treating a disease in a subject, comprising the step of: administering to the subject a therapeutically effective amount of the GAS6 fusion protein therapeutic drug described above.

[0105] It can be understood that the GAS6 fusion protein is not limited to being synthesized in cells by DNA, cDNA, mRNA, in cells or secreted outside cells.

[0106] The application uses a single-chain variable fragment (scFv) that recognizes tumor cell antigens to replace the Gla domain (recognizes PtdSer on the surface of apoptotic cells) in the human GAS6 sequence and cuts off the LG domain of GAS6 (binds to TAM receptors to induce receptor dimerization and activation) to obtain a GAS6 fusion protein, scFv-GAS6c. The GAS6 fusion protein provided by the application bridges the phagocytosis mediated by the GAS6-TAM pathway.

[0107] The scFv in the GAS6 fusion protein can specifically recognize tumor cell antigens, and the GAS6c in the GAS6 fusion protein interacts with TAM RTK to make phagocytes phagocytize tumor cells. Therefore, the GAS6 fusion protein of the application can specifically recognize tumor cell antigens and is suitable for phagocytizing various tumor cells and has strong broad-spectrum. For example, the GAS6 fusion protein drug of the application can be used for osteoarthritis, Crohn's disease, Parkinson's disease, Alzheimer's disease, lupus erythematosus, aging-related diseases, etc.

[0108] The embodiments of the application will be described in detail below with reference to the examples. It should be understood that the examples are only used to illustrate the application and not to limit the scope of the application. The experimental methods in the following examples are not specified, and the guidelines given in the application are preferred. The experimental manual or conventional conditions in the art can also be used, or the conditions suggested by the manufacturer can be used, or the known experimental methods in the art can be used.

[0109] In the following specific examples, the amount of the raw material components is measured, and slight deviations within the weighing accuracy range can exist if not otherwise specified. The temperature and time parameters allow for acceptable deviations caused by instrument testing accuracy or operation accuracy.

[0110] It should be understood that in various embodiments of the application, the size of the sequence number of the above-mentioned processes does not mean the order of execution, and the execution order of the processes should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the application.

[0111] Example 1 scFv-GAS6c protein

[0112] The single-chain variable fragment (scFv) recognizing tumor cell antigens is used to replace the Gla domain in the human GAS6 sequence, and the LG domain of GAS6, amino acids 298-678, is cut off to generate scFv-GAS6c. The specific modification schematic diagram is shown in FIG. 1.

[0113] The gene fragments with myc tag, GAS6 signal peptide, single-chain variable region (scFv) of anti-HER2 or other antigens, linker, GAS6c, and EPM are sequentially connected, and the corresponding nucleotide sequences are obtained by artificial synthesis method or PCR method, and are inserted into a lentivirus pCDH-SFFV-copGFP-EF1a-puro vector or an adenovirus vector, and the recombinant plasmid is identified by enzyme digestion.

[0114] Next, the plasmid was extracted using the endotoxin-free small-scale extraction kit from Omega, and the extracted plasmid was used to transiently transfect HEK-293T cells using jetPRIME. The supernatant was collected as the conditioned medium. It should be noted that the plasmid extraction and transfection methods used in the present application are only specific examples, and the extraction methods and transfection protocols include but are not limited to the above methods. In order to verify the mediation killing effect of the GAS6 fusion protein disclosed in the present application, the scFv-GAS6c mediated phagocytosis and killing of N87 or N87-Fluc, or other tumor target cells by human primary macrophages were verified by co-culturing with the conditioned medium in vitro.

[0115] Further, in order to verify the influence of the GAS6 fusion protein disclosed in the present application on the mediation killing effect of different phenotypes of macrophages, after stimulating human primary macrophages to be M1 phenotype using IFNγ or LPS or stimulating human primary macrophages to be M2 phenotype using IL4, the scFv-GAS6c mediated phagocytosis and killing of N87 or N87-Fluc, or other tumor target cells by human primary macrophages or other monocytes were verified by co-culturing with the conditioned medium in vitro, and the GAS6 fusion protein disclosed in the present application achieved mediation killing of different phenotypes of macrophages independent of the phenotype of macrophages.

[0116] Further, in order to verify the influence of the GAS6 fusion protein disclosed in the present application on the mediation killing effect of different types of effector cells, the scFv-GAS6c mediated phagocytosis and killing of N87 or N87-Fluc, or other tumor target cells by monocytes or natural killer cells were verified by co-culturing with the conditioned medium in vitro, and the scFv-GAS6c disclosed in the present application mediated phagocytosis and killing of N87 or N87-Fluc, or other tumor target cells by human monocytes or natural killer cells, which was not limited to the type of effector cells.

[0117] Further, in order to verify the influence of the GAS6 fusion protein disclosed in the present application on the mediation killing effect of different types of effector cells, the scFv-GAS6c mediated phagocytosis and killing of N87 or N87-Fluc, or other tumor target cells by monocytes or natural killer cells were verified by co-culturing with the conditioned medium in vitro, and the scFv-GAS6c disclosed in the present application mediated phagocytosis and killing of N87 or N87-Fluc, or other tumor target cells by human monocytes or natural killer cells, which was not limited to the type of effector cells.

[0118] In summary, scFv-GAS6c mediates phagocytic killing of tumor cells based on interaction with TAM RTKs, and the phagocytes are neither restricted to M1 nor M2 phenotype of macrophages, nor to effector cells.

[0119] More specifically, when the target of the scFv of the scFv-GAS6c is HER2 (it should be clear that the scFv can be any scFv that targets the cell to be killed, and is not limited to HER2). Further, the scFv-GAS6c targeting HER2 is prepared as follows:

[0120] Plasmid construction of pCDH-SFFV-copGFP-EF1a-myc-HER2 scFv-GAS6c:

[0121] The gene fragments with myc tag, GAS6 signal peptide, anti-HER2 single-chain variable region (scFv), GAS6c, EPM were sequentially connected, and all sequences were human.

[0122] The amino acid sequence of HER2-scFv-GAS6c-EPM is shown in SEQ ID NO. 7; the amino acid sequence of HER2-scFv-GAS6c is shown in SEQ ID NO. 8.

[0123] The gene sequence fragments obtained above were connected to a lentiviral expression vector, synthesized by General Biosystems, to obtain a plasmid pCDH-SFFV-copGFP-EF1a-myc-HER2 scFv-GAS6c that can express scFv targeting HER2 target and interact with TAM receptor GAS6.

[0124] The plasmid map is shown in Figure 2. The vector pCDH-SFFV-copGFP-EF1a-myc-HER2 scFv-GAS6c uses the EF1a promoter to initiate the expression of HER2-scFv-GAS6c.

[0125] Further, the steps of synthesizing the plasmid are as follows:

[0126] The synthesized plasmid was transiently transfected into 293FT cells, and the supernatant and cells were collected at 24h, 48h, and 72h after transfection, respectively.

[0127] 1. The transient transfection steps are as follows:

[0128] (1) 293FT cells were passaged into 6-well plates (2mL complete DMEM), 6 / 7x10 5 cells per well, and transfected the next day.

[0129] (2) 4 μg plasmid 200 μL PRIME Buffer; vortex.

[0130] (3) Add 8 μL PRIME into the above mixture, vortex 10 s, stand at room temperature for 10 min.

[0131] (4) Change the medium (2 mL complete DMEM) 4 h after transfection.

[0132] (5) Collect the supernatant 24 h after transfection, and supplement the medium (2 mL complete DMEM).

[0133] (6) Collect the supernatant 48 h after transfection, and supplement the medium (2 mL complete DMEM).

[0134] (7) Collect the supernatant and cells 72 h after transfection.

[0135] (8) Construct the plasmid capable of expressing myc-HER2 scFv-GAS6c by using the above construction method.

[0136] 2. Further, detect the expression of myc-HER2 scFv-GAS6c in the cells and supernatant after transfection by WB, and the steps are as follows:

[0137] Lysate the cells, quantify the protein, and perform SDS-PAGE gel electrophoresis

[0138] (1) Gel preparation: use One-Step PAGE Gel Fast Preparation Kit (10%).

[0139] (2) Sample preparation: add 5x Laemmli buffer (5x Laemmli buffer: 0.15 M Tris-HCl pH 6.8, 5% SDS, 25% Glycerol and 0.05% Bromophenol blue) containing 3% β-mercaptoethanol and 200 mM DTT (β-mercaptoethanol and DTT are added fresh) into the cell lysate, 95°C metal bath for 8 min, 12000 rpm centrifugation for 2 min, and the supernatant is used for loading.

[0140] (3) Loading.

[0141] (4) Gel running: 80 V constant voltage electrophoresis.

[0142] (5) Transferring: PVDF membrane activation: place the membrane in methanol and soak for 5 min. Wet transfer conditions: 80 mA transfer for 180 min.

[0143] (6) Blocking: Put the PVDF membrane into the blocking solution (5% sugar-free soy milk / skim milk powder with TBST as solvent), incubate at room temperature for 1-2 h.

[0144] (7) Primary antibody: Incubate overnight at 4°C. The primary antibody is diluted with the blocking solution, and the antibody dilution ratio is 1:1000.

[0145] Washing: Incubate with TBST at room temperature for 10 min each time, for a total of 3 times.

[0146] (8) Secondary antibody: Incubate at room temperature for 1 h, and the secondary antibody is diluted with the blocking solution, with a dilution ratio of 1:2000.

[0147] Washing: Incubate with TBST at room temperature for 10 min each time, for a total of 3 times.

[0148] (9) Exposure.

[0149] Further, the mediated killing effect of the scFv-GAS6c fusion protein on different types of effector cells, i.e., human primary macrophage in vitro phagocytosis and killing N87 experiment, is verified, and the results are shown in FIG. 4.

[0150] FIG. 4 is the result of HER2-GAS6c fusion protein-mediated human primary macrophage in vitro phagocytosis of N87: using normal 293FT complete medium and 293FT complete medium transfected with pCDH-SFFV-copGFP-EF1a-copGFP as negative controls, compared with the control group, the HER2-GAS6c fusion protein promotes the phagocytosis of human primary macrophages to N87. Based on the experimental data described above, it is confirmed that the scFv-GAS6c disclosed in the present application promotes the phagocytosis of tumor cells.

[0151] 1. Human primary macrophage in vitro phagocytosis of N87 experiment

[0152] (1) Count 3x10 5 Plate in a 6-well plate;

[0153] (2) After 24 h, when the hMDM adheres, use Cell Tracker Deep Red for adherent staining, with a working concentration of 1 μM, 106cells are added with 1 μL of staining solution, and incubate at 37°C for 2 h in the dark.

[0154] (3) Discard the supernatant, wash twice with PBS, and then add serum-containing 1640 medium for continued incubation in the dark.

[0155] (4) Count the hMDM cells and tumor cells, respectively, and co-culture with E:T = 1:3, using the above-mentioned conditioned medium collected after transfection to incubate in a 37°C incubator in the dark for 18 h.

[0156] (5) After co-culture, discard the supernatant, wash once with PBS, add 1 ml trypsin for digestion, neutralize with 2 ml complete medium, centrifuge to collect cell pellets, and wash once with PBS.

[0157] (6) Resuspend with 500 μL PBS, and perform flow analysis.

[0158] 2. Human primary macrophage in vitro killing N87-Fluc experiment:

[0159] (1) Plate hMDM according to E:T ratio, E:T = 1:1, 2:1, 3:1;

[0160] (2) After 24 h, when hMDM adheres, add 4 x 10 4 N87-Fluc to each well, and co-culture for 18 h using the above-mentioned conditioned medium collected after transfection.

[0161] (3) After co-culture, discard the culture medium, add 100 μl serum-free 1640 medium containing D-luciferin potassium (working concentration 0.3 μg / μl) to each well, incubate at 37°C in the dark for 10 min, and then expose to light for development.

[0162] Further, the GAS6 fusion protein described in the application can enhance the in vitro phagocytosis of adenovirus, and the results are shown in Figure 5.

[0163] Figure 5 is the result of in vitro phagocytosis of N87-Fluc after RAW264.7 is infected with adenovirus mediated by HER2-GAS6c fusion protein: since the mechanism of CAR-M targeting HER2 is that the ITAM of the intracellular region of the FC receptor plays a role, which is different from the mechanism of TAM, the synergistic effect of the two realizes that the macrophages have stronger phagocytosis and killing of target cells. Especially under the premise that the transcription level of the GAS6 binding receptor AXL is significantly up-regulated by the CAR-Ms constructed by adenovirus. Therefore, it is evaluated whether the secreted fusion protein of HER2-GAS6 can further enhance the effect of adenovirus infected HER2 CAR-M. The experimental results show that in any culture medium, the phagocytosis ability of CAR-M is higher than that of the non-transduced RAW group and the empty RAW-Ad5F35-mCherry group, and compared with the phagocytosis of CAR-Ms on N87-Fluc under the conditions of complete culture medium and control culture medium, the phagocytosis effect mediated by the conditioned medium of HER2-GAS6c fusion protein is more significant.

[0164] Further, the GAS6 fusion protein disclosed in the application is verified by a RAW264.7 stable strain overexpressing AXL.

[0165] Further, the construction steps of the RAW264.7 stable strain overexpressing AXL are as follows:

[0166] Construction of RAW264.7 stable strain overexpressing AXL:

[0167] 1. Plasmid synthesis; pCDH-CMV-AXL-EF1-puro plasmid was synthesized by General Biosystems (Company);

[0168] 2. Package lentivirus;

[0169] (1) Take jetPRIME out of the 4°C refrigerator, restore to room temperature, and mix well before use.

[0170] 293FT cells were transferred to a 6-well plate (2 mL of complete DMEM), 6 / 7 x 10 5 cells per well, and transfected the next day

[0171] (2) Envelope plasmid-pMD2.G: 0.5 μg, packaging plasmid-pSPAX2: 1.5 μg, backbone plasmid: 2 μg of plasmid mixed with 200 μL PRIME Buffer; vortex.

[0172] (3) Add 8 μL PRIME to the above mixture, vortex for 10 s, and stand at room temperature for 10 min.

[0173] (4) Change the liquid (2 mL of complete DMEM) 4 h after transfection.

[0174] (5) Change the liquid 24 h after transfection, and then supplement the medium (2 mL of complete DMEM).

[0175] (6) Collect the virus supernatant 48 h / 72 h after transfection. After centrifugation at 300g for 5 min, pass through a 0.45 μM filter, label, and store at -80°C.

[0176] 3. Construction of stable strain

[0177] (1) RAW264.7 cells were inoculated into a 12-well plate one day in advance.

[0178] (2) Add the packaged lentivirus at a ratio of 1:1, i.e., 500 μl of complete DMEM medium and 500 μl of crude virus.

[0179] (3) Change the liquid to fresh complete medium 24 h later.

[0180] (4) Observe the fluorescence 48 h later, and start adding drugs for screening after the appearance of fluorescence.

[0181] 4. Plasmid synthesis

[0182] 5. Package lentivirus

[0183] Further, the phagocytosis and killing N87 experiment steps of the RAW264.7 stable strain overexpressing AXL are as follows:

[0184] (1) Plasmid synthesis; the specific operation steps are the same as above.

[0185] (2) The synthesized plasmid transiently transfects 293FT cells, and the supernatant and cells are collected at 24h, 48h, and 72h after transfection, respectively, and the specific operation steps are the same as above.

[0186] (3) WB verifies the expression of myc-HER2 scFv-GAS6c in the cells and supernatant after transfection, and the specific operation is the same as above.

[0187] Further, the RAW264.7 AXL stable strain phagocytosis N87 experiment steps in vitro are as follows:

[0188] 1, count RAW264.7 AXL 3x10 5 Plated in a 6-well plate.

[0189] 2, after 24h, the RAW264.7 AXL adheres, and CellTracker DeepRed is used for adherent staining, the working concentration is 1 μM, 10 6 Add 1 μL staining solution to the cells, and incubate at 37°C for 2h in the dark.

[0190] 3, discard the supernatant, wash twice with PBS, and then add serum-free 1640 medium for continued incubation in the dark.

[0191] 4, count the RAW264.7 AXL cells and tumor cells, respectively, and co-culture at E:T=1:3, use the same conditioned medium collected after transfection in the 37°C incubator in the dark for 18h.

[0192] 5, after co-culture, discard the supernatant, wash once with PBS, add 1ml trypsin for digestion, neutralize with 2ml complete medium, centrifuge to collect the cell pellet, and wash once with PBS.

[0193] 6, add 500 μL PBS to resuspend and perform flow analysis.

[0194] Further, the RAW264.7 AXL stable strain killing N87-Fluc experiment steps in vitro are as follows:

[0195] 1, count and plate RAW264.7 AXL according to E:T ratio, E:T=1:1, 2:1, 3:1.

[0196] 2, after the RAW264.7 AXL adheres, add 4x10 4N87-Fluc, and co-cultured with the above-mentioned post-transfection harvested conditioned medium for 18h.

[0197] 3. After co-culture, the culture medium was discarded, 100ul serum-free 1640 medium containing D-luciferin potassium (working concentration 0.3ug / ul) was added to each well, and after incubation at 37℃ in the dark for 10min, it was exposed to light for development.

[0198] In a specific embodiment, the GAS6 fusion protein is further prepared into an scFv-GAS6c fusion protein antibody drug.

[0199] The present application also provides an scFv-GAS6c fusion protein antibody drug. After the scFv-GAS6c fusion protein is purified by protein purification technology, it is used as an antibody drug to treat cancer.

[0200] The HER2-scFv-GAS6c-EPM fusion protein is connected using a G4S linker, and the amino acid sequence of the G4S linker is shown in SEQ ID NO. 5.

[0201] Through in vitro experiments, human primary macrophages or other effector cells are co-cultured with N87 or N87-Fluc or other tumor target cells, and the purified scFv-GAS6c fusion protein is added for experimental verification, which proves that the scFv-GAS6c fusion protein mediates the phagocytosis and killing of human primary macrophages targeting N87 or N87-Fluc or other tumor target cells. A mouse gastric cancer orthotopic model is established by N87-Fluc tumor cells, and the scFv-GAS6c fusion protein protein drug is delivered for treatment, which proves that the scFv-GAS6c fusion protein protein drug mediates the phagocytosis and killing of N87-Fluc, providing a potential strategy for treating gastric cancer or other cancers.

[0202] Further, the present application also provides an MSC-based scFv-GAS6c delivery method.

[0203] Verification of killing based on MSC delivery of scFv-GAS6c

[0204] According to the embodiments of the present application, the nucleotide sequence of TRP1-TA99-scFv-GAS6c is obtained by artificial synthesis method or PCR method.

[0205] The plasmid map of TRP1-TA99-scFv-GAS6c is shown in Figure 3.

[0206] The TRP1-scFv-GAS6c-EPM fusion protein is connected using a G3S linker, and the amino acid sequence is shown in SEQ ID NO. 6.

[0207] The nucleic acid sequence of TRP1-TA99-scFv-GAS6c-EPM is shown as SEQ ID NO. 9, and the amino acid sequence of TRP1-scFv-GAS6c is shown as SEQ ID NO. 10.

[0208] The recombinant plasmid is subjected to enzyme digestion identification on the lentivirus pCDH-SFFV-copGFP-EF1a-puro vector or other vectors such as adenovirus. The results show that the TRP1-TA99-scFv-GAS6c coding sequence is correctly inserted into the predetermined position of the plasmid. Next, the plasmid is extracted using the endotoxin-free small-scale extraction kit of Omega Company, and the lentivirus or adenovirus is packaged. The MSC infected with the TRP1-scFv-GAS6c virus is verified for the ability to phagocytose or kill B16 or B16-Fluc or other tumor target cells in vitro after co-cultured with human primary macrophages, B16 or B16-Fluc or other tumor cells, and the factor expression after co-culture is detected. The MSC cells infected with the scFv-GAS6c virus are intravenously injected to treat melanoma in the melanoma in vivo animal experiment.

[0209] In summary, since the MSC can promote tissue repair and regeneration by releasing growth factors and cytokines, and help recruit other cells to the injury site, regulate the immune system and other superior properties, as a nutrient cell, the scFv-GAS6c can be used to treat many other diseases such as osteoarthritis, Crohn's disease, Parkinson's disease, etc. Further, the application also provides an evaluation method, i.e., killing of tumor by the stable strain overexpressing AXL.

[0210] According to the embodiments of the application, the binding affinity of GAS6 to AXL in TAM (TYRO3, AXL and MERTK) is higher than that to TYRO3 and MERTK. Therefore, the application adopts the construction of a stable strain overexpressing AXL, which can be used as a tool cell instead of mononuclear cells or mononuclear macrophages and other cells with limited sources to perform experimental evaluation.

[0211] Firstly, the CDS sequence of AXL gene was searched on NCBI, and the nucleotide sequence was obtained by artificial synthesis method or PCR method, and inserted into the lentivirus pCDH-SFFV-copGFP-EF1a-puro vector. The recombinant plasmid was identified by enzyme digestion. Next, the plasmid was extracted using the endotoxin-free small extraction kit of Omega company, and the extracted plasmid was packaged into lentivirus using jetPRIME, and a stable strain was constructed. At the same time, the lentivirus vector of scFv-GAS6c was constructed, and the HEK-293T cells were transiently transfected after the plasmid was extracted using jetPRIME, and the supernatant was collected as the conditioned medium. Through the stable strain overexpressing AXL, the conditioned medium was co-cultured to perform in vitro phagocytosis or killing of B16 or B16-Fluc, or other tumor target cell experiments, and it was verified that scFv-GAS6c mediated the phagocytosis and killing of B16 or B16-Fluc, or other tumor target cells by the stable strain overexpressing AXL.

[0212] In summary, it is proved that the stable strain overexpressing AXL can be used as a tool cell to replace monocytes or mononuclear macrophages to verify the phagocytosis and killing ability of tumor cells in vitro.

[0213] Further, the expression of TRP1-TA99-scFv-GAS6c in the cells and supernatant after transient transfection was detected by WB, and the steps were as above, and the results were as shown in FIG. 6.

[0214] FIG. 6 is the expression of TRP1-TA99-scFv-GAS6c fusion protein in the cells and supernatant after transient transfection:

[0215] The HER2-GAS6c fusion protein can be connected to HER2-positive tumor cells at one end and to macrophages at the other end, thereby serving as a supernatant additive to play a bridging role, so that the macrophages target and bind to HER2-positive tumor cells through this double-headed connecting molecule, thereby eliminating the target cells. The expression of HER2-GAS6c protein in the cells and supernatant after transient transfection of pCDH-SFFV-copGFP-EF1a-HER2-GAS6c to 293FT was detected by WB, and the results proved that HER2-GAS6 was normally secreted.

[0216] More specifically, the ability of RAW264.7 to phagocytize B16 in vitro was verified by the conditioned medium of TRP1-TA99-scFv-GAS6c, and the results were as shown in FIG. 7.

[0217] FIG. 7 is the result of TRP1-TA99-GAS6c fusion protein mediated RAW264.7 phagocytizing B16 in vitro:

[0218] The culture 293FT complete medium and 293FT complete medium transfected with pCDH-SFFV-copGFP-EF1a-copGFP were used as negative controls, and the pCDH-SFFV-copGFP-EF1a-GAS6 conditional medium expressing full-length GAS6 was used as a positive control. Compared with the control group, the TRP1-TA99-GAS6c fusion protein significantly promoted the phagocytosis of B16 by RAW264.7 cells. Based on the above experimental data, it is confirmed that the scFv-GAS6c disclosed in the present application promotes the phagocytosis of tumor cells.

[0219] The above-described embodiments only express several embodiments of the present application, facilitate specific and detailed understanding of the technical scheme of the present application, but cannot be understood as a limitation on the scope of patent protection. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which are within the scope of protection of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various modifications or modifications to the present application, and the equivalent forms obtained are also within the scope of protection of the present application. It should also be understood that those skilled in the art can obtain technical solutions on the basis of the technical solutions provided by the present application through logical analysis, reasoning or limited experiments, which are within the scope of protection of the appended claims of the present application. Therefore, the scope of protection of the patent of the present application should be based on the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A GAS6 fusion protein, characterized by, from N-terminal to C-terminal in turn includes an antigen-specific binding protein, a linker and a GAS6c; the amino acid sequence of the GAS6c is shown as SEQ ID NO.

1.

2. The GAS6 fusion protein of claim 1, wherein the C-terminal of the GAS6 fusion protein further includes a fragment encoded by EPM gene shown as SEQ ID NO.

2.

3. The GAS6 fusion protein of claim 1, wherein the amino acid sequence of the scFv is shown as SEQ ID NO. 3 or SEQ ID NO.

4.

4. The GAS6 fusion protein of claim 1, wherein the N-terminal of the GAS6 fusion protein further includes a signal peptide shown as SEQ ID NO.

11. The signal peptide includes one or more of GAS6, CSF2RA and hCD8A.

5. The GAS6 fusion protein of claim 1, wherein The linker includes one or more of flexible linkers.

6. The GAS6 fusion protein of claim 5, wherein The amino acid sequence of the linker is shown as SEQ ID NO. 5 or SEQ ID NO.

6.

7. The GAS6 fusion protein according to any one of claims 1 to 6, wherein The antigen-specific binding protein includes one or more of single-chain antibodies, ligands and antibodies modified into antibody binding types.

8. The GAS6 fusion protein according to any one of claims 1 to 6, wherein The amino acid sequence of the single-chain antibody is shown as SEQ ID NO. 7 or SEQ ID NO.

8.

9. The GAS6 fusion protein according to any one of claims 1 to 6, wherein The amino acid sequence of the ligand is shown as SEQ ID NO. 9 or SEQ ID NO.

10.

10. The GAS6 fusion protein according to any one of claims 1 to 6, wherein The GAS6 fusion protein is delivered by MSCs.

11. A nucleic acid molecule, characterized in that, The nucleic acid molecule of claim 11; 12. A recombinant vector, characterized in that, The recombinant vector is a eukaryotic expression vector or a prokaryotic expression vector. The recombinant cell expresses the GAS6 fusion protein of any one of claims 1 to 9, or contains the nucleic acid molecule of claim 11 or the recombinant vector of claim 12; 13. A recombinant cell, wherein, The recombinant cell is a eukaryotic cell or a prokaryotic cell. The method comprises:

14. A method of producing a GAS6 fusion protein, comprising, The method comprises: The pharmaceutical composition contains the GAS6 fusion protein of any one of claims 1 to 9 and pharmaceutically acceptable adjuvants.

15. A pharmaceutical composition comprising, The pharmaceutical composition further includes immune cells.

16. The pharmaceutical composition of claim 15, wherein, The immune cells include one or more of monocytes or macrophages, dendritic cells, natural killer cells or natural killer T cells.

17. The pharmaceutical composition of claim 16, wherein, The macrophages include M1 type macrophages or M2 type macrophages. The method comprises:

18. A method of treating a disease in a subject, comprising, The method comprises: The method comprises: The method comprises:

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