Engineered GPI anchored signal peptide, and fusion protein thereof and use thereof

Abstract

Provided is an engineered GPI anchored signal peptide. Compared with an original native GPI anchored signal peptide, the engineered GPI anchored signal peptide has an N-terminal histidine replaced with an uncharged non-polar amino acid having a C3-C6 linear or branched alkyl side chain, and contains a flexible hydrophobic amino acid combination at a C-terminus thereof. Further provided are a fusion protein containing a functional peptide fragment connected to the engineered GPI anchored signal peptide of the present application, and a use of the engineered GPI anchored signal peptide and the fusion protein.

Description

Engineered GPI anchored signal peptide, fusion protein thereof and application TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and medicine. Specifically, the present application relates to an engineered novel GPI anchored signal peptide, a fusion protein comprising the novel GPI anchored signal peptide, and the design, preparation and application of the former. BACKGROUND

[0002] Glycosyl-phosphatidyl inositol-anchored proteins (GPI-APs) are a class of proteins widely present on the surface of eukaryotic cell membranes, which do not contain transmembrane domains and cytoplasmic regions, and the carboxylic acid end is anchored on the eukaryotic cell membrane through the GPI anchor structure generated in the cytoplasm. The N- and C-terminal of GPI-APs both have signal peptide sequences, the N-terminal signal peptide is the protein membrane export signal peptide, and the C-terminal signal peptide can induce the combination of anchored proteins and GPI anchor structure, so it is called GPI anchored signal peptide. The C-terminal signal peptide is a segment of uncharged hydrophobic amino acids, usually composed of 15-30 amino acids. The GPI anchor structure is highly conserved, composed of phosphoethanolamine, core sugar chain and phosphoinositol glycolipid chain. GPI-APs in mammals mainly participate in the interaction between cells and matrix, and GPI-APs are involved in the life activities of macrophage anti-inflammatory response, T cell activation, complement cascade amplification, cell proliferation, leukocyte extravasation, tumor invasion and metastasis in mammals; and also play an important role in spermatogenesis, development and capacitation (PNAS, 2020, 117(36) 22061-22067).

[0003] Domestic and foreign researches show that the extracellular segment of functional proteins can be effectively fixed on the cell membrane surface by using GPI anchored signal peptide to induce the connection of GPI anchor structure generated in the cytoplasm, thereby exerting biological effects. McHugh et al. first proved that GPI / CD80 anchored on mouse tumor cells can enhance the immune function of mice and clear tumors (Cancer Res. 1999, 59(10):2433-2437). The GPI / mouse B7.1 fusion protein designed by Yi Pingyong et al. has strong anti-tumor effect (Cancer. 2005, 103(7):1519-1528). The research on constructing a renal cancer vaccine by fusing the IL-12 gene sequence with the GPI anchored signal peptide sequence of ESAT-6 has also made certain progress. The GPI anchoring system has unique anchoring efficiency, and in theory, any membrane surface protein can be anchored on the cell membrane surface in the form of GPI, thereby exerting biological effects.

[0004] However, the current GPI technology system has obvious limitations in application research: first, it is generally limited to discovering existing natural GPI anchor signal sequences, without exploring the creation of new and efficient GPI anchor sequences; second, it is generally only used to anchor membrane proteins on various cell membranes, without studying the unique biological effects of anchoring secreted proteins on cell membranes; third, there is a lack of systematic engineering research on cell membrane surface modification using GPI.

[0005] In summary, exploring a GPI anchoring system that can anchor membrane-bound and secreted proteins on the surfaces of various cell membranes, and then anchoring various bioactive factors on the surfaces of various types of cells to mediate specific biological responses and exert biological effects, is of great significance to cell engineering research and cell membrane modification and has high potential clinical application value. SUMMARY

[0006] The present application provides a new type of GPI anchoring signal peptide with enhanced anchoring efficiency after engineering, related connecting peptides, encoding molecules, cells, products, and applications thereof.

[0007] In some aspects of the present application, an engineered glycosylated phosphatidylinositol (GPI) anchoring signal peptide is provided, wherein the N-terminal histidine of the engineered GPI anchoring signal peptide is replaced with a non-charged non-polar amino acid with a C3-C6 straight-chain or branched-chain alkyl side chain, and a combination of flexible hydrophobic amino acids is included at the C-terminus of the engineered GPI anchoring signal peptide.

[0008] In some aspects of the present application, a protein molecule is provided, comprising: (a) an engineered GPI anchoring signal peptide of the present application; and (b) a functional peptide segment connected to the engineered GPI anchoring signal peptide in (a).

[0009] In some aspects of the present application, a nucleic acid molecule is provided, which encodes an engineered GPI anchoring signal peptide or a protein molecule of the present application.

[0010] In some aspects of the present application, a cell is provided, comprising a nucleic acid molecule of the present application; or having an engineered GPI anchoring signal peptide or a protein molecule of the present application anchored to the cell membrane thereof.

[0011] In some aspects of the present application, a product is provided, comprising one or more substances selected from the group consisting of: an engineered GPI anchoring signal peptide, a protein molecule, a nucleic acid molecule, and / or a cell of the present application.

[0012] In some aspects of the present application, there are provided uses of the engineered GPI anchor signal peptide, protein molecule, nucleic acid molecule, cell and / or product of the present application for disease diagnosis, prevention and / or treatment.

[0013] In some aspects of the present application, there is provided a method for disease diagnosis, prevention and / or treatment, comprising administering to a subject in need thereof a diagnosis, prevention and / or treatment effective amount of the engineered GPI anchor signal peptide, protein molecule, nucleic acid molecule, cell and / or product of the present application.

[0014] In some aspects of the present application, there are provided uses of the engineered GPI anchor signal peptide, protein molecule, nucleic acid molecule, cell and / or product of the present application for disease diagnosis, prevention and / or treatment.

[0015] Those skilled in the art can make any combinations of the foregoing technical solutions and technical features without departing from the inventive concept and protection scope of the present application. Other aspects of the present application are obvious to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0016] The present application is further described below in conjunction with the accompanying drawings, which are shown only for the purpose of illustrating embodiments of the present application and not for limiting the scope of the present application.

[0017] Figure 1: pTT5-hPD-L1 / new GPI anchor signal peptide fusion gene expression vector.

[0018] Figures 2A-2D: Comparison of the new GPI anchor signal peptide (new GPI) and the original anchor signal peptide (CD73 GPI) in anchoring human PD-L1 to the CHO cell membrane surface: Figure 2A: day 3; Figure 2B: day 4; Figure 2C: day 5; Figure 2D: day 6.

[0019] Figure 2E: Efficiency test of the N-terminal double histidine-substituted anchor signal peptide (double histidine substituted with valine) in anchoring human PD-L1 to the CHO cell membrane surface: left panel: day 3; right panel: day 6.

[0020] Figure 3: The new GPI anchor signal peptide anchoring human IL-2 to the Hela cell membrane surface.

[0021] Figure 4: The new GPI anchor signal peptide anchoring human CD80 to the 293t cell membrane surface.

[0022] Figure 5: The new GPI anchor signal peptide anchoring human IL-2 to the 293t cell membrane surface.

[0023] Fig. 6A-6B: Comparison of the new GPI anchor signal peptide sequence and CD55 GPI anchor signal peptide induced anchoring effect.

[0024] Fig. 6A: New GPI anchor signal peptide (3 days and 7 days);

[0025] Fig. 6B: CD55 GPI anchor signal peptide (3 days and 7 days).

[0026] Fig. 7: Effect study of lentiviral vector packaging IL-4 / new GPI fusion factor transfection of H1299 cells.

[0027] Fig. 8A-8B: Effect study of lentiviral vector packaging hCD80 / new GPI fusion factor transfection of DC cells:

[0028] Fig. 8A: Immature DCs cultured for the fourth day;

[0029] Fig. 8B: Lentiviral vector packaging hCD80 / new GPI fusion factor transfection of DC cells.

[0030] Fig. 9A-9B: hCD80 / new GPI anchor modified DCs stimulate T cell activation proliferation:

[0031] Fig. 9A: Effect of control DCs without membrane cell modification on T cell proliferation;

[0032] Fig. 9B: Effect of hCD80 / new GPI anchor modified DCs on T cell proliferation. DETAILED DESCRIPTION

[0033] To solve the deficiencies mentioned in the background art, the present application provides an engineered new GPI anchor signal peptide, which has a broad-spectrum and high-efficiency anchoring function, and provides a new application path for cell membrane surface modification and cell engineering research.

[0034] The inventors of the present application have creatively engineered the natural GPI anchor signal peptide through long-term and in-depth research, and obtained a new GPI anchor signal peptide; and on this basis, further constructed an expression vector of the new GPI anchor signal peptide and its connecting peptide with a functional factor, transformed cells and products. Through detection of the anchoring efficiency of membrane-bound and secreted factors on the surface of various cell membranes, membrane modification effect and function determination of various cells, etc., it is verified that the new GPI anchor signal peptide of the present application can be used to anchor various functional factors on the surface of different cell membranes.

[0035] All numerical ranges herein are intended to expressly include all values and sub-ranges falling within the range endpoints. Combinations of features or steps can be used in any combination. Individual features disclosed in the specification can be used in any combination. The various features disclosed in the specification can be used in any combination. Unless otherwise stated, the features disclosed in the specification are not to be interpreted as being equivalent or identical.

[0036] All numerical ranges herein are intended to expressly include all values and sub-ranges falling within the range endpoints. For example, 1 to 3 includes the endpoints 1 and 3, the specific integer values between 1 and 3 (e.g., 2) and the fractional values (e.g., 1.2, 1.5, 1.8, 2.1, 2.3, 2.4, 2.8, etc.) between the integer values (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.), and sub-ranges (e.g., 1 to 2.3, 2.1 to 2.5, 1.8 to 2.1, etc.) thereof.

[0037] As used herein, “containing,” “having,” or “including” includes “comprising,” “consisting essentially of,” “consisting of,” and “made of”; “consisting essentially of,” “consisting of,” and “made of” are subsumed by “containing,” “having,” or “including.”

[0038] GPI anchor signal peptide and coding sequence thereof

[0039] As used herein, the terms “GPI anchor signal peptide,” “anchor signal peptide” are used interchangeably to refer to a C-terminal signal peptide derived from a glycosylated phosphatidylinositol-anchored protein, which can induce the binding of the anchored protein to a GPI anchor structure, thereby enabling anchoring on the eukaryotic cell membrane.

[0040] As used herein, the terms “original GPI anchor signal peptide,” “native GPI anchor signal peptide,” or “original native GPI anchor signal peptide” are used interchangeably to refer to a GPI anchor signal peptide that has not been engineered.

[0041] The native GPI anchor signal peptide can be, for example, the anchor signal peptide in a protein such as CD73, CD55, ESAT-6, etc., such as the native GPI anchor signal peptide shown in SEQ ID NO: 1 or 15. GPI anchor signal peptides known in the art or predicted using GPI anchor protein prediction software or websites (e.g., PredGPI) can also be used to predict and further determine GPI anchor proteins and their signal peptides. For example, PredGPI predicts the presence of GPI anchors and their omega sites in eukaryotes by HMM model and SVM.

[0042] The term "(engineered) GPI anchor signal peptide" or "novel GPI anchor signal peptide" or "signal peptide of the present application" refers to a novel GPI anchor signal peptide obtained by engineering a native GPI anchor signal peptide according to the engineering scheme described in the present application. The engineering of the present application includes replacing (e.g., by mutation) the N-terminal histidine of the original native GPI anchor signal peptide with a non-charged non-polar amino acid with a side chain of C3-C6 linear or branched alkyl, and adding a combination of flexible hydrophobic amino acids at the C-terminus of the original native GPI anchor signal peptide.

[0043] The novel GPI anchor signal peptide of the present application can be obtained by engineering based on the anchor signal peptide in a protein such as CD73, CD55, ESAT-6, etc. In some embodiments, the novel GPI anchor signal peptide of the present application is obtained by engineering based on the native GPI anchor signal peptide shown in SEQ ID NO: 1 or 15. In some embodiments, the novel GPI anchor signal peptide of the present application can be a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or 22, or can be encoded by a nucleotide molecule comprising the nucleotide sequence shown in SEQ ID NO: 4 or 23.

[0044] The novel signal peptide of the present application can be produced by chemical synthesis, or using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacterial, yeast, higher animal, insect, and mammalian cells), and can also be obtained by site-directed mutagenesis or other known molecular biology techniques.

[0045] As used herein, the terms "signal peptide gene", "signal peptide-encoding sequence / molecule" can be used interchangeably, and all refer to a nucleotide molecule capable of encoding and expressing a GPI anchor signal peptide. In some embodiments, the coding sequence of the original GPI anchor signal peptide can be obtained from public databases, or obtained by prediction software or websites. For example, in some embodiments, the GPI anchor signal peptide-encoding sequence of CD73 shown in SEQ ID NO: 2 or the GPI anchor signal peptide-encoding sequence of CD55 shown in SEQ ID NO: 16 can be used.

[0046] In some embodiments, the engineered GPI anchor signal peptide-encoding sequence of the present application can be constructed, which can be optionally codon-optimized. In some embodiments, the novel GPI anchor signal peptide of the present application can be encoded by, for example, the coding sequence of SEQ ID NO: 4 or 23. Molecules that hybridize to these sequences under stringent conditions, or family gene molecules highly homologous to the above molecules can also be used, as long as they can correctly express the desired signal peptide under suitable conditions.

[0047] As used herein, the term "stringent conditions" means: (1) hybridization and washing under low ionic strength and high temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90%, more preferably more than 95%. For example, the sequence can be the complement of the sequence defined in (a).

[0048] The full-length nucleotide sequence of the signal peptide gene of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed according to the nucleotide sequence disclosed herein, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequence.

[0049] Without being bound by theory, the applicants surprisingly found that the novel GPI anchor signal peptide of the present application structurally incorporates a side chain-rich amino acid site near the N-terminus by mutation, adds a flexible amino acid group with strong extension at the C-terminus, ensures the integrity of the omega site (i.e., the GPI-anchor attachment site) and the amino acid sequence near the omega site in the original GPI anchor signal peptide sequence, and strengthens the extension of the original anchor signal peptide sequence structure, thereby producing a more superior anchoring effect, including the broad spectrum of anchoring cells, the broad spectrum of anchoring objects (e.g., functional factors connected to the novel GPI anchor signal peptide), and the durability of the anchoring effect.

[0050] Linker peptides comprising GPI anchor signal peptides

[0051] The present application also relates to a protein molecule comprising the novel GPI anchor signal peptide of the present application, which comprises: (a) the engineered GPI anchor signal peptide of the present application; and (b) a functional peptide segment connected to the engineered GPI anchor signal peptide described in (a).

[0052] The functional peptide segment linked to the GPI anchor signal peptide engineered in the present application can be selected according to actual needs. In some embodiments, the functional peptide segment includes but is not limited to a secretory peptide, an intracellular peptide, an intracellular segment or an extracellular segment of a transmembrane peptide, a membrane-bound peptide. In some embodiments, the functional peptide segment can be derived from but is not limited to an antibody or its antigen-binding fragment or its targeted binding peptide segment (e.g., a peptide segment containing a binding epitope), a ligand, a receptor, a cytokine (such as a lymphokine, a monokine, an interleukin, an interferon, a colony-stimulating factor, a tumor necrosis factor, a transforming growth factor).

[0053] In some embodiments, the functional peptide segment can be derived from one or more antibodies or functional fragments thereof or binding fragments thereof selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-Artemin antibody, an anti-CD19, CD44, CD47 or CD123 antibody, an anti-STING antibody.

[0054] In some embodiments, the functional peptide segment can be derived from one or more cytokines selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15; CD80, CD86, GCSF, MCSF, GMCSF, SCF, EPO; IFN-α, INN-β, IFN-γ; FNF-ɑ, TNF-β; TGF-β1, TGF-β2, TGF-β3, TGFβ1β2, BMP; GRO / MGSA, PF-4, CTAP-III, IP-10, ENA-78; MIP-1α, MIP-1β, RANTES, MCP-1, MCP-2, MCP-3, I-309; EGF, PDGF, FGF, HGF, IGF-I, IGF-II, LIF, NGF, OSM, PDECGF, TGF-ɑ, VEGF.

[0055] In some embodiments, the functional peptide segment is selected from the group consisting of a PD-L1 extracellular segment (e.g., a peptide segment with an amino acid sequence as shown in SEQ ID NO: 6), IL-2 (e.g., a peptide segment with an amino acid sequence as shown in SEQ ID NO: 2), a CD80 extracellular segment (e.g., a peptide segment with an amino acid sequence as shown in SEQ ID NO: 13), IL-4 (e.g., a peptide segment with an amino acid sequence as shown in SEQ ID NO: 19).

[0056] The engineered GPI anchor signal peptide of the present application can be connected to the functional peptide segment in the form of direct connection, connection through a linker molecule, coupling or fusion, etc. The connection mode can be selected as needed. In some embodiments, the protein molecule of the present application further comprises other active molecules, such as cytotoxins, etc., connected to (a) or (b).

[0057] The novel GPI anchor signal peptide of the present application can be connected to various functional moieties to form a protein molecule to anchor the functional moiety to the membrane of various target cells to exert its function. In some embodiments, a dendritic cell comprising the protein molecule on its cell membrane is provided, which can more efficiently present antigens and / or promote T cell proliferation.

[0058] Vectors and cells

[0059] The present application also relates to a vector comprising the novel GPI anchor signal peptide gene or the linker peptide gene of the present application, and a host cell generated by genetic engineering using the vector.

[0060] The coding sequence of the present application can be used to express or produce the novel GPI anchor signal peptide or the linker peptide by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally, the following steps are involved:

[0061] (1) transforming or transducing a suitable host cell with a polynucleotide molecule encoding the novel GPI anchor signal peptide or the linker peptide of the present application, or with a recombinant expression vector containing the polynucleotide;

[0062] (2) culturing the host cell in a suitable medium to produce the desired signal peptide or linker peptide.

[0063] In the present application, the terms "vector" and "recombinant expression vector" are used interchangeably and refer to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses or other vectors well known in the art. In general, any plasmid and vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0064] Methods well known to those skilled in the art can be used to construct an expression vector containing a coding sequence and suitable transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively connected to a suitable promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In the present application, the pTT5 vector, pcDNA3.1 vector, pIRES2-EGFP vector, AdMaxTM Expression system.

[0065] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0066] Vectors containing the appropriate DNA sequences and appropriate promoters or control sequences can be used to transform suitable host cells to enable them to express proteins or peptides. Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples include: *Escherichia coli*, *Streptomyces*, *Agrobacterium*; fungal cells such as yeast; and animal cells. In this invention, CHO, A549, *E. coli* cells, and mouse dendritic cells are preferably used as host cells.

[0067] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. An enhancer is a cis-acting factor of DNA, typically approximately 10 to 300 base pairs, that acts on the promoter to enhance gene transcription. Those skilled in the art will understand how to select appropriate vectors, promoters, enhancers, and host cells.

[0068] The recombinant peptides in the above methods can be anchored to the cell membrane. If desired, the recombinant proteins can be separated and purified by various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0069] Also provided in the present application are dendritic cells modified with the novel GPI anchor signal peptide or the linker peptide of the present application. In some embodiments, the surface of the dendritic cells comprises the engineered GPI anchor signal peptide of the present application anchored to the cell membrane thereof or a protein molecule comprising a functional polypeptide linked to the signal peptide. In some embodiments, the functional polypeptide anchored to the surface of the dendritic cells modified with the novel GPI anchor signal peptide of the present application is increased by more than 20%, for example, 20-400%, such as 30-300%, 50-200%, or any range or value therebetween, as compared to the unmodified dendritic cells. In some embodiments, the promotion of T cell proliferation by the dendritic cells modified with the novel GPI anchor signal peptide of the present application is increased by more than 20%, for example, 20-400%, such as 30-300%, 50-200%, or any range or value therebetween, as compared to the unmodified dendritic cells.

[0070] In some embodiments, the cells useful in the present application can be selected from in vivo cells, in vitro cells, or adoptive cells; and / or the cells are production cells, therapeutic cells, immunological cells, or detection cells. In some embodiments, the cells are dendritic cells, such as mature dendritic cells sensitized with a target antigen, optionally further presenting the target antigen, such as a tumor antigen, a viral antigen inducing anti-viral effect, on the surface thereof.

[0071] Pharmaceutical, pharmaceutical composition or kit

[0072] The present application also provides a pharmaceutical, pharmaceutical composition or kit comprising an effective amount of the linker peptide of the present application linked with the novel GPI anchor signal peptide or a coding sequence or a vector or a cell thereof, and a pharmaceutically or immunologically acceptable carrier. As used herein, the term "active substance" or "active substance of the present application" are used interchangeably to refer to the linker peptide of the present application linked with the novel GPI anchor signal peptide or a coding sequence or a vector or a cell thereof having disease diagnosis, prevention and / or treatment activity.

[0073] The novel GPI anchor signal peptide of the present application can anchor a desired functional substance on the membrane of a cell or a vesicle (such as an exosome) or other surface with GPI anchor structure, and exert the function of the functional substance. For example, the immune molecule (such as CD80) linked thereto can be anchored on the membrane of a tumor cell by the novel GPI anchor signal peptide of the present application to enhance the immune function and eliminate the tumor; or the targeting molecule linked thereto can be anchored on the membrane of an exosome vesicle by the novel GPI anchor signal peptide of the present application to enhance the targeting of the exosome, thereby improving the accuracy of the targeted delivery of the contents of the exosome.

[0074] In some embodiments, the medicaments of the present application can be used for the diagnosis, prevention and / or treatment of one or more diseases selected from the group consisting of malignant tumors, benign tumors, endocrine diseases (such as nutritional and metabolic diseases, immune diseases (e.g., autoimmune diseases), diseases of the blood and hematopoietic organs, mental diseases, nervous system diseases, diseases of the eye and adnexa, diseases of the ear and mastoid, circulatory system diseases, respiratory system diseases, digestive system diseases, urogenital system diseases, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, injuries, poisoning, and the like.

[0075] As used herein, the terms "containing" or "including" include "comprising," "consisting essentially of," and "consisting of." As used herein, the term "pharmaceutically acceptable" means a substance that is appropriate for use with humans and / or animals without undue adverse side effects such as toxicity, irritation, and allergic response, commensurate with a reasonable benefit / risk ratio, and is acceptable to the medical diagnostic, medical therapeutic, and pharmaceutical communities.

[0076] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier for therapeutic agents, including various excipients and diluents. The term refers to carriers that are nontoxic to the subject to be treated and that do not interact deleteriously with the active ingredient. Suitable carriers are well known to persons of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences, Mack Pub. Co., N.J. 1991.

[0077] In the compositions, the pharmaceutically acceptable carrier can contain liquids such as water, saline, glycerol, and ethanol. Additionally, auxiliary substances can be present in the carrier such as fillers, disintegrating agents, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffering substances, and the like. Generally, these substances are present from about 0.1% to about 10.0% by weight of the composition. Typically, these substances can be formulated into a non-toxic, inert, and pharmaceutically acceptable aqueous vehicle medium, wherein the pH is generally about 5 to about 8, and preferably, the pH is about 6 to about 8.

[0078] As used herein, the term "unit dosage form" refers to a dosage form of the composition of the present application prepared for the convenience of administration in a single administration, including but not limited to various solid agents (such as tablets), liquid agents, capsules, sustained-release agents.

[0079] It is to be understood that the effective dose of the active substance used can vary depending on the severity of the subject to be administered or treated. The specific case is determined on an individual basis for the subject (e.g., the subject's weight, age, physical condition, the effect desired to be achieved), which is within the range of judgment of a skilled physician.

[0080] The compositions of the present application can be in solid (e.g., granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid (e.g., oral solution) or other suitable forms. The administration route can be: (1) direct naked DNA or protein injection; (2) cDNA, mRNA and protein are linked to transferrin / poly-L-lysine complex to enhance their biological effects; (3) cDNA, mRNA and protein are linked to positively charged lipids to overcome the difficulty of crossing the cell membrane caused by the negative charge of the phosphate backbone; (4) cDNA, mRNA and protein are encapsulated by liposomes to mediate their entry into cells, which is conducive to the smooth entry of macromolecules and protects them from the hydrolysis of various extracellular enzymes; (5) cDNA, mRNA and protein are transported by immunoliposomes to specifically transport them to target tissues and target cells; (6) cDNA, mRNA and protein are transfected into transgenic cells (e.g., fibroblasts) in vitro; (7) electroporation, i.e., cDNA, mRNA and protein are introduced into target cells by means of electric current.

[0081] In addition, the compositions of the present application can also contain other active substances for diagnosis, improvement and treatment of related diseases.

[0082] Exemplary embodiments

[0083] The present application specifically provides the following exemplary embodiments:

[0084] 1. An engineered glycosylated phosphatidylinositol (GPI) anchor signal peptide, wherein the N-terminal histidine of the engineered GPI anchor signal peptide is replaced with a non-charged non-polar amino acid having a C3-C6 linear or branched alkyl side chain, and a flexible hydrophobic amino acid combination is contained at the C-terminal end thereof, as compared to the original native GPI anchor signal peptide.

[0085] 2. The engineered GPI anchor signal peptide according to embodiment 1, wherein the non-charged non-polar amino acid is independently selected from the group consisting of valine (V), leucine (L) and isoleucine (I), for example valine (V); and / or

[0086] wherein the non-charged non-polar amino acid replaces the first histidine, the second histidine or both at the N-terminal end of the original native GPI anchor signal peptide; and / or

[0087] the flexible hydrophobic amino acid combination consists of one or two amino acid residues selected from the group consisting of glycine (G) and serine (S); and / or

[0088] the flexible hydrophobic amino acid combination has a length of 5-20 amino acid residues.

[0089] 3. The engineered GPI anchor signal peptide of embodiment 1, wherein the combination of flexible hydrophobic amino acids is a combination of amino acids in a ratio of 1-3: 1 of G and S, for example selected from the group consisting of: (GS) n , (GGS) n , (GGGS) n wherein n is an integer from 3 to 10; and / or

[0090] wherein the original native GPI anchor signal peptide is derived from a protein or polypeptide selected from the group consisting of: CD73, CD55, ESAT-6, CD59, PLAP, CD56; and / or

[0091] the original native GPI anchor signal peptide has an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 15; or is encoded by a nucleotide molecule comprising a sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 16.

[0092] 4. The engineered GPI anchor signal peptide of embodiment 1, comprising a peptide stretch according to formula (I):

[0093] STGS-Xa1-C-Xa2-GSFSLIFLSLWAVIFVLYQ-X b formula (I)

[0094] wherein Xa1and X a2 each independently represents an uncharged non-polar amino acid with a side chain being a C3-C6linear or branched alkyl group, for example an amino acid residue selected from the group consisting of valine (V), leucine (L) and isoleucine (I);

[0095] X b represents a combination of flexible hydrophobic amino acids.

[0096] 5. The engineered GPI anchor signal peptide of embodiment 1, comprising a peptide stretch having an amino acid sequence according to SEQ ID NO: 3 or 22, or is encoded by a nucleotide molecule comprising a sequence according to SEQ ID NO: 4 or 23.

[0097] 6. A protein molecule comprising:

[0098] (a) the engineered GPI anchor signal peptide according to any one of embodiments 1-5; and

[0099] (b) a functional peptide stretch linked to the engineered GPI anchor signal peptide according to (a).

[0100] 7. The protein molecule of embodiment 6, wherein the functional peptide segment is selected from the group consisting of a secretory peptide, an intracellular peptide, an intracellular segment or an extracellular segment of a transmembrane peptide, a membrane-bound peptide; and / or

[0101] the functional peptide segment is selected from the group consisting of an antibody or an antigen-binding fragment thereof or a binding peptide segment thereof targeted to (e.g. comprising a binding epitope), a ligand, a receptor, a cytokine (e.g. a lymphokine, a monokine, an interleukin, an interferon, a colony-stimulating factor, a tumor necrosis factor, a transforming growth factor); and / or

[0102] the linkage is a direct junction of (a) and (b), a linkage via a linker molecule, a coupling or a fusion;

[0103] for example:

[0104] the antibody is selected from one or more of the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-Artemin antibody, an anti-CD19, CD44, CD47 or CD123 antibody, an anti-STING antibody; and / or

[0105] the cytokine is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15; CD80, CD86, GCSF, MCSF, GMCSF, SCF, EPO; IFN-alpha, INN-beta, IFN-gamma; FNF-alpha, TNF-beta; TGF-beta1, TGF-beta2, TGF-beta3, TGFbeta1beta2, BMP; GRO / MGSA, PF-4, CTAP-III, IP-10, ENA-78; MIP-1 alpha, MIP-1 beta, RANTES, MCP-1, MCP-2, MCP-3, I-309; EGF, PDGF, FGF, HGF, IGF-I, IGF-II, LIF, NGF, OSM, PDECGF, TGF-alpha, VEGF; and / or

[0106] the protein molecule further comprises another active molecule linked to (a) or (b), e.g. a cytotoxin; and / or

[0107] the disease is a disease of a mammal, e.g. a human or a non-human primate, a livestock mammal, a pet, an experimental animal; and / or

[0108] the protein molecule comprises an amino acid molecule as set forth in SEQ ID NO: 6, 10, 13 and / or 19 linked to an amino acid molecule as set forth in SEQ ID NO: 3 or 22.

[0109] 8. A nucleic acid molecule encoding the engineered GPI anchor signal peptide of any one of embodiments 1-5 or the protein molecule of any one of embodiments 6-7.

[0110] For example, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 4, 8, 11, 14, 20, 23.

[0111] 9. A cell comprising the nucleic acid molecule of embodiment 8; or having anchored to its cell membrane the engineered GPI anchor signal peptide of any one of embodiments 1-5 or the protein molecule of any one of embodiments 6-7.

[0112] For example, the cell is selected from the group consisting of an antigen presenting cell (e.g. a dendritic cell), an epithelial cell, a neural cell, a red blood cell, a white blood cell, a platelet, a phagocyte (e.g. a neutrophil, a basophil, an eosinophil, etc.), a B lymphocyte, an effector B cell, a memory B cell, a T lymphocyte, a memory T cell, an effector T cell, a cardiac muscle cell, a smooth muscle cell, a skeletal muscle cell, a cardiac muscle cell, an osteoblast, a glial cell, a hepatocyte, a kidney cell, a gland cell, an endocrine cell (e.g. a thyroid cell, a thymus cell, an islet B cell, a pancreatic islet cell); for example, a CHO cell, an A549 cell, a Hela cell, an E. coli cell, a yeast cell; and / or,

[0113] The cell is a normal cell (e.g. an immune cell) or a diseased cell (e.g. a tumor cell); and / or,

[0114] The cell is an in vivo cell, an in vitro cell or an adoptive cell; and / or

[0115] The cell is a production cell, a therapeutic cell, an immunological cell, a detection cell.

[0116] 10. The cell of embodiment 9, wherein the cell is a dendritic cell, and wherein:

[0117] The dendritic cell is derived from a bone marrow cell, an umbilical cord blood cell or a peripheral blood mononuclear cell; and / or

[0118] The dendritic cell is derived from a mammal, e.g. a human, a non-human primate, a murine; and / or

[0119] The dendritic cell is a mature dendritic cell sensitized with an antigen of interest, e.g. which further presents on its surface an antigen of interest, e.g. a tumor antigen, a viral antigen inducing an anti-viral effect.

[0120] 11. A product comprising one or more substances selected from the group consisting of the engineered GPI anchor signal peptide according to any one of embodiments 1-5, the protein molecule according to any one of embodiments 6-7, the nucleic acid molecule according to embodiment 8, or the cell according to embodiment 9 or 10;

[0121] For example, the product is selected from the group consisting of a dendritic cell vaccine, an adoptive cell therapy drug, an effector cell stimulator, or a combination thereof.

[0122] For example, the product comprises the dendritic cell according to embodiment 13 or the specific T cells induced with the dendritic cell.

[0123] 12. Use of the engineered GPI anchor signal peptide according to any one of embodiments 1-5, the protein molecule according to any one of embodiments 6-7, the nucleic acid molecule according to embodiment 8, the cell according to embodiment 9 or 10, or the product according to embodiment 11, for the manufacture of a diagnostic, prophylactic and / or therapeutic drug for a disease.

[0124] For example, the disease is selected from the group consisting of a malignant tumor, a benign tumor, an endocrine disease (such as a nutrition and metabolism disease, an immunological disease (e.g. an autoimmune disease)), a blood and hematopoietic organ disease, a mental disease, a nervous system disease, an eye and accessory organ disease, an ear and mastoid disease, a circulatory system disease, a respiratory system disease, a digestive system disease, a urogenital system disease, a skin and subcutaneous tissue disease, a musculoskeletal system and connective tissue disease, an injury, a poisoning; and / or

[0125] The drug is selected from the group consisting of an antibody drug, an exosome delivery drug, a vaccine.

[0126] Examples

[0127] The present application is further described in conjunction with the following examples. It should be understood that these examples are only used to illustrate the present application and should not be used to limit the scope of the present application. Those skilled in the art can make appropriate modifications and variations to the present application, and these modifications and variations are within the scope of the present application.

[0128] The experimental methods in the following examples, for which specific conditions are not indicated, can use conventional methods in the art, for example, refer to Molecular Cloning: A Laboratory Manual (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989) or follow the conditions suggested by the supplier. The sequencing method of DNA is a conventional method in the art, which can also be provided by a commercial company.

[0129] Unless otherwise indicated, percentages and parts are by weight. 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. Furthermore, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The preferred methods and materials described herein are intended to be illustrative only and are not intended to be limiting.

[0130] Example 1. Design of novel GPI anchor signal peptide sequence and construction of vector

[0131] Based on the CD73 GPI anchor signal peptide protein, a novel GPI anchor signal peptide sequence was designed, and its vector was constructed (as shown in Figure 1).

[0132] (I) Experimental materials

[0133] The pTT5 vector and competent cells were purchased from Jiangsu Jinserui Biotechnology Co., Ltd.

[0134] Gene synthesis and primer synthesis services were provided by Jiangsu Jinserui Biotechnology Co., Ltd.

[0135] Restriction endonucleases and T4 DNA ligase were purchased from Takara Co.

[0136] LB medium and ampicillin were purchased from Shanghai Biotechnology Co.

[0137] (II) Experimental methods

[0138] The novel GPI anchor signal peptide sequence (SEQ ID NO: 3 and 4) designed according to the original CD73 GPI anchor signal peptide protein sequence (SEQ ID NO: 1 and 2) was synthesized by Jinserui Company. The DNA sequence of hPD-L1 / new GPI (SEQ ID NO: 8) was synthesized, and Not I and EcoR I enzyme cutting sites were carried at both ends. The hPD-L1 / GPI fragment synthesized by gene was modified by Not I and EcoR I double enzyme cutting, and then was connected with the pTT5 vector digested by Not I and EcoR I under the action of ligase at 4℃ for 12h. The competent cells DH5α were transformed, and the positive clones were picked and cultured in LB medium (Amp+). The plasmid was extracted in large quantities, and after sequencing by Jinserui Company, it was stored at -80℃.

[0139] The construction of eukaryotic expression vectors of other functional protein / GPI fusion factor sequences was carried out according to the above method.

[0140] (III) Experimental results

[0141] Through research and analysis, we designed a new GPI anchor signal peptide sequence based on CD73, in which the single His replaces the amino acid sequence of the new GPI anchor signal peptide as shown in SEQ ID NO: 3, and its coding sequence is as shown in SEQ ID NO: 4; the double His replaces the amino acid sequence of the new GPI anchor signal peptide as shown in SEQ ID NO: 22, and its coding sequence is as shown in SEQ ID NO: 23.

[0142] • CD73 amino acid sequence (SEQ ID NO: 21):

[0143] • Original CD73 GPI anchor signal peptide protein sequence (SEQ ID NO: 1 = amino acids 549-574 of SEQ ID NO: 21):

[0144] • Coding sequence of original CD73 GPI anchor signal peptide (SEQ ID NO: 2)

[0145] • New CD73 GPI anchor signal peptide protein sequence (SEQ ID NO: 3):

[0146] • Coding sequence of new CD73 GPI anchor signal peptide (SEQ ID NO: 4):

[0147] • N-terminal double-replaced histidine CD73 GPI anchor signal peptide protein sequence (SEQ ID NO: 22):

[0148] • Coding sequence of new CD73 GPI anchor signal peptide (SEQ ID NO: 23):

[0149] Specifically, we select the original GPI anchor signal peptide sequence of CD73 (amino acids 549-574 of CD73) to be modified, mutate a histidine at the N-terminus of the anchor signal peptide to a flexible amino acid, and add a combination of flexible, hydrophobic amino acids at the C-terminus of the sequence, design a new CD73 / GPI anchor signal peptide sequence. The new GPI anchor signal peptide sequence is structurally mutated with a side chain-rich amino acid site Val at the front end, and a strong flexible amino acid group is added at the C-terminus, ensuring the integrity of the 9th (amino acid 557 of the mature CD73 protein) serine site (which is one of the omega sites (i.e. GPI-anchor attachment site)) and the amino acid sequence near the omega site, strengthening the extension of the original anchor signal peptide sequence structure, and is expected to have better anchoring performance.

[0150] We further constructed a eukaryotic expression vector of pTT5-hPD-L1 / new GPI (as shown in Figure 1), and after sequencing, it was proved to be correctly constructed. We can use this to determine the anchoring function of the PD-L1 fusion factor eukaryotic vector carrying the GPI anchor signal peptide.

[0151] The construction method of N-terminal double replacement new CD73 / GPI and its eukaryotic expression vector is the same as above, except that the 7th histidine is also replaced with valine.

[0152] Example 2. Comparison of the anchoring effect of hPD-L1 / new GPI and hPD-L1 / CD73 GPI on the membrane of CHO cells

[0153] The eukaryotic expression vector of pTT5-hPD-L1 / new GPI was used to transfect CHO cells, and flow cytometry detection verified that the anchoring performance of hPD-L1 / new GPI on the membrane of CHO cells was better than that of hPD-L1 / CD73 GPI (as shown in Figures 2A-2D). At the same time, we also verified the ability of CD73 GPI with the 5th and 7th histidines at the N-terminus replaced with valine (N-terminal double replacement new CD73 / GPI) to anchor hPD-L1 on the membrane of CHO cells. The anchoring ability of the double-replacement new CD73 / GPI did not reach the anchoring performance of the single-His-replacement new CD73 / GPI, but was significantly better than the original CD73 / GPI.

[0154] (I) Experimental materials

[0155] The PE-labeled primary antibody (mouse anti-human) for flow cytometry detection of hPD-L1 was purchased from Abcom Company;

[0156] JetPEI plasmid transfection reagent was purchased from Polyplus;

[0157] CHO cell line, 1640 medium, fetal bovine serum were purchased from Thermo Fisher Scientific company;

[0158] Flow cytometer was purchased from BD company;

[0159] The eukaryotic expression vectors pTT5-hPD-L1 / new GPI, pTT5-hPD-L1 / double replacement CD73 GPI and pTT5-hPD-L1 / CD73 GPI were constructed according to the method of Example 1.

[0160] (ii) Experimental method

[0161] The eukaryotic expression vectors pTT5-hPD-L1 / new GPI, pTT5-hPD-L1 / double replacement CD73 GPI and pTT5-hPD-L1 / CD73 GPI prepared according to the method of Example 1 were used. CHO cells were plated in a 6-well plate at a density of 3x105 cells / well. After 18 hours, 1 μg of each plasmid was transfected into the cells using JetPEI plasmid transfection reagent. At 72 h, 96 h, 120 h and 144 h after transfection, samples were taken from each well, 100 μl of which was added to an anti-hPD-L1 antibody labeled with PE. The mixture was incubated at 4°C for 20 min, washed twice with PBS, and then subjected to flow cytometry for phenotype analysis to detect the expression of hPD-L1 on the cell membrane.

[0162] (iii) Experimental results

[0163] We selected the extracellular region of human PD-L1 molecule (SEQ ID NO: 5 and 6) to be fused with a novel GPI anchoring signal peptide sequence and the original CD73 / GPI anchoring signal peptide sequence, respectively, to construct pTT5-hPD-L1-original CD73 GPI anchoring signal peptide fusion gene (SEQ ID NO: 7) and pTT5-hPD-L1-new GPI anchoring signal peptide fusion gene (SEQ ID NO: 8) expression vectors, respectively, and transfect CHO cells. Flow cytometry detection found that the fusion proteins expressed by the two fusion genes could be detected on the CHO membrane. On the 3rd, 4th, 5th and 6th day after transfection, the expression rates of PD-L1-GPI / CD73 were 72.8%, 70.6%, 64.7% and 54.4%, respectively; and the expression rates of PD-L1 / new GPI were 94.4%, 91.9%, 91.5% and 91.2%, respectively. The results showed that after the fusion factor was expressed intracellularly in the CHO cells, the generated hPD-L1 / GPI fusion protein migrated and attached to the CHO cell membrane by means of the GPI anchor structure. The anchoring effect induced by the novel GPI anchoring signal peptide was better than that of the original CD73 GPI anchoring signal peptide sequence, and the sustained effect was better. The N-terminal double-substituted histidine for valine in the new CD73 / GPI had a hPD-L1 membrane expression rate of 89.1% on the 3rd day and 87.7% on the 6th day, which was better than the original CD73 / GPI, but not as good as the new CD73 / GPI.

[0164] Example 3. Anchoring effect of IL-2 / new GPI on Hela cell membrane

[0165] Hela cells were transfected with the eukaryotic expression vector pTT5-IL-2 / new GPI, and the expression efficiency of IL-2 / new GPI on the Hela cell membrane was verified by flow cytometry (the results are shown in Figure 3).

[0166] (I) Experimental materials

[0167] The APC-labeled primary antibody (mouse anti-human) for flow cytometry detection of hIL-2 was purchased from Abcom Company;

[0168] JetPEI plasmid transfection reagent was purchased from Polyplus;

[0169] Hela cells, 1640 culture medium and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Company;

[0170] The flow cytometer was purchased from BD Company;

[0171] The eukaryotic expression vector pTT5-hIL-2 / new GPI was constructed according to the method of Example 1.

[0172] (II) Experimental method

[0173] The eukaryotic expression vector pTT5-hIL-2 / neoGPI was constructed by the method of Example 1, and Hela cells were plated in a 6-well plate at a density of 3x10 5 The two plasmids were transfected into the Hela cells at 1 μg / well using the JetPEI plasmid transfection reagent 14 hours after plating. The samples were taken 96 hours after transfection, and 100 μl of each sample was added to the APC-labeled anti-hIL-2 primary antibody and incubated at 4°C for 20 minutes. After washing twice with PBS, the samples were subjected to flow cytometry for phenotype analysis to detect the expression of hIL-2 on the cell membrane.

[0174] (III) Experimental results

[0175] The human IL-2 gene sequence (SEQ ID NO: 9 and 10) was selected to construct the pTT5-hIL-2 / neoGPI anchor signal peptide fusion gene (SEQ ID NO: 11) expression vector with the novel GPI anchor signal peptide sequence. The Hela cells were transfected with the liposome, and flow cytometry detection found that IL-2 was expressed on the Hela membrane. The expression rate of IL-2 / neoGPI was 35.2% on the fourth day after transfection, and the control group of Hela cell membrane surface basically detected no IL-2. The results show that the fusion protein generated after the expression of the fusion factor in the Hela cells is attached to the Hela cell membrane by means of the GPI anchor structure. The anchoring effect of the novel GPI anchor signal peptide can anchor the secreted IL-2 on the tumor cells, and also explores a new way for the preparation of tumor vaccine to enhance the immune response.

[0176] Example 4. Anchoring effect of hCD80 / neoGPI on the 293t cell membrane

[0177] The 293t cells were transfected with the pTT5-hCD80 / neoGPI eukaryotic expression vector, and the expression efficiency of hCD-80 / GPI on the 293t cell membrane was verified by flow cytometry detection (the results are shown in Figure 4).

[0178] (I) Experimental materials

[0179] The FITC-labeled primary antibody (mouse anti-human) for flow cytometry detection of hCD80 was purchased from Abcom Company;

[0180] The JetPEI plasmid transfection reagent was purchased from Polyplus;

[0181] The 293t cells, 1640 culture medium, and fetal bovine serum FBS were purchased from Thermo Fisher Scientific Company;

[0182] The flow cytometer was purchased from BD Company;

[0183] The eukaryotic expression vector pTT5-hCD80 / neoGPI was constructed according to the method of Example 1.

[0184] (ii) Experimental method

[0185] The eukaryotic expression vectors pTT5-hCD80 / neoGPI and pTT5-hPD-L1 / CD73GPI prepared according to the method of Example 1 were used to transfect 293t cells at a density of 3x10 5 5 The eukaryotic expression vectors pTT5-hCD80 / neoGPI and pTT5-hPD-L1 / CD73GPI prepared according to the method of Example 1 were used to transfect 293t cells at a density of 3x10

[0186] (iii) Experimental results

[0187] We selected the extracellular segment of human CD80 molecule (SEQ ID NO: 12 and 13) and the novel CD73 / GPI anchoring signal peptide sequence to construct the pTT5-hCD80 / neoGPI fusion gene (SEQ ID NO: 14) expression vector, and transfect 293t cells. Flow cytometry detection found that the CD80 molecule expressed by the fusion gene could be detected on the 293 cell membrane. On the third day after transfection, the expression rate of CD80 on the membrane surface of the original 293t cells was 0.44%, and the expression rate of CD80 after transfection of the hCD80 / neoGPI fusion gene reached 75.7%. The above results show that the fusion protein can be attached to the 293t cell membrane by the GPI anchor structure induced by the novel GPI anchoring signal peptide after intracellular expression in the 293t cell expression system.

[0188] Example 5. Anchoring effect of hIL-2 / neoGPI on the 293t cell membrane

[0189] The 293t cells were transfected with the eukaryotic expression vector pTT5-IL-2 / neoGPI, and the expression efficiency of hIL-2 / neoGPI on the 293t cell membrane was verified by flow cytometry detection (results shown in Figure 5).

[0190] (i) Experimental materials

[0191] The PE-labeled primary antibody (mouse anti-human) for flow cytometry detection of hIL-2 was purchased from Abcom Company;

[0192] JetPEI plasmid transfection reagent was purchased from Polyplus;

[0193] 293t cells, 1640 culture medium, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific Company.

[0194] Flow cytometry was purchased from BD company;

[0195] The eukaryotic expression vector pTT5-IL-2 / new GPI was constructed according to the method of Example 1, and the expression vector used was the same as in Example 3.

[0196] (ii) Experimental method

[0197] The eukaryotic expression vector pTT5-hIL-2 / new GPI prepared by the method of Example 1 was used, and 293t cells were plated in a 6-well plate at a density of 3x10 5 6 / well, and 1 μg of plasmid was transfected per well using JetPEI plasmid transfection reagent 16 hours later. 72 hours after transfection, samples were taken in turn, and 100 μl of sample was taken per well. The sample was labeled with PE-labeled anti-hCD80 primary antibody at 4°C for 25 min, washed with PBS twice, and the expression of human IL-2 on the cell membrane surface was detected by flow cytometry.

[0198] (iii) Experimental results

[0199] In order to verify the broad-spectrum of the new GPI anchor signal peptide in inducing anchoring of immune molecules, the sequence of the secreted cytokine human IL-2 was selected, and the pTT5-hIL-2 / new GPI anchor signal peptide fusion gene expression vector was constructed. After transfection of 293t cells, flow cytometry detection found that the hIL-2 expressed by the fusion gene could be detected on the 293 cell membrane. The expression rate of IL-2 on the membrane surface of the original 293t cells was 2.11% on the third day after transfection, and the expression rate of IL-2 on the membrane surface of the 293t cells transfected with the hIL-2 / new GPI fusion gene reached 99.8%.

[0200] The above experimental results show that after the intracellular expression of the fusion gene in the 293t cell expression system is a fusion protein, the secreted factor IL-2 can be anchored on the membrane surface of the engineered cells by means of the GPI anchor structure. In combination with the anchoring efficiency of IL-2 / new GPI on the Hela cell membrane in Example 3, it is proved that the new GPI can effectively anchor the secreted factor on the membrane surface of various cells.

[0201] Example 6. Comparison of the anchoring effects of hPD-L1 / new GPI and hPD-L1 / CD55 GPI on the CHO cell membrane

[0202] CHO cells were transfected with the eukaryotic expression vector pTT5-hPD-L1 / new GPI and the eukaryotic expression vector pTT5-hPD-L1 / CD55 GPI, respectively, and flow cytometry detection verified that the anchoring effect mediated by the new GPI was better than that of the commonly used CD55 GPI (results shown in Figures 6A and 6B).

[0203] (i) Experimental materials

[0204] PE-labeled primary antibody for detecting hPD-L1 by flow cytometry was purchased from Abcom;

[0205] JetPEI plasmid transfection reagent was purchased from Polyplus;

[0206] CHO cells, 1640 medium, fetal bovine serum were purchased from Thermo Fisher Scientific;

[0207] Flow cytometer was purchased from BD;

[0208] The eukaryotic expression vectors pTT5-hPD-L1 / new GPI and pTT5-hPD-L1 / CD55 GPI were constructed according to the method of Example 1.

[0209] (B) Experimental method

[0210] The eukaryotic expression vectors pTT5-hPD-L1 / new GPI and pTT5-hPD-L1 / CD55 GPI prepared according to the method of Example 1 were used. CHO cells were plated in 6-well plates at a density of 3x10 5 per well, and 18 hours later, 1 μg of each plasmid was transfected into the cells using JetPEI plasmid transfection reagent. At 72 h and 168 h after transfection, samples were taken from each well, and 100 μl of each sample was added to a tube containing PE-labeled anti-hPD-L1 primary antibody. The mixture was incubated at 4°C for 20 min, and then washed twice with PBS. The samples were then analyzed by flow cytometry to detect the expression of hPD-L1 on the surface of CHO cells.

[0211] (C) Experimental results

[0212] The CD55 / GPI anchor signal peptide sequence (SEQ ID NO: 15; its coding sequence is shown as SEQ ID NO: 16) is commonly used in current cell biology experiments. We selected a new GPI anchor signal peptide sequence and constructed the pTT5-hPD-L1 / new CD73 GPI anchor signal peptide fusion gene expression vector and the pTT5-hPD-L1-CD55 / GPI anchor signal peptide fusion gene (SEQ ID NO: 17) expression vector. After transfection of CHO cells, flow cytometry detection showed that the expression rates of human PD-L1 on the surface of CHO cells were 89.5% and 89.7% at 3 days and 7 days after transfection of the hPD-L1-new CD73 / GPI anchor signal peptide fusion gene (Figure 6A), and the expression rates of PD-L1 on the surface of CHO cells were 80.8% and 72.7% at 3 days and 7 days after transfection of the hPD-L1-CD55 / GPI anchor signal peptide fusion gene (Figure 6B).

[0213] The above results show that the anchoring effect of the new CD73 / GPI anchor signal peptide is better and more persistent than that of the CD55 / GPI anchor signal peptide.

[0214] Example 7. Construction of IL-4 / new GPI fusion factor lentiviral expression vector and verification of anchoring function

[0215] An IL-4 / new GPI fusion factor lentiviral expression vector was constructed, and its anchoring function was verified (the results are shown in Figure 7).

[0216] (I) Experimental materials

[0217] pLVX-CMV lentiviral plasmid vector, packaging vector, and competent cells were purchased from Shanghai Shengbo Biomedical Technology Co., Ltd.

[0218] Lentiviral expression vector construction was provided by Shanghai Shengbo Biomedical Technology Co., Ltd.

[0219] Gene synthesis and primer synthesis were provided by Jiangsu Jinerswai Biological Technology Co., Ltd.

[0220] 293t cells, H1299 cells, and serum-free medium were purchased from Thermo Fisher Scientific.

[0221] FITC-labeled hIL-4 antibody was purchased from Abcom.

[0222] (II) Experimental methods

[0223] After amplification of the designed and synthesized IL-4 / new GPI sequence, we submitted it to Shanghai Shengbo Biomedical Technology Co., Ltd. for construction of a lentiviral expression vector, which was sequenced and verified before lentivirus packaging. ViraPower Packaging Mix and lentivirus vector were transfected into 293 cells, and after 48 h, the supernatant was collected. Virus titer determination was performed by using a dilution method to infect 29t3 cells. H1299 cells were transfected with serum-free medium at a MOI of 10, and after 72 h, the cell pellets were collected by centrifugation. APC-labeled hIL-4 antibody was added and incubated at 4°C for 25 min. After washing twice with PBS, the phenotype was analyzed by flow cytometry to detect the expression of IL-4 on the membrane surface of H1299 cells. TM Packaging Mix and lentivirus vector were transfected into 293 cells, and after 48 h, the supernatant was collected. Virus titer determination was performed by using a dilution method to infect 29t3 cells. H1299 cells were transfected with serum-free medium at a MOI of 10, and after 72 h, the cell pellets were collected by centrifugation. APC-labeled hIL-4 antibody was added and incubated at 4°C for 25 min. After washing twice with PBS, the phenotype was analyzed by flow cytometry to detect the expression of IL-4 on the membrane surface of H1299 cells.

[0224] (III) Experimental results

[0225] To further verify the ability of the new GPI anchor signal peptide to induce anchoring in different expression systems and different cells, we selected the secreted cytokine human IL-4 sequence (SEQ ID NO: 18 and 19), constructed a hIL-4 / new GPI anchor signal peptide fusion gene (SEQ ID NO: 20) lentiviral expression vector, transfected H1299 cells, and found that the expressed hIL-4 could be detected on the H1299 cell membrane by flow cytometry. Three days after transfection, the expression rate of IL-4 on the membrane surface of the original H1299 cells was 0.19%, and the expression rate of IL-4 on the membrane surface of the H1299 cells transfected with the new IL-4-GPI fusion gene reached 44.3%.

[0226] The above results show that the fusion gene can also anchor the secreted factor IL-4 on the cell membrane surface with the help of the GPI anchor structure in the viral vector expression system, further confirming the universal anchoring ability of the new GPI.

[0227] Example 8. Construction of CD80 / new GPI fusion factor lentiviral expression vector and verification of anchoring function

[0228] A CD80 / new GPI fusion factor lentiviral expression vector was constructed, and the anchoring function of the fusion factor was verified (as shown in Figures 8A and 8B).

[0229] (I) Experimental materials

[0230] pLVX-CMV lentiviral plasmid vector, packaging vector, and competent cells were purchased from Shanghai Shengbo Biomedical Technology Co., Ltd.; gene synthesis and primer synthesis were completed by Jiangsu Jinerswai Biological Technology Co., Ltd.; 293t cells, 1640 culture medium, fetal bovine serum, serum-free culture medium were purchased from Thermo Fisher Scientific company; hGM-CSF, hIL-4 were purchased from RD company; FITC-labeled human CD80 monoclonal antibody was purchased from Abcom company; human peripheral blood mononuclear cells were obtained from healthy adult volunteers.

[0231] (II) Experimental methods

[0232] Freshly isolated peripheral blood leukocytes from healthy volunteers were obtained by density gradient centrifugation with lymphocyte separation medium to obtain peripheral blood mononuclear cells (PBMCs), which were suspended in complete culture medium (RPMI 1640 containing 10% fetal bovine serum). The cells were then centrifuged at 1x10 7Cells were plated at 1 cell / well in 6-well plates and incubated at 37°C in 5% CO2 for 2 hours, and then non-adherent cells were removed with pre-warmed medium. The adherent cells were cultured in complete medium containing human recombinant GM-CSF (50 ng / ml) and human recombinant IL-4 (10 ng / ml) at 37°C in 5% CO2. After the self-designed and synthesized hCD80 / new / GPI sequence was amplified, it was sent to Shanghai Shengbo Biomedicine Technology Co., Ltd. for construction of a lentivirus expression vector, and after sequencing verification, lentivirus packaging was performed. ViraPower TM Packaging Mix and lentivirus vector were transfected into 293t cells, and the supernatant was collected after 48 hours. The virus titer was determined by using a dilution method to infect 293t cells. The immature DC cells cultured in serum-free medium to the fourth day were transfected according to a gradient of MOI 50, and the cells were collected by centrifugation after 72 hours. FITC-labeled primary antibodies were added, and the mixture was incubated at 4°C for 25 minutes. After washing twice with PBS, the phenotype was analyzed by flow cytometry, and the expression of CD80 on the membrane surface of DC cells was detected.

[0233] (III) Experimental results

[0234] We used a human peripheral blood-derived DC induction culture system to culture immature DCs (as shown in FIG. 8A), constructed a hCD80 / new / GPI fusion gene lentivirus expression vector, transfected human immature DC cells, and found by flow cytometry detection after 72 hours that the hCD80 expressed by the fusion gene could be detected on the DC cell membrane. On the third day after transfection, the expression rate of CD80 on the membrane surface of the original DC cells was 25.4%, and the expression rate of CD80 on the membrane surface of the DC cells transfected with the CD80 / new / GPI fusion gene was 97.4% at an MOI of 50, indicating that the GPI anchor structure can efficiently anchor overexpressed CD80 on the membrane surface of DC cells.

[0235] Example 9. DCs modified by co-stimulatory molecules-GPI anchoring significantly promote T cell proliferation in MLR reaction

[0236] The promoting effect of DCs modified by co-stimulatory molecules-GPI anchoring on T cell proliferation was detected by mixed lymphocyte reaction (MLR reaction) (as shown in FIGS. 9A-9B).

[0237] (I) Experimental materials

[0238] DCs transfected with lentivirus packaged hCD80 / new / GPI were prepared as in Examples 7 and 8; T cells were obtained from healthy adult volunteers; flow cytometry antibodies (primary antibodies) for hCD3, hCD4, and hCD8 were purchased from Abcom Company; 1640 medium, fetal bovine serum, BSA, and cell culture plates were purchased from Thermo Fisher Scientific Company.

[0239] (ii) Experimental Methods

[0240] We selected the lentivirus packaged hCD80 / new GPI anchor modified DC prepared by the aforementioned method, and stimulated the membrane modified DC with OVA antigen (Shanghai Xingboseng Company), and then co-incubated with CFSE labeled T cells (DC:T was 1:3) for 5 days, and FACE was used to detect the proliferation of T cells stimulated by membrane modified DC.

[0241] (iii) Experimental Results

[0242] We selected the lentivirus packaged hCD80 / new GPI anchor modified DC prepared by the aforementioned method, and stimulated the membrane modified DC with OVA antigen, and then co-incubated with T cells for 5 days, and detected the proliferation of T cells stimulated by membrane modified DC. The results showed that after co-incubation of DC and T cells for 5 days, the proliferation efficiency of T cells stimulated by membrane modified DC reached 61.1% (Figure 9A), while the proliferation efficiency of T cells stimulated by unmodified control DC was only 31% (Figure 9B).

[0243] The above results prove that the co-stimulatory molecule membrane modified DC has strong stimulating T cell proliferation ability. It shows that the GPI membrane modified DC has the potential to enhance the immune response ability, and has the value of application and development.

[0244] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. In addition, it should be understood that various modifications or changes can be made to the present application by those skilled in the art after reading the above teaching of the present application, and these equivalent forms also fall within the scope defined by the claims attached to the present application.

[0245] Appendix: Sequence information (capital letters are amino acid sequences, and lowercase letters are nucleotide sequences)

Claims

1. An engineered glycosylated phosphatidylinositol (GPf) anchor signal peptide, wherein the N-terminal histidine of the engineered GPI anchor signal peptide is replaced with an uncharged non-polar amino acid having a C3-C6 linear or branched alkyl side chain, and comprises a flexible hydrophobic amino acid combination at its C-terminus, as compared to the original native GPI anchor signal peptide.

2. The engineered GPI anchor signal peptide of claim 1, wherein, the uncharged non-polar amino acid is independently selected from the group consisting of valine (V), leucine (L), and isoleucine (I), such as valine (V); and / or wherein the uncharged non-polar amino acid replaces the first histidine, the second histidine, or both, from the N-terminus of the original native GPI anchor signal peptide; and / or the flexible hydrophobic amino acid combination consists of one or two amino acid residues selected from the group consisting of glycine (G) and serine (S); and / or the flexible hydrophobic amino acid combination has a length of 5-20 amino acid residues.

3. The engineered GPI anchor signal peptide of claim 1, wherein, The combination of flexible hydrophobic amino acids is a combination of amino acids in a ratio of 1-3: 1 of G and S, for example selected from the group consisting of (GS) n , (GGS) n , (GGGS) n wherein n is an integer from 3 to 10; and / or wherein the original native GPI anchor signal peptide is derived from a protein or polypeptide selected from the group consisting of CD73, CD55, ESAT-6, CD59, PLAP, CD56; and / or the original native GPI anchor signal peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 15; or is encoded by a nucleotide molecule comprising a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:

16.

4. The engineered GPI anchor signal peptide of claim 1, comprising a peptide segment represented by Formula (I): STGS-Xa1-C-Xa2-GSFSLIFLSLWAVIFVLYQ-X b Formula (I) wherein Xa1and X a2 each independently represents an uncharged non-polar amino acid with a side chain that is a C3-C6straight chain or branched alkyl group, for example an amino acid residue selected from valine (V), leucine (L) and isoleucine (I); X b represents a combination of flexible hydrophobic amino acids.

5. The engineered GPI anchor signal peptide of claim 1, comprising a peptide segment having an amino acid sequence as set forth in SEQ ID NO: 3 or 22, or is encoded by a nucleotide molecule comprising a sequence as set forth in SEQ ID NO: 4 or 23.

6. A protein molecule comprising: (a) the engineered GPI anchor signal peptide of any one of claims 1-5; and (b) a functional peptide segment linked to the engineered GPI anchor signal peptide of (a).

7. The proteinaceous molecule of claim 6, wherein, the functional peptide segment is selected from the group consisting of a secretory peptide, an intracellular peptide, an intracellular segment or an extracellular segment of a transmembrane peptide, a membrane-bound peptide; and / or the functional peptide segment is selected from the group consisting of an antibody or an antigen-binding fragment thereof or a targeted binding peptide segment thereof (e.g., a peptide segment comprising a binding epitope), a ligand, a receptor, a cytokine (such as a lymphokine, a monokine, an interleukin, an interferon, a colony-stimulating factor, a tumor necrosis factor, a transforming growth factor); and / or the linkage is a direct junction, a linkage via a linker molecule, a conjugation, or a fusion of (a) and (b); for example: the antibody is one or more selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-Artemin antibody, an anti-CD19, CD44, CD47, or CD123 antibody, an anti-STING antibody; and / or the antibody is one or more selected from the group consisting of an anti-PD1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-Artemin antibody, an anti-CD19, CD44, CD47, or CD123 antibody, an anti-STING antibody; and / or the cytokine is selected from the group consisting of: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15; CD80, CD86, GCSF, MCSF, GMCSF, SCF, EPO; IFN-alpha, INN-beta, IFN-gamma; FNF-alpha, TNF-beta; TGF-beta1, TGF-beta2, TGF-beta3, TGFbeta1beta2, BMP; GRO / MGSA, PF-4, CTAP-III, IP-10, ENA-78; MIP-1 alpha, MIP-1 beta, RANTES, MCP-1, MCP-2, MCP-3, I-309; EGF, PDGF, FGF, HGF, IGF-I, IGF-II, LIF, NGF, OSM, PDECGF, TGF-alpha, VEGF; and / or the protein molecule further comprises another active molecule linked to (a) or (b), such as a cytotoxin; and / or the disease is a disease of a mammal, such as a human or non-human primate, a livestock mammal, a pet, an experimental animal; and / or the protein molecule comprises an amino acid molecule as set forth in SEQ ID NO: 6, 10, 13, and / or 19 linked to an amino acid molecule as set forth in SEQ ID NO: 3 or 22.

8. A nucleic acid molecule encoding the engineered GPI-anchored signal peptide of any one of claims 1-5 or the protein molecule of any one of claims 6-7; for example, the nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 4, 8, 11, 14, 20, 23.

9. A cell comprising the nucleic acid molecule of claim 8; or having anchored to its cell membrane the engineered GPI-anchored signal peptide of any one of claims 1-5 or the protein molecule of any one of claims 6-7; for example, the cell is selected from the group consisting of: an antigen presenting cell (such as a dendritic cell), an epithelial cell, a neural cell, a red blood cell, a white blood cell, a platelet, a phagocyte (such as a neutrophil, a basophil, an eosinophil, etc.), a B lymphocyte, an effector B cell, a memory B cell, a T lymphocyte, a memory T cell, an effector T cell, a cardiac muscle cell, a smooth muscle cell, a skeletal muscle cell, a cardiac muscle cell, an osteoblast, a glial cell, a hepatocyte, a kidney cell, a gland cell, an endocrine cell (such as a thyroid cell, a thymus cell, an islet B cell, a pancreatic islet cell); for example, a CHO cell, an A549 cell, a Hela cell, an E. coli cell, a yeast cell; and / or, the cell is a normal cell (such as an immune cell) or a diseased cell (such as a tumor cell); and / or, the cell is an in vivo cell, an in vitro cell, or an adoptive cell; and / or the cell is a production cell, a therapeutic cell, an immunological cell, a detection cell. ​ 10. The cell of claim 9, wherein the cell is a dendritic cell, and wherein: the dendritic cell is derived from a bone marrow cell, a cord blood cell, or a peripheral blood mononuclear cell; and / or the dendritic cell is derived from a mammal, such as a human, a non-human primate, a murine; and / or the dendritic cell is a mature dendritic cell that is pulsed with an antigen of interest, such as a tumor antigen, a viral antigen that induces an anti-viral effect, and / or the like, such that the surface of the dendritic cell also presents the antigen of interest.

11. A product comprising one or more substances selected from the group consisting of: the engineered GPI-anchored signal peptide of any one of claims 1-5, the protein molecule of any one of claims 6-7, the nucleic acid molecule of claim 8, or the cell of claim 9 or 10; for example, the product is selected from the group consisting of: a dendritic cell vaccine, an adoptive cell therapy drug, an effector cell stimulator, or a combination thereof; for example, the product comprises or is induced by the dendritic cell of claim 13.

12. Use of the engineered GPI-anchored signal peptide of any one of claims 1-5, the protein molecule of any one of claims 6-7, the nucleic acid molecule of claim 8, the cell of claim 9 or 10, or the product of claim 11, for the manufacture of a medicament for the diagnosis, prevention, and / or treatment of a disease; for example, the disease is selected from the group consisting of: a malignant tumor, a benign tumor, an endocrine disease (such as a nutritional and metabolic disease, an immunological disease (such as an autoimmune disease)), a disease of the blood and blood-forming organs, a mental disease, a nervous system disease, an eye and adnexa disease, an ear and mastoid disease, a circulatory system disease, a respiratory system disease, a digestive system disease, a genitourinary system disease, a skin and subcutaneous tissue disease, a musculoskeletal system and connective tissue disease, an injury, a poisoning; and / or the medicament is selected from the group consisting of: an antibody drug, an exosome delivery drug, a vaccine.

Images