Engineering GPI anchoring signal peptide and fusion protein and application thereof

Abstract

The invention provides an engineered GPI anchoring signal peptide as well as a fusion protein and application thereof. Specifically, the invention provides an engineered glycosylated phosphatidylinositol (GPI) anchoring signal peptide, and compared with an original natural GPI anchoring signal peptide, the N-terminal histidine of the engineered GPI anchoring signal peptide is replaced with an uncharged non-polar amino acid of which the side chain is C3-C6 straight chain or branched chain alkyl, and the side chain of the GPI anchoring signal peptide is replaced with an uncharged non-polar amino acid of which the side chain is C3-C6 straight chain or branched chain alkyl. And comprises a flexible hydrophobic amino acid combination at its C-terminus. The invention further provides a protein molecule which comprises a functional peptide fragment connected with the GPI anchoring signal peptide engineered and modified by the invention. The GPI anchoring signal peptide disclosed by the invention has an enhanced anchoring effect, can be connected with various functional peptide fragments and can be anchored to the surface of a cell membrane in a large amount and enduringly, so that the GPI anchoring signal peptide has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine. Specifically, this invention relates to a novel engineered GPI-anchored signal peptide, a fusion protein comprising the novel GPI-anchored signal peptide, and the design, preparation, and application of the former. Background Technology

[0002] Glycosyl-phosphatidyl inositol-anchored proteins (GPI-APs) are a class of proteins ubiquitous on the surface of eukaryotic cell membranes. They lack transmembrane and cytoplasmic domains, and their C-terminus is anchored to the eukaryotic cell membrane via a GPI anchor structure generated within the cytoplasm. Both the N-terminus and C-terminus of GPI-anchored proteins possess signal peptide sequences. The N-terminal signal peptide is the protein's exit signal peptide, while the C-terminal signal peptide induces the anchored protein to bind to the GPI anchor structure; hence, it is called the GPI-anchored signal peptide. This C-terminal signal peptide is a non-electrolyte hydrophobic amino acid sequence, typically composed of 15-30 amino acids. The GPI anchor structure is highly conserved, consisting of phosphatidylinositol phosphate, a core glycan chain, and a phosphatidylinositol glycolipid chain. GPI-anchored proteins in mammals are mainly involved in cell-cell or cell-matrix interactions. GPI-anchored proteins participate in life activities in mammals such as macrophage anti-inflammatory response, T cell activation, complement cascade amplification, cell proliferation, leukocyte exudation, tumor invasion and metastasis; they also play an important role in spermatogenesis, development and capacitation (PNAS, 2020, 117(36)22061-22067).

[0003] Domestic and international studies have shown that by using GPI-anchored signal peptides to induce intracellular GPI anchor structures, the extracellular segments of functional proteins can be continuously and effectively fixed to the cell membrane surface, exerting biological effects. McHugh et al. first demonstrated that anchoring GPI / CD80 to mouse tumor cells can enhance mouse immune function and clear tumors (Cancer Res. 1999, 59(10):2433-2437). Yi Pingyong et al. designed a GPI / mouse B7.1 fusion protein with strong anti-tumor activity (Cancer. 2005, 103(7):1519-1528). Research on constructing renal cancer vaccines by fusing the IL-12 gene sequence with the GPI-anchored signal peptide sequence of ESAT-6 has also made some progress. The GPI anchoring system has unique anchoring efficiency, and theoretically, any membrane surface protein can be anchored to the cell membrane surface in the form of GPI, thereby exerting biological effects.

[0004] However, current research on the application of GPI technology still has obvious limitations: First, it is generally limited to discovering existing natural GPI anchoring signal sequences, without exploring and creating new and efficient GPI anchoring sequences; second, it generally only uses the GPI system to anchor membrane proteins to various cell membranes, and lacks research on the unique biological effects of secretory proteins anchoring to cell membranes; third, there is a lack of systematic engineering research on using GPI to modify cell membrane surfaces.

[0005] In summary, exploring GPI anchoring systems capable of broadly anchoring membrane-bound and secreted proteins to various cell membrane surfaces, and subsequently anchoring multiple bioactive factors to the surfaces of various cell types to mediate specific biological responses and exert biological effects, is of great significance for cell engineering research and cell membrane modification, and also has high potential clinical application value. Summary of the Invention

[0006] This application provides a novel GPI anchoring signal peptide with enhanced anchoring efficacy after engineering modification, its related linking peptides, encoding molecules, cells, products, and applications.

[0007] In some aspects of this application, an engineered glycosylated phosphatidylinositol (GPI) anchoring signal peptide is provided, wherein, compared with the original natural GPI anchoring signal peptide, the N-terminal histidine of the engineered GPI anchoring signal peptide is replaced with an uncharged nonpolar amino acid with a C3-C6 straight-chain or branched alkyl side chain, and its C-terminus contains a flexible hydrophobic amino acid combination.

[0008] In some aspects of this application, a protein molecule is provided comprising: (a) an engineered GPI-anchored signal peptide of this application; and (b) a functional peptide linked to the engineered GPI-anchored signal peptide described in (a).

[0009] In some aspects of this application, a nucleic acid molecule is provided that encodes the engineered GPI-anchored signal peptide or protein molecule of this application.

[0010] In some aspects of this application, a cell is provided that contains the nucleic acid molecules of this application; or has an engineered GPI-anchored signal peptide or protein molecule of this application anchored to its cell membrane.

[0011] In some aspects of this application, a product is provided comprising one or more substances selected from the group consisting of: engineered GPI-anchored signal peptides, protein molecules, nucleic acid molecules, and / or cells of the present application.

[0012] In some aspects of this application, the application of the engineered GPI-anchored signal peptides, protein molecules, nucleic acid molecules, cells and / or products of this application is provided for use in disease diagnosis, prevention and / or treatment.

[0013] In some aspects of this application, a method for diagnosing, preventing, and / or treating a disease is provided, the method comprising administering to a subject in need an effective amount of the engineered GPI-anchored signal peptide, protein molecule, nucleic acid molecule, cell, and / or product of this application for diagnosis, prevention, and / or treatment.

[0014] In some aspects of this application, an engineered GPI-anchored signal peptide, protein molecule, nucleic acid molecule, cell and / or product is provided for disease diagnosis, prevention and / or treatment.

[0015] Those skilled in the art can combine the foregoing technical solutions and features in any way without departing from the inventive concept and protection scope of this invention. Other aspects of this invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0016] The present invention will be further described below with reference to the accompanying drawings, which are shown only for illustrating embodiments of the present invention and are not intended to limit the scope of the present invention.

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

[0018] Figure 2A-2D Comparison of the anchoring of human PD-L1 to the surface of CHO cells by the novel GPI-anchored signal peptide (new GPI) and the original anchored signal peptide (CD73GPI): Figure 2A Day 3; Figure 2B Day 4; Figure 2C Day 5; Figure 2D Day 6.

[0019] Figure 2E Efficacy of N-terminal double-histidine-substituted anchoring signal peptide (double-histidine replaced with valine) in anchoring human PD-L1 to the CHO cell membrane surface: Left: Day 3; Right: Day 6.

[0020] Figure 3 A novel GPI-anchored signal peptide anchors human IL-2 to the surface of HeLa cell membranes.

[0021] Figure 4 A novel GPI-anchored signal peptide anchors human CD80 to the surface of 293t cell membranes.

[0022] Figure 5 A novel GPI-anchored signal peptide anchors human IL-2 to the surface of 293t cell membranes.

[0023] Figures 6A-6B Comparison of the anchoring effect induced by the novel GPI anchoring signal peptide sequence and the CD55GPI anchoring signal peptide:

[0024] Figure 6A Novel GPI-anchored signal peptide (days 3 and 7);

[0025] Figure 6B CD55GPI-anchored signal peptide (days 3 and 7).

[0026] Figure 7 Effects of lentiviral vector-packaged IL-4 / novel GPI fusion factor transfected into H1299 cells.

[0027] Figures 8A-8B Effects of lentiviral vector packaging of hCD80 / novel GPI fusion factor transfected into dendritic cells:

[0028] Figure 8A : Cultivating immature DCs on the fourth day;

[0029] Figure 8B Lentiviral vector packaging of hCD80 / novel GPI fusion factor transfected dendritic cells.

[0030] Figures 9A-9B hCD80 / new GPI-anchored modified DCs stimulate T cell activation and proliferation.

[0031] Figure 9A Effect of unmodified control DCs on T cell proliferation;

[0032] Figure 9B Effects of hCD80 / new GPI-anchored DCs on T cell proliferation. Detailed Implementation

[0033] To address the shortcomings mentioned in the background art, this invention provides a novel engineered GPI-anchored signal peptide with broad-spectrum and efficient anchoring function, offering a new application path for cell membrane surface modification and cell engineering research.

[0034] Through long-term and in-depth research, the inventors of this application have innovatively engineered natural GPI-anchored signal peptides to obtain novel GPI-anchored signal peptides. Based on this, they further constructed expression vectors for the novel GPI-anchored signal peptides and their linker peptides to functional factors, and transformed cells and products. The anchoring efficiency of membrane-bound and secreted factors on various cell membrane surfaces, as well as the membrane modification effects and functional assays for various cell types, have verified that the novel GPI-anchored signal peptides of this application can be used to broadly anchor various functional factors to different cell membrane surfaces.

[0035] All numerical ranges provided herein are intended to clearly include all values ​​falling between the endpoints of the range and the range of values ​​between them. Features mentioned in the invention or embodiments may be combined. All features disclosed in this specification may be used in any combination form, and each feature disclosed in the specification may be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0036] All numerical ranges provided in this document are intended to clearly include all numerical values ​​falling between the endpoints of the range and the range of numerical values ​​between them. For example, 1 to 3 includes endpoints 1 and 3, specific integer numerical points 2 and non-integer numerical points (e.g., but not limited to: 1.2, 1.5, 1.8, 2.1, 2.3, 2.4, 2.8, etc.), and its subranges (e.g., but not limited to: 1 to 2, 2 to 3, 1 to 1.2, 1.5 to 1.8, etc.).

[0037] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0038] GPI-anchored signal peptide and its coding sequence

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

[0040] As used herein, the terms “original GPI-anchored signaling peptide,” “natural GPI-anchored signaling peptide,” or “original natural GPI-anchored signaling peptide” are used interchangeably and refer to GPI-anchored signaling peptides that have not been engineered.

[0041] Natural GPI-anchoring signal peptides can be, for example, anchoring signal peptides in proteins such as CD73, CD55, and ESAT-6, such as the natural GPI-anchoring signal peptides shown in SEQ ID NO:1 or 15. GPI-anchoring signal peptides known in the art can also be used, or GPI-anchoring protein prediction software or websites (such as PredGPI) can be employed to predict and further identify GPI-anchored proteins and their signal peptides. For example, PredGPI uses an HMM model and nuclear SVM to predict the presence of GPI anchors and their ω sites in eukaryotes.

[0042] The terms "engineered GPI-anchored signal peptide," "novel GPI-anchored signal peptide," or "signal peptide of this application" refer to novel GPI-anchored signal peptides obtained by engineering a natural GPI-anchored signal peptide according to the modification scheme described in this application. The engineering modification of this application includes: replacing (e.g., by mutation) the N-terminal histidine of the original natural GPI-anchored signal peptide with an uncharged, nonpolar amino acid with a C3-C6 straight-chain or branched alkyl side chain, and adding a flexible, hydrophobic amino acid combination to the C-terminus of the original natural GPI-anchored signal peptide.

[0043] The novel GPI-anchored signal peptide of this application can be obtained by modifying anchoring signal peptides in proteins such as CD73, CD55, and ESAT-6. In some embodiments, the novel GPI-anchored signal peptide of this application is obtained by modifying the natural GPI-anchored signal peptide shown in SEQ ID NO:1 or 15. In some embodiments, the novel GPI-anchored signal peptide of this application may be a polypeptide containing the amino acid sequence shown in SEQ ID NO:3 or 22, or may be encoded by a nucleotide molecule containing the nucleotide sequence shown in SEQ ID NO:4 or 23.

[0044] The novel signal peptides of the present invention can be produced by chemical synthesis or by using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher animals, insects and mammalian cells), or by site-directed mutagenesis or other known molecular biology techniques.

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

[0046] In some embodiments, the engineered GPI-anchored signal peptide coding sequence of this application can be constructed, and codon optimization can be optionally performed on it. In some embodiments, coding sequences such as SEQ ID NO:4 or 23 can be used to encode the novel GPI-anchored signal peptide of this application. Molecules that hybridize with these sequences under stringent conditions, or family gene molecules that are 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 "strict conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2 × SSC, 0.1% SDS, 60°C; or (2) hybridization with the addition of a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the similarity between the two sequences is at least 50%, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, more preferably 95%. For example, the sequence may be a complementary sequence to the sequence defined in (a).

[0048] The full-length nucleotide sequence or fragment thereof of the signal peptide gene of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art as templates.

[0049] Beyond theoretical limitations, the applicant unexpectedly discovered that the novel GPI-anchored signal peptide of this application incorporates a side-chain-rich amino acid site near the N-terminus through mutation, and adds a highly extensible flexible amino acid sequence at the C-terminus. This ensures the integrity of the ω site (i.e., the GPI-anchor attachment site) and the amino acid sequence near the ω site in the original GPI-anchored signal peptide sequence, enhancing the extensibility of the original anchoring signal peptide sequence structure, thereby producing a superior anchoring effect, including broad-spectrum cell anchoring, broad-spectrum anchoring targets (e.g., functional factors linked to the novel GPI-anchored signal peptide), and durable anchoring action.

[0050] Linking peptides containing GPI-anchored signaling peptides

[0051] This application also relates to protein molecules comprising the novel GPI-anchored signal peptide of this application, comprising: (a) the engineered GPI-anchored signal peptide of this application; and (b) a functional peptide linked to the engineered GPI-anchored signal peptide described in (a).

[0052] The functional peptides linked to the GPI-anchored signal peptide engineered in this application can be selected according to actual needs. In some embodiments, the functional peptides include, but are not limited to: secreted peptides, intracellular peptides, intracellular or extracellular segments of transmembrane peptides, and membrane-bound peptides. In some embodiments, the functional peptides may be derived from, but are not limited to: antibodies or their antigen-binding fragments or their targeted binding peptides (e.g., peptides containing binding epitopes), ligands, receptors, and cytokines (such as lymphokines, monokines, interleukins, interferons, colony-stimulating factors, tumor necrosis factor, and transforming growth factor).

[0053] In some embodiments, the functional peptide may be derived from one or more antibodies selected from the group consisting of: anti-PD1 antibody, anti-PD-L1 antibody, anti-TIGIT antibody, anti-Artemin antibody, anti-CD19, CD44, CD47 or CD123 antibody, and anti-STING antibody.

[0054] In some embodiments, the functional peptide may 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-α, TNF -β; TGF-β1, TGF-β2, TGF-β3, TGFβ1β2, BMP; GRO / MGSA, PF-4, CTAP-III, IP-10, ENA-78; MIP-1α, MIP-1β, RA NTES, MCP-1, MCP-2, MCP-3, I-309; EGF, PDGF, FGF, HGF, IGF-I, IGF-II, LIF, NGF, OSM, PDECGF, TGF-α, VEGF.

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

[0056] The engineered GPI-anchored signal peptide and functional peptide of this application can be linked directly, through a linker molecule, coupled, or fused. The linking method can be selected as needed. In some embodiments, the protein molecule of this application also contains other active molecules, such as cytotoxins, linked to (a) or (b).

[0057] The novel GPI-anchored signal peptide of this application can be linked to various functional moieties to form protein molecules that anchor the functional moieties to various target cell membranes to perform their functions. In some embodiments, dendritic cells containing the protein molecules on their cell membranes are provided, which are capable of more efficiently presenting antigens and / or promoting T cell proliferation.

[0058] carriers and cells

[0059] This application also relates to a vector containing the novel GPI-anchored signal peptide gene or linker peptide gene of this application, and a host cell generated by genetic engineering using the vector.

[0060] Using conventional recombinant DNA technology (Science, 1984; 224:1431), the coding sequence of this invention can be used to express or produce novel recombinant GPI-anchored signal peptides or linker peptides. Generally, the following steps are involved:

[0061] (1) Use the polynucleotide molecule encoding the novel GPI-anchored signal peptide or linker peptide of this application, or use the recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell.

[0062] (2) Host cells cultured in a suitable culture medium to produce the desired signal peptide or linker peptide.

[0063] In this invention, the terms "vector" and "recombinant expression vector" are used interchangeably, referring to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translation control elements.

[0064] Methods well known to those skilled in the art can be used to construct expression vectors containing coding sequences and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In this invention, pTT5 vector, pcDNA3.1 vector, pIRES2-EGFP vector, and AdMax vector are preferred. TM 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] Furthermore, this application also provides dendritic cells modified with the novel GPI-anchored signal peptide or linker peptide of this application. In some embodiments, the surface of the dendritic cells contains the engineered GPI-anchored signal peptide of this application anchored to its cell membrane, or a protein molecule containing a functional polypeptide linked to said signal peptide. In some embodiments, the functional polypeptide anchored on the surface of dendritic cells modified with the novel GPI-anchored signal peptide of this application is increased by more than 20% compared to unmodified dendritic cells, for example, 20-400%, for example, 30%-300%, 50%-200%, or any range or value between these values. In some embodiments, the promoting effect of dendritic cells modified with the novel GPI-anchored signal peptide of this application on T cell proliferation is increased by more than 20%, for example, 20-400%, for example, 30%-300%, 50%-200%, or any range or value between these values ​​compared to unmodified dendritic cells.

[0070] In some embodiments, the cells used in this application may be selected from in vivo cells, in vitro cells, or adoptive cells; and / or the cells may be production cells, therapeutic cells, immunization cells, or detection cells. In some embodiments, the cells are dendritic cells, such as mature dendritic cells sensitized with a target antigen, optionally also presenting the target antigen on their surface, such as tumor antigens or viral antigens that induce antiviral effects.

[0071] Drugs, pharmaceutical compositions or kits

[0072] This invention also provides a medicament, pharmaceutical composition, or kit containing an effective amount of the linker peptide of this application linked with a novel GPI-anchored signal peptide, or its coding sequence, or a vector or cell, and a pharmaceutically or immunologically acceptable vector. As used herein, the terms "active substance" and "active substance of the invention" are used interchangeably and refer to a linker peptide linked with a novel GPI-anchored signal peptide, or its coding sequence, or a vector or cell, having disease diagnostic, preventive, and / or therapeutic activity.

[0073] The novel GPI-anchored signal peptide of this application can be used to anchor desired functional substances to cell membranes or vesicle (e.g., exosome) membranes or other surfaces with GPI-anchored structures, thereby exerting the function of the functional substance. For example, the novel GPI-anchored signal peptide of this application can be used to anchor immune molecules (such as CD80) linked to it to the tumor cell membrane to enhance immune function and eliminate tumors; it can also be used to anchor targeting molecules linked to it to the exosome vesicle membrane to enhance the targeting of exosomes, thereby improving the accuracy of exosome targeted delivery of its contents.

[0074] In some embodiments, the medicament of this application may be used to diagnose, prevent and / or treat one or more diseases selected from the group consisting of: malignant tumors, benign tumors, endocrine diseases (such as nutritional and metabolic diseases), immune diseases (such as autoimmune diseases), blood and hematopoietic organ diseases, mental illnesses, nervous system diseases, eye and appendage diseases, ear and mastoid diseases, circulatory system diseases, respiratory system diseases, digestive system diseases, genitourinary system diseases, skin and subcutaneous tissue diseases, musculoskeletal system and connective tissue diseases, injuries, poisoning, etc.

[0075] As used herein, the terms “containing” or “including” include “comprising,” “consistently composed of,” and “composed of.” As used herein, a “pharmaceutically acceptable” ingredient is a substance suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. As used herein, the term “effective amount” refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0076] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0077] Pharmaceutically acceptable carriers in the composition may contain liquids such as water, saline, glycerol, and ethanol. Additionally, these carriers may contain auxiliary substances such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffers, etc. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, with a pH usually around 5-8, preferably around 6-8.

[0078] As used herein, the term "unit dosage form" refers to a dosage form in which the compositions of the present invention are prepared for a single dose for ease of administration, including but not limited to various solid dosage forms (such as tablets), liquid dosage forms, capsules, and sustained-release formulations.

[0079] It should be understood that the effective dose of the active substance used can vary depending on the severity of the condition of the patient being treated. The specific dosage is determined based on the individual patient's circumstances (e.g., weight, age, physical condition, and desired outcome), within the judgment of a skilled physician.

[0080] The compositions of the present invention can be in solid form (such as granules, tablets, lyophilized powder, suppositories, capsules, sublingual tablets) or liquid form (such as oral liquid) or other suitable forms. The administration routes can be: (1) direct injection of naked DNA or protein; (2) linking cDNA, mRNA, and protein with a transferrin / poly-L-lysine complex to enhance their biological effects; (3) forming complexes of cDNA, mRNA, and protein with positively charged lipids to overcome the difficulty of crossing cell membranes caused by the negative charge of the phosphate backbone; (4) encapsulating cDNA, mRNA, and protein in liposomes to mediate their entry into cells, which facilitates the smooth entry of macromolecules and protects them from hydrolysis by various extracellular enzymes; (5) using immunoliposomes to transport cDNA, mRNA, and protein specifically to target tissues and cells; (6) transfecting cDNA, mRNA, and protein in vitro into regenerating cells (such as fibroblasts); (7) electroporation, i.e., introducing cDNA, mRNA, and protein into target cells using an electric current.

[0081] In addition, the compositions of the present invention may also contain other active substances for diagnosing, improving and treating related diseases.

[0082] Example

[0083] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make appropriate modifications and variations to the present invention, and such modifications and variations are all within the scope of the present invention.

[0084] Experimental methods not specifically described in the following examples can be performed using conventional methods in the art, such as those described in *Molecular Cloning: A Laboratory Manual* (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989) or according to the conditions recommended by the supplier. DNA sequencing methods are conventional in the art and can also be provided by commercial companies.

[0085] Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0086] Example 1. Design and vector construction of novel GPI-anchored signal peptide sequences

[0087] A novel GPI-anchored signal peptide sequence was designed based on the CD73 GPI-anchored signal peptide protein, and its vector was constructed (e.g., Figure 1 (As shown).

[0088] (I) Experimental Materials

[0089] pTT5 vector and competent cells were purchased from Jiangsu Genscript Biotech Co., Ltd.

[0090] Gene synthesis and primer synthesis services were provided by Jiangsu GenScript Biotech Co., Ltd.

[0091] Restriction endonucleases and T4 DNA ligase were purchased from Takara.

[0092] LB culture medium and ampicillin antibiotic were purchased from Shanghai Sangon Biotech Co., Ltd.

[0093] (II) Experimental Methods

[0094] A novel GPI anchoring signal peptide sequence (SEQ ID NO: 3 and 4) was designed based on the original CD73 GPI anchoring signal peptide protein sequence (SEQ ID NO: 1 and 2). The hPD-L1 / new GPI DNA sequence (SEQ ID NO: 8) was synthesized by GenScript, with Not I and EcoRI restriction sites at both ends. The synthesized hPD-L1 / GPI fragment was modified by double digestion with Not I and EcoRI, and then ligated with a Not I and EcoRI-digested pTT5 vector at 4°C for 12 hours using ligase. The ligation was performed after transformation into competent DH5α cells, and positive clones were selected and cultured on LB medium (Amp...). + The culture was performed, plasmids were extracted in large quantities, sequenced by GenScript, and stored at -80℃.

[0095] The construction of eukaryotic expression vectors for other functional protein / GPI fusion factor sequences was carried out according to the above method.

[0096] (III) Experimental Results

[0097] Through research and analysis, we designed novel GPI-anchored signal peptide sequences based on CD73. The amino acid sequence of the novel GPI-anchored signal peptide with single His substitution is shown in SEQ ID NO:3, and its coding sequence is shown in SEQ ID NO:4. The amino acid sequence of the novel GPI-anchored signal peptide with double His substitution is shown in SEQ ID NO:22, and its coding sequence is shown in SEQ ID NO:23.

[0098] • CD73 amino acid sequence (SEQ ID NO:21):

[0099] MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLLKEAKF PILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRK VPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENL AAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKF STGSHCHGSFSLIFLSLWAVIFVLYQ

[0100] ● Original CD73GPI anchoring signal peptide sequence (amino acids 549-574 of SEQ ID NO:1 = SEQ ID NO:21):

[0101] STGS H CHGSFSL IFLSLWAVIFVLYQ

[0102] ● The coding sequence of the original CD73GPI-anchored signal peptide (SEQ ID NO:2)

[0103] tctaccggctctcactgccatggctctttctccctgatcttcctgtctctgtgggccgtgatctttgtgctgtac cag

[0104] ● Novel CD73GPI anchored signal peptide sequence (SEQ ID NO:3):

[0105] STGS V CHGSFSL IFLSLWAVIFVLYQ GSGSGSGS

[0106] ●The coding sequence of the novel CD73GPI-anchored signal peptide (SEQ ID NO:4):

[0107] agtactggtagtgtgtgccatggtagttttagcctgatcttcctgtccctgtgggccgtgatcttcgtgctgtac cagggctctggctctggctctggctct

[0108] ●The CD73GPI-anchored signal peptide sequence with double N-terminal histidine substitutions (SEQ ID NO:22):

[0109] STGS V C V GSFSL IFLSLWAVIFVLYQ GSGSGSGS

[0110] ●The coding sequence of the novel CD73GPI-anchored signal peptide (SEQ ID NO:23):

[0111] agtactggtagtgtgtgcgtgggtagttttagcctgatcttcctgtccctgtgggccgtgatcttcgtgctgtac cagggctctggctctggctctggctct

[0112] Specifically, we selected the original GPI anchoring signal peptide sequence of CD73 (SEQ ID NO:21) (amino acids 549-574 of CD73) and modified it. We mutated a histidine at the N-terminus of the anchoring signal peptide to a flexible amino acid and added a flexible, hydrophobic amino acid combination at the C-terminus of the sequence to design a novel CD73 / GPI anchoring signal peptide sequence. Structurally, this novel GPI anchoring signal peptide sequence has a Val amino acid site rich in side chains mutated at the front end and a highly extensible flexible amino acid combination added at the C-terminus. This ensures the integrity of the serine site at position 9 (amino acid 557 of the mature CD73 protein) in the original GPI anchoring signal peptide sequence (this amino acid is one of the amino acids of the ω site (i.e., GPI-anchor attachment site) and the amino acid sequence near the ω site, enhancing the extensibility of the original anchoring signal peptide sequence structure and is expected to have better anchoring efficiency.

[0113] We further constructed a eukaryotic expression vector for pTT5-hPD-L1 / new GPI (e.g., Figure 1As shown in the figure, sequencing confirmed the correct construction. We can use this to determine the anchoring function of the PD-L1 fusion factor eukaryotic vector carrying the GPI-anchored signal peptide.

[0114] The construction method of the new N-terminal double-replacement CD73 / GPI and its eukaryotic expression vector is the same as above, except that the 7th histidine is also replaced with valine.

[0115] Example 2. Comparison of the anchoring effects of hPD-L1 / new GPI and hPD-L1 / CD73GPI on CHO cell membranes

[0116] CHO cells were transfected with the eukaryotic expression vector pTT5-hPD-L1 / new GPI. Flow cytometry analysis confirmed that the anchoring efficacy of hPD-L1 / new GPI on the CHO cell membrane was superior to that of hPD-L1 / CD73GPI (e.g., pTT5-hPD-L1 / new GPI). Figures 2A-2D (As shown). We also verified the ability of CD73GPI with both histidine residues at the N-terminus 5 and 7 positions replaced by valine (N-terminal double-replacement new CD73 / GPI) to anchor hPD-L1 on the CHO cell membrane. Although the anchoring ability of the double-replacement new CD73 / GPI did not reach the anchoring efficiency of the single His-replacement new CD73 / GPI, it was still significantly better than the original CD73 / GPI.

[0117] (I) Experimental Materials

[0118] The PE-labeled primary antibody (mouse anti-human) for hPD-L1 was purchased from Abcom and detected by flow cytometry.

[0119] JetPEI plasmid transfection reagent was purchased from Polyplus;

[0120] CHO cell line, 1640 medium, and fetal bovine serum were purchased from Thermo Fisher Scientific.

[0121] The flow cytometer was purchased from BD.

[0122] The eukaryotic expression vectors of pTT5-hPD-L1 / new GPI, pTT5-hPD-L1 / double-substituted CD73GPI, and pTT5-hPD-L1 / CD73GPI were constructed according to Example 1.

[0123] (II) Experimental Methods

[0124] The eukaryotic expression vectors pTT5-hPD-L1 / new GPI, pTT5-hPD-L1 / double-substituted CD73GPI, and pTT5-hPD-L1 / CD73GPI constructed and prepared in Example 1 above were used to express CHO cells at a rate of 3 × 10⁻⁶. 5The cells were seeded at a density of 1 μg / well in 6-well plates. After 18 hours, two plasmids were transfected at a density of 1 μg / well using JetPEI plasmid transfection reagent. Samples were taken at 72 h, 96 h, 120 h and 144 h after transfection. 100 μl of sample was taken from each well, and PE-labeled anti-hPD-L1 primary antibody was added. The cells were labeled at 4 °C for 20 min, washed twice with PBS, and phenotypic analysis was performed by flow cytometry to detect the expression of hPD-L1 on the cell membrane surface.

[0125] (III) Experimental Results

[0126] We selected extracellular regions of the human PD-L1 molecule (SEQ ID NO:5 and 6) and fused them with novel GPI-anchored signal peptide sequences and original CD73 / GPI-anchored signal peptide sequences, respectively, to construct expression vectors for pTT5-hPD-L1-original CD73GPI-anchored signal peptide fusion genes (SEQ ID NO:7) and pTT5-hPD-L1-novel GPI-anchored signal peptide fusion genes (SEQ ID NO:8), respectively. These vectors were transfected into CHO cells. Flow cytometry analysis revealed that the fusion proteins expressed by both fusion genes were detectable on the CHO membrane. On days 3, 4, 5, and 6 post-transfection, the expression rates of PD-L1-GPI / CD73 were 72.8%, 70.6%, 64.7%, and 54.4%, respectively; while the expression rates of PD-L1 / novel GPI reached 94.4%, 91.9%, 91.5%, and 91.2%, respectively. The results showed that after the fusion factor was expressed intracellularly in CHO cells, the generated hPD-L1 / GPI fusion protein migrated and attached to the CHO cell membrane via a GPI anchor structure. The anchoring effect induced by the novel GPI-anchored signal peptide was better and more sustained than that of the original CD73 / GPI anchored signal peptide sequence. The new CD73 / GPI with N-terminal double histidine substitutions for valine showed an hPD-L1 membrane expression rate of 89.1% on day 3 and 87.7% on day 6. Although its hPD-L1 anchoring efficacy was not as high as the novel CD73 / GPI, it was still significantly better than the original CD73 / GPI.

[0127] Example 3. Anchoring effect of IL-2 / new GPI on HeLa cell membrane

[0128] HeLa cells were transfected with the pTT5-IL-2 / new GPI eukaryotic expression vector, and the expression efficiency of IL-2 / new GPI on the HeLa cell membrane was verified by flow cytometry (results are shown in the figure). Figure 3 (As shown).

[0129] (I) Experimental Materials

[0130] The APC-labeled primary antibody (mouse anti-human) for flow cytometry detection of hIL-2 was purchased from Abcom.

[0131] JetPEI plasmid transfection reagent was purchased from Polyplus;

[0132] HeLa cells, 1640 medium, and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific.

[0133] The flow cytometer was purchased from BD.

[0134] The eukaryotic expression vector pTT5-hIL-2 / new GPI was constructed according to the method in Example 1.

[0135] (II) Experimental Methods

[0136] The pTT5-hIL-2 / new GPI eukaryotic expression vector, constructed using the method described in Example 1 above, was used to express HeLa cells at a concentration of 3 × 10⁻⁶ cells / mL. 5 The cells were seeded at a density of 1 μg / well in 6-well plates. After 14 hours, two plasmids (1 μg / well) were transfected with JetPEI plasmid transfection reagent. 96 hours after transfection, samples were taken sequentially, with 100 μl of each sample added to each well. The sample was then incubated at 4°C for 20 minutes with APC-labeled anti-hIL-2 primary antibody, washed twice with PBS, and subjected to flow cytometry for phenotypic analysis to detect the expression of hIL-2 on the cell membrane surface.

[0137] (III) Experimental Results

[0138] We constructed a pTT5-hIL-2 / novel GPI-anchored signal peptide fusion gene expression vector (SEQ ID NO: 11) using the human IL-2 gene sequence (SEQ ID NO: 9 and 10) and a novel GPI-anchored signal peptide sequence. Liposome transfection of HeLa cells revealed IL-2 expression on the HeLa cell membrane. On day 4 post-transfection, the expression rate of IL-2 / novel GPI was 35.2%, while IL-2 was virtually undetectable on the surface of HeLa cells in the control group. This result indicates that after the fusion factor is expressed in HeLa cells, the resulting fusion protein migrates and attaches to the HeLa cell membrane via a GPI anchoring structure. The anchoring effect of the novel GPI-anchored signal peptide can anchor secreted IL-2 to tumor cells, exploring a new avenue for the development of tumor vaccines that enhance immune responses.

[0139] Example 4. Anchoring effect of hCD80 / new GPI on 293t cell membranes

[0140] 293t cells were transfected with the eukaryotic expression vector pTT5-hCD80 / new GPI. Flow cytometry was used to verify the expression efficiency of hCD-80 / GPI on the 293t cell membrane (results are shown in the figure). Figure 4 (As shown).

[0141] (I) Experimental Materials

[0142] The FITC-labeled primary antibody (mouse anti-human) for hCD80 detection by flow cytometry was purchased from Abcom.

[0143] JetPEI plasmid transfection reagent was purchased from Polyplus;

[0144] 293T cells, 1640 culture medium, and fetal bovine serum FBS were purchased from Thermo Fisher Scientific.

[0145] The flow cytometer was purchased from BD.

[0146] The eukaryotic expression vector pTT5-hCD80 / new GPI was constructed according to the method in Example 1.

[0147] (II) Experimental Methods

[0148] The pTT5-hCD80 / new GPI eukaryotic expression vector and the pTT5-hPD-L1 / CD73GPI eukaryotic expression vector prepared using the method described in Example 1 above were used to express 293t cells at a rate of 3 × 10⁻⁶. 5 Plasmids were seeded at a density of 1 μg / well in 6-well plates. After 16 hours, plasmids were transfected at a density of 1 μg / well using JetPEI plasmid transfection reagent. 72 hours after transfection, samples were taken sequentially, with 100 μl of each sample added to each well. FITC-labeled anti-hCD80 primary antibody was added, and the samples were incubated at 4°C for 25 minutes. The cells were washed twice with PBS, and phenotypic analysis was performed by flow cytometry to detect the expression of hCD80 on the cell membrane surface.

[0149] (III) Experimental Results

[0150] We selected the extracellular segment of human CD80 (SEQ ID NO: 12 and 13) and a novel CD73 / GPI anchoring signal peptide sequence to construct the pTT5-hCD80 / novel GPI fusion gene expression vector (SEQ ID NO: 14). This vector was transfected into 293t cells. Flow cytometry analysis revealed that the CD80 molecules expressed by the fusion gene were detectable on the 293t cell membrane. On day 3 post-transfection, the expression rate of CD80 on the original 293t cell membrane was 0.44%, while the expression rate reached 75.7% after transfection with the hCD80 / novel GPI fusion gene. These results indicate that after intracellular expression of the fusion protein in the 293t cell expression system, CD80 molecules can attach to the 293t cell membrane via a GPI anchoring structure induced by the novel GPI anchoring signal peptide.

[0151] Example 5. Anchoring effect of hIL-2 / new GPI on 293t cell membranes

[0152] 293t cells were transfected with the pTT5-IL-2 / new GPI eukaryotic expression vector. Flow cytometry was used to verify the expression efficiency of hIL-2 / new GPI on the 293t cell membrane (results are shown in the figure). Figure 5 (As shown).

[0153] (I) Experimental Materials

[0154] The PE-labeled primary antibody (mouse anti-human) for flow cytometry detection of hIL-2 was purchased from Abcom.

[0155] JetPEI plasmid transfection reagent was purchased from Polyplus;

[0156] 293T cells, 1640 culture medium, and fetal bovine serum FBS were purchased from Thermo Fisher Scientific.

[0157] The flow cytometer was purchased from BD.

[0158] The eukaryotic expression vector of pTT5-IL-2 / new GPI was constructed according to the method in Example 1, and the expression vector used was the same as in Example 3.

[0159] (II) Experimental Methods

[0160] The pTT5-hIL-2 / new GPI eukaryotic expression vector, constructed using the method described in Example 1 above, was used to express 293t cells at a rate of 3 × 10⁻⁶. 5 Plasmids were seeded at a density of 1 μg / well in 6-well plates. After 16 hours, plasmids were transfected at a density of 1 μg / well using JetPEI plasmid transfection reagent. 72 hours after transfection, samples were taken sequentially, with 100 μl of each sample added to each well. The sample was then labeled with PE-labeled anti-hCD80 primary antibody and incubated at 4°C for 25 minutes. The cells were washed twice with PBS and subjected to flow cytometry for phenotypic analysis to detect the expression of human IL-2 on the cell membrane surface.

[0161] (III) Experimental Results

[0162] To verify the broad-spectrum nature of the novel GPI-anchored signal peptide inducing anchored immune molecules, we selected the secreted cytokine human IL-2 sequence and constructed a pTT5-hIL-2 / novel GPI-anchored signal peptide fusion gene expression vector. This vector was transfected into 293t cells. Flow cytometry analysis revealed that hIL-2 expressed by the fusion gene was detectable on the 293t cell membrane. On day 3 post-transfection, the expression rate of IL-2 on the surface of the original 293t cell membrane was 2.11%, while the expression rate of IL-2 on the surface of the 293t cell membrane reached 99.8% after transfection with the hIL-2 / novel GPI fusion gene.

[0163] The above experimental results indicate that after the fusion gene is expressed intracellularly as a fusion protein in the 293t cell expression system, the secretory factor IL-2 can be anchored to the surface of engineered cell membranes via the GPI anchoring structure. Combined with the anchoring efficiency of IL-2 / new GPI on the HeLa cell membrane in Example 3, this demonstrates that the new GPI can effectively anchor secretory factors to the surfaces of various cell membranes.

[0164] Example 6. Comparison of the anchoring effects of hPD-L1 / new GPI and hPD-L1 / CD55GPI on CHO cell membranes

[0165] CHO cells were transfected with the eukaryotic expression vectors pTT5-hPD-L1 / new GPI and pTT5-hPD-L1 / CD55GPI, respectively. Flow cytometry analysis verified that the anchoring effect mediated by the new GPI was superior to that of the commonly used CD55GPI (results are shown in Figure 1). Figure 6A and 6B (As shown).

[0166] (I) Experimental Materials

[0167] The PE-labeled primary antibody (mouse anti-human) for hPD-L1 was purchased from Abcom and was used for flow cytometry detection.

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

[0169] CHO cells, 1640 medium, and fetal bovine serum were purchased from Thermo Fisher Scientific.

[0170] The flow cytometer was purchased from BD.

[0171] The eukaryotic expression vectors pTT55-hPD-L1 / new GPI and pTT5-hPD-L1 / CD55GPI were constructed according to the method in Example 1.

[0172] (II) Experimental Methods

[0173] The pTT5-hPD-L1 / new GPI eukaryotic expression vector and the pTT5-hPD-L1 / CD55GPI eukaryotic expression vector were constructed using the method described in Example 1 above. CHO cells were then used at a concentration of 3 × 10⁻⁶ cells / year. 5 The cells were seeded at a density of 1 μg / well in 6-well plates. After 18 hours, two plasmids (1 μg / well) were transfected with JetPEI plasmid transfection reagent. Samples were taken at 72 h and 168 h after transfection, and 100 μl of each sample was added to each well. The cells were labeled with PE-conjugated anti-hPD-L1 primary antibody and incubated at 4 °C for 20 minutes. The cells were washed twice with PBS and the expression of hPD-L1 on the surface of CHO cell membranes was detected by flow cytometry.

[0174] (III) Experimental Results

[0175] Currently, the CD55 / GPI anchoring signal peptide sequence (SEQ ID NO:15; its coding sequence is shown in SEQ ID NO:16) is commonly used in cell biology experiments. We selected a novel GPI anchoring signal peptide sequence and constructed expression vectors for the pTT5-hPD-L1 / novel CD73GPI anchoring signal peptide fusion gene and pTT5-hPD-L1-CD55 / GPI anchoring signal peptide fusion gene (SEQ ID NO:17), respectively. These were transfected into CHO cells. Flow cytometry analysis revealed that the expression rates of human PD-L1 on the CHO cell membrane surface were 89.5% and 89.7% on days 3 and 7 after transfection with the hPD-L1-novel CD73 / GPI anchoring signal peptide fusion gene, respectively. Figure 6A The expression rates of PD-L1 on the cell membrane surface of CHO cells transfected with the hPD-L1-CD55 / GPI anchoring signal peptide fusion gene were 80.8% and 72.7%, respectively. Figure 6B ).

[0176] The above results indicate that the anchoring effect mediated by the novel CD73 / GPI anchoring signal peptide is superior and more durable than that mediated by the CD55 / GPI anchoring signal peptide.

[0177] Example 7. Construction and anchoring function verification of IL-4 / novel GPI fusion factor lentiviral expression vector

[0178] A lentiviral expression vector for the IL-4 / novel GPI fusion factor was constructed, and its anchoring function was validated (results are shown in Figure 1). Figure 7 (As shown).

[0179] (I) Experimental Materials

[0180] pLVX-CMV lentiviral plasmid vector, packaging vector, and competent cells were purchased from Shanghai Shengbo Biomedical Technology Co., Ltd.

[0181] The lentiviral expression vector was constructed by Shanghai Shengbo Biomedical Technology Co., Ltd.

[0182] Gene synthesis and primer synthesis were provided by Jiangsu GenScript Biotech Co., Ltd.

[0183] 293t cells, H1299 cells, and serum-free culture medium were purchased from Thermo Fisher Scientific.

[0184] The FITC-labeled hIL-4 antibody was purchased from Abcom.

[0185] (II) Experimental Methods

[0186] After amplifying the synthesized IL-4 / novel GPI sequence, we entrusted Shanghai Shengbo Biomedical Technology Co., Ltd. to construct a lentiviral expression vector. Following sequencing verification, the vector was packaged into a lentiviral expression vector and used to package ViraPower.TM Packaging Mix and lentiviral vector were transfected into 293 cells. The supernatant was collected after 48 hours, and the viral titer was determined by serial dilution of 29t3 cells. H1299 cells were transfected with serum-free medium at MOI 10. After 72 hours, the cell pellet was collected by centrifugation, and APC-labeled hIL-4 antibody was added. The cells were incubated at 4°C for 25 minutes, washed twice with PBS, and phenotypic analysis was performed by flow cytometry to detect the expression of IL-4 on the H1299 cell membrane.

[0187] (III) Experimental Results

[0188] To further verify the ability of the novel GPI-anchored signal peptide to induce and anchor different factors in different expression systems and cells, we selected the secreted cytokine human IL-4 sequence (SEQ ID NO: 18 and 19) to construct a lentiviral expression vector for the hIL-4 / novel GPI-anchored signal peptide fusion gene (SEQ ID NO: 20). This vector was transfected into H1299 cells. Flow cytometry analysis revealed that hIL-4 expressed by the fusion gene could be detected on the H1299 cell membrane. On day 3 post-transfection, the expression rate of IL-4 on the surface of the original H1299 cell membrane was 0.19%, while the expression rate of IL-4 on the surface of the H1299 cell membrane reached 44.3% after transfection with the novel IL-4-GPI fusion gene.

[0189] The above results indicate that the fusion gene can also anchor the secretory factor IL-4 to the cell membrane surface through the GPI anchoring structure in the viral vector expression system, further confirming the universality of the anchoring ability induced by the novel GPI.

[0190] Example 8. Construction and anchoring function verification of CD80 / novel GPI fusion factor lentiviral expression vector

[0191] A lentiviral expression vector for the CD80 / novel GPI fusion factor was constructed, and the anchoring function of the fusion factor was validated (e.g., Figure 8A and 8B (As shown).

[0192] (I) Experimental Materials

[0193] 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 performed by Jiangsu Genscript Biotech Co., Ltd.; 293T cells, 1640 medium, fetal bovine serum, and serum-free medium were purchased from Thermo Fisher Scientific; hGM-CSF and hIL-4 were purchased from RD Biotech; FITC-labeled human CD80 primary antibody was purchased from Abcom; human peripheral blood mononuclear cells were obtained from healthy adult volunteers.

[0194] (II) Experimental Methods

[0195] Freshly isolated, anticoagulated peripheral blood leukocytes from healthy volunteers were collected and centrifuged using a density gradient in a lymphocyte separation medium to obtain peripheral blood mononuclear cells (PBMCs). These PBMCs were then resuspended in complete culture medium (RPMI 1640 containing 10% fetal bovine serum) at a concentration of 1 x 10⁻⁶ cells / mL. 7 Cells were seeded per well in 6-well plates and incubated at 37°C with 5% CO2 for 2 hours. Non-adherent cells were removed with pre-warmed culture medium. Adherent cells were cultured at 37°C with 5% CO2 in complete medium containing recombinant human GM-CSF (50 ng / ml) and recombinant human IL-4 (10 ng / ml). The self-designed and synthesized hCD80 / novel GPI sequence was amplified and then used by Shanghai Shengbo Biomedical Technology Co., Ltd. to construct a lentiviral expression vector. After sequencing verification, lentiviral packaging was performed, and ViraPower was used. TM PackagingMix and lentiviral vectors were transfected into 293t cells. The supernatant was collected after 48 hours, and viral titer was determined using a serial dilution method. Immature dendritic cells (DCs) cultured to day 4 were transfected with serum-free medium and cultured using an MOI gradient of 50. Cell pellets were collected by centrifugation after 72 hours, and FITC-labeled primary antibody was added. The cells were incubated at 4°C for 25 minutes, washed twice with PBS, and phenotypic analysis was performed using flow cytometry to detect CD80 expression on the DC cell membrane.

[0196] (III) Experimental Results

[0197] We used a human peripheral blood-derived DC induction culture system to culture immature DCs (such as...). Figure 8A As shown in the figure, a lentiviral expression vector for the hCD80 / new / GPI fusion gene was constructed and transfected into immature human dendritic cells (DCs). Flow cytometry analysis 72 hours later revealed that hCD80 expressed by the fusion gene could be detected on the DC cell membrane. On day 3 after transfection, the expression rate of CD80 on the surface of the original DC cell membrane was 25.4%, while the expression rate of CD80 on the surface of the DC cell membrane transfected with the CD80 / new GPI fusion gene at MOI50 reached 97.4%, indicating that the GPI anchor structure can efficiently anchor the overexpressed CD80 to the surface of the DC cell membrane.

[0198] Example 9. DCs modified with co-stimulatory molecule-GPI anchoring significantly promoted T cell proliferation in the MLR response.

[0199] The promoting effect of co-stimulatory molecule-GPI-anchored dendritic cells (DCs) on T cell proliferation was detected by mixed lymphocyte reaction (MLR). Figures 9A-9B (As shown).

[0200] (I) Experimental Materials

[0201] Lentiviral-packaged hCD80 / new GPI transfected DCs were prepared as described in Examples 7 and 8; T cells were obtained from healthy adult volunteers; flow cytometry antibodies (primary antibodies) for hCD3, hCD4, and hCD8, and CFSE were purchased from Abcom; 1640 medium, fetal bovine serum, BSA, and cell culture plates were purchased from Thermo Fisher Scientific.

[0202] (II) Experimental Methods

[0203] We selected hCD80 / new GPI-anchored dendritic cells (DCs) packaged with lentivirus prepared by the aforementioned method, and stimulated these membrane-modified DCs with OVA antigen (Shanghai Xinbosheng Company). Then, we co-incubated them with CFSE-labeled T cells (DC:T ratio of 1:3) for 5 days, and used FACE to detect the proliferation of T cells stimulated by the membrane-modified DCs.

[0204] (III) Experimental Results

[0205] We selected hCD80 / new GPI-anchored dendritic cells (DCs) packaged with lentivirus prepared using the aforementioned method, stimulated these membrane-modified DCs with OVA antigen, and then co-incubated them with T cells for 5 days. The results showed that after 5 days of co-incubation with T cells, the membrane-modified DCs achieved a T cell proliferation efficiency of 61.1% (…). Figure 9A The efficiency of unmodified control group DCs in promoting T cell proliferation was only 31%. Figure 9B ).

[0206] The above results demonstrate that DCs modified with co-stimulatory molecular membranes possess strong efficacy in stimulating T cell proliferation. This indicates that DCs modified with GPI membranes have the potential to enhance immune response efficacy and have application development value.

[0207] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0208] Appendix: Sequence Information (Uppercase letters represent amino acid sequences, lowercase letters represent nucleotide sequences)

[0209]

[0210]

[0211]

[0212]

Claims

1. An engineered glycosylated phosphatidylinositol (GPI) anchoring signal peptide, wherein, compared with the original natural GPI anchoring signal peptide, the N-terminal histidine of the engineered GPI anchoring signal peptide is replaced with an uncharged nonpolar amino acid with a C3-C6 straight-chain or branched alkyl side chain, and its C-terminus contains a flexible hydrophobic amino acid combination.

2. The engineered GPI-anchored signal peptide as described in claim 1, wherein, The uncharged nonpolar amino acid is independently selected from: valine (V), leucine (L), and isoleucine (I), for example, valine (V); and / or Wherein, the uncharged nonpolar amino acid replaces the first histidine, the second histidine, or both of the N-terminus of the original natural GPI-anchored signal peptide; and / or The flexible hydrophobic amino acid combination is composed of one or two amino acid residues selected from the group consisting of glycine (G) and serine (S); and / or The length of the flexible hydrophobic amino acid combination is 5-20 amino acid residues.

3. The engineered GPI-anchored signal peptide as described in claim 1, wherein, The flexible hydrophobic amino acid combination is an amino acid combination in which the ratio of G to S is 1 to 3:1, for example, it is selected from the following group: (GS) n (GGS) n (GGGS) n Where n is an integer from 3 to 10; and / or The original natural GPI-anchored signal peptide is derived from proteins or peptides selected from the group consisting of: CD73, CD55, ESAT-6, CD59, PLAP, CD56; and / or The original natural GPI-anchored 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 containing a sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:

16.

4. The engineered GPI-anchored signal peptide as described in claim 1, comprising the peptide segment represented by formula (I): STGS-Xa1-C-Xa2-GSFSLIFLSLWAVIFVLYQ-X b Equation (I) where Xa 1 and X a2 Each of these represents an uncharged nonpolar amino acid with a C3-C6 straight-chain or branched alkyl side chain, such as an amino acid residue selected from valine (V), leucine (L), and isoleucine (I). X b This represents a flexible, hydrophobic amino acid combination.

5. The engineered GPI-anchored signal peptide as described in claim 1, comprising a peptide segment with an amino acid sequence as shown in SEQ ID NO:3 or 22, or encoded by a nucleotide molecule comprising a sequence as shown in SEQ ID NO:4 or 23.

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

7. The protein molecule of claim 6, wherein, The functional peptides are selected from: secreted peptides, intracellular peptides, intracellular or extracellular segments of transmembrane peptides, membrane-bound peptides; and / or The functional peptide is selected from: antibodies or their antigen-binding fragments or their targeted binding peptides (e.g., peptides containing binding epitopes), ligands, receptors, cytokines (e.g., lymphokines, monokines, interleukins, interferons, colony-stimulating factors, tumor necrosis factor, transforming growth factor); and / or The connection is a direct connection between (a) and (b), a connection through a connector molecule, a coupling, or a fusion; For example: The antibody is selected from one or more of the following groups: anti-PD1 antibody, anti-PD-L1 antibody, anti-TIGIT antibody, anti-Artemin antibody, anti-CD19, CD44, CD47 or CD123 antibody, anti-STING antibody; and / or The cytokine is selected from: 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-1 4. IL-15; CD80, CD86, GCSF, MCSF, GMCSF, SCF, EPO; IFN-α, INN-β, IFN-γ; FNF-α, 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-α, VEGF; and / or The protein molecule also contains other active molecules, such as cytotoxins, linked to (a) or (b); and / or The disease is a mammalian disease, such as a disease of humans or non-human primates, livestock mammals, pets, or laboratory animals; and / or The protein molecule comprises amino acid molecules described in SEQ ID NO:6, 10, 13 and / or 19 linked to the amino acid molecule shown in SEQ ID NO:3 or 22.

8. A nucleic acid molecule encoding an engineered GPI-anchored signal peptide as described in any one of claims 1-5 or a protein molecule as described in any one of claims 6-7; For example, the nucleic acid molecule comprises nucleotide sequences 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 an engineered GPI-anchored signal peptide of any one of claims 1-5 or a protein molecule of any one of claims 6-7 anchored to its cell membrane; For example, the cells are selected from: antigen-presenting cells (e.g., dendritic cells), epithelial cells, nerve cells, erythrocytes, leukocytes, platelets, phagocytes (e.g., neutrophils, basophages, etc.). Eosinophils, etc.), B lymphocytes, effector B cells, memory B cells, T lymphocytes, memory T cells, effector T cells, cardiomyocytes, smooth muscle cells, skeletal muscle cells, osteoblasts, glial cells, hepatocytes, kidney cells, glandular cells, endocrine cells (such as thyroid cells, thymocytes, pancreatic B cells, and islet cells); for example, CHO cells, A549 cells, HeLa cells, Escherichia coli cells, yeast cells; and / or, The cells are normal cells (such as immune cells) or diseased cells (such as tumor cells); and / or, The cells are in vivo cells, in vivo cells, or adoptive cells; and / or The cells mentioned are production cells, therapeutic cells, immune cells, and detection cells.

10. The cell of claim 9, wherein the cell is a dendritic cell, and wherein: The dendritic cells are derived from bone marrow cells, umbilical cord blood cells, or peripheral blood mononuclear cells; and / or The dendritic cells are derived from mammals, such as humans, non-human primates, and mice; and / or The dendritic cells are mature dendritic cells sensitized with target antigens, such as tumor antigens or viral antigens that induce antiviral effects, which are also presented on their surface.

11. A product comprising one or more substances selected from the group consisting of: an engineered GPI-anchored signal peptide as claimed in any one of claims 1-5, a protein molecule as claimed in any one of claims 6-7, a nucleic acid molecule as claimed in claim 8, or a cell as claimed in claim 9 or 10; For example, the product is selected from: dendritic cell vaccines, adoptive cell therapy drugs, effector cell stimulants, or combinations thereof; For example, the product may contain dendritic cells as described in claim 13 or specific T cells induced by said dendritic cells.

12. The application of the engineered GPI-anchored signal peptide as described in any one of claims 1-5, the protein molecule as described in any one of claims 6-7, the nucleic acid molecule as described in claim 8, the cell as described in claim 9 or 10, or the product as described in claim 11, for the preparation of diagnostic, preventive, and / or therapeutic drugs for diseases; For example, the diseases are selected from: malignant tumors, benign tumors, endocrine diseases (such as nutritional and metabolic diseases, immune diseases (such as autoimmune diseases)), blood and hematopoietic organ diseases, mental illnesses, nervous system diseases, eye and appendage diseases, ear and mastoid diseases, circulatory system diseases, respiratory system diseases, digestive system diseases, genitourinary system diseases, skin and subcutaneous tissue diseases, musculoskeletal system and connective tissue diseases, injuries, poisoning; and / or The drugs are selected from: antibody drugs, exosome delivery drugs, and vaccines.

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