Cd164 fusion proteins and uses thereof

Abstract

The invention described herein is a CD 164 mucin domain fusion protein joined to a heterologous protein, e.g., a heterologous protein having a therapeutic function, and is beneficial for extending serum half-life. Methods of using the fusion proteins of the invention are also provided.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 202180009089.6, filed on November 8, 2021, entitled "CD164 fusion protein and its application". Background of the Invention

[0002] Cell surface mucins are large transmembrane glycoproteins involved in protecting the respiratory epithelium from pathogen infection and regulating cell signal transduction and transcription. Unlike large mucins, CD164 contains small mucin domains less than 60 amino acids in length. To date, the function of the mucin domain in CD164 remains unclear. Mice lacking the CD164 gene survive without a distinct phenotype. Human CD164 is an adhesive, salivary mucin-like membrane protein. It is a type I membrane protein, virtually ubiquitous in tissues, primarily located in intracellular endosomes and lysosomes (Ihrke 2000, Chan 2000). Functional analysis suggests that CD164 may play a helper role in the docking of CD34+ cells with bone marrow matrix tissue. CD164 contains two mucin domains (I and II), linked by a cysteine-rich non-mucin domain, followed by a transmembrane domain and an intracellular domain (Doyonnas 2000). CD164's mucin domain I consists of 37 amino acids and can theoretically be post-translational modified at 3 N-glycosylation sites and 9 O-glycosylation sites.

[0003] Several monoclonal antibodies targeting the anti-adhesion domain I can bind to glycosidic epitopes, preventing CD34+ cells from attaching to bone marrow stromal reticulum cells. Desialylation of CD164 reduces CD34+ cell attachment; therefore, terminal glycosylation such as sialylation may be important for maintaining the “stem cell-like” characteristics of CD34+ cells (Doyonnas 2000). Furthermore, CD164 may be involved in CXCR4 signaling-related migration responses (F or des 2007). In another assay system, transfection of 293T cells with the CD164 Fc fusion protein inhibited the formation of multinucleated myotubes in response to differentiation signals (Lee et al., 2001).

[0004] In all cases, CD164 function was sensitive to treatment with sialidase or O-glycopeptidase, indicating that glycosylation modification of CD164 is essential for its mediating intercellular signal transduction binding.

[0005] The human CD164 mucin domain II, composed of 56 amino acids, can theoretically undergo post-translational modifications at two N-glycosylation sites and 23 O-glycosylation sites. However, there is no evidence that the epitopes of CD164 mucin domain II are involved in functional binding. Instead, CD164 transcript analysis revealed that splicing variants lacking exon 4 or exon 5 removed most of the CD164 mucin domain II without affecting CD164 function (Chan 2001). Therefore, the CD164 mucin domain II forms a brush-like stem supporting a crown-like structure composed of CD164 mucin domain I and cysteine-rich regions, participating in intercellular interactions.

[0006] The cysteine-rich region between domains I and II of the CD164 mucin contains 52 amino acids, of which 8 cysteine ​​residues can form disulfide bridges. This domain also contains 4 putative N-linked glycosylation sites, as well as potentially additional O-glycosylation sites.

[0007] In summary, if CD164 is fully glycosylated, glycosylation can contribute 70% of its molecular weight. Glycosylated CD164 of 90 kDa can be observed in human bone marrow cells, CD34+ purified umbilical cord blood cells, or cultured bone marrow stromal reticulum cells, confirming significant glycosylation modification in the 174-amino acid residue polypeptide of mature human CD164 (Doyonnas 2000). Invention Overview

[0008] One aspect of the present invention is to provide a fusion protein comprising: (1) a polypeptide having an amino acid sequence that is at least 90% identical to sequence SEQ ID NO:1 or 2, and (2) a heteropeptide.

[0009] In some embodiments, the polypeptide consists of an amino acid sequence numbered SEQ ID NO:1 or 2.

[0010] In some embodiments, (1) is the C-terminus of (2). In this case, (1) is SEQ ID NO:2

[0011] In some embodiments, (1) is the -N end of (2). In this case, (1) is SEQ ID NO:1

[0012] In some embodiments, the fusion protein further includes (3), a second polypeptide containing an amino acid sequence that is at least 90% identical to sequence SEQ ID NO:1 or 2, wherein (1) and (3) each contain a different sequence from SEQ ID NO:1 or 2.

[0013] In some embodiments, the SEQ ID NO:1 peptide is fused to the N-terminus of a heterologous peptide, and the SEQ ID NO:2 peptide is fused to the C-terminus of a heterologous peptide.

[0014] In some embodiments, the heteropeptide is a therapeutic peptide.

[0015] In some embodiments, the serum or circulating half-life of the therapeutic peptide (human or mouse) is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% shorter than that of the fusion protein.

[0016] In some embodiments, the heterologous peptides include fibroblast growth factor 21 (FGF21), follicle-stimulating hormone (FSH), myeloid-derived growth factor (MYDGF), fibroblast growth factor-binding protein 3 (FGFBP3), natriuretic peptide B, incretin peptide, glucagon-like peptide-1 (GLP-1), gonadotropin-releasing hormone, secretin, leucine, emfovitin, glucagon, divalirudin, serum hormones, adrenocorticotropic hormone tetrapeptide, insulin-like growth factor (IGF), parathyroid hormone, or amylin.

[0017] Fusion proteins include O and / or N chain glycosylation.

[0018] In some embodiments, the fusion protein includes sialylation.

[0019] In some embodiments, the fusion protein also includes a linker peptide between (1) and (2).

[0020] In some embodiments, (1) in the fusion protein is the C-terminus of (2), (1) may be SEQ ID NO:2, and the heteropeptide is MYDGF or a functional fragment thereof.

[0021] In some embodiments, the amino acid sequence of the fusion protein is at least 90% identical to any one of the amino acid sequences of SEO ID NOs:3-8.

[0022] In some embodiments, the amino acid sequence of the fusion protein is SEQ ID NO:3.

[0023] Another aspect of the present invention provides a polynucleotide encoding the fusion protein of the present invention.

[0024] In some embodiments, the polynucleotide is expressed in the target host cell after codon optimization.

[0025] In some embodiments, the target host cell is a human cell, a rodent cell (such as a mouse cell), or a non-human mammal cell.

[0026] Another aspect of the present invention provides a vector comprising the polynucleotides of the present invention.

[0027] In some embodiments, the vector is an expression vector.

[0028] In some embodiments, the vector is a plasmid.

[0029] Another aspect of the present invention provides a host cell comprising the fusion protein of the present invention, the polynucleotide of the present invention, or the vector of the present invention.

[0030] In some embodiments, the host cell is a tissue culture cell.

[0031] In some embodiments, the host cell is a CHO K-1 cell (ATCC#CCL61) or a CHO DG44 cell or a CHODXB-11 cell, a Namalwa cell (e.g., ATCC#CRL-1432), a HeLa cell (ATCC#CCL-2), a HEK293 cell (ATCC#CCL-1573), a WI-38 cell (ATCC#CCL-75), a MRC-5 cell (ATCC#CCL-171), a HepG2 cell (ATCC#HB-8065), a 3T3 cell (ATCC#CCL-92), an L-929 cell (ATCC#CCL-1), a Myeloma (e.g., NS / O) cell, a BHK-21 cell (ATCC#CCL-10), a COS-7 cell (ATCC#CCL-1651), or a Vero cell (ATCC#CCL-81) or a derivative thereof.

[0032] In some embodiments, the host cell is a CHO K-1 cell (ATCC#CCL61) or a derivative thereof, or a HEK293 cell (ATCC#CCL-1573) or a derivative thereof.

[0033] Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the fusion protein of the present invention, the polynucleotide of the present invention or the carrier of the present invention, and pharmaceutically acceptable additives or excipients.

[0034] In some embodiments, the pharmaceutical composition is formulated for intravenous injection.

[0035] Another aspect of the present invention provides a method for prolonging the serum / circulating half-life of a protein, comprising fusing the protein into a polypeptide having at least 90% homology to the amino acid sequence of SEQ ID NO:1 or 2.

[0036] In some embodiments, the polypeptide consists of the amino acid sequence of SEQ ID NO:1 or 2.

[0037] In some embodiments, the protein is fused to the N-terminus of SEQ ID NO:2.

[0038] In some embodiments, the protein is fused to the polypeptide via a linker peptide.

[0039] In another aspect, the present invention provides a method for treating a disease, disorder, or unhealthy state in a subject in need, the method comprising administering to the subject an effective dose of the fusion protein of the present invention, the polynucleotide of the present invention, or the carrier of the present invention, wherein the disease, disorder, or unhealthy state can be treated by the heterologous polypeptide.

[0040] In some embodiments, the disease, disorder, or unhealthy state is selected from a disease group that includes tissue damage, cardiovascular disease, inflammatory disease or disorder, and kidney disease.

[0041] In some embodiments, tissue damage is an acute injury, such as myocardial infarction or stroke.

[0042] In some embodiments, the tissue damage is a chronic injury, such as kidney damage caused by diabetes.

[0043] In some embodiments, cardiovascular diseases are selected from the groups of myocardial infarction, arteriosclerosis, hypertension, angina pectoris, hyperlipidemia, and heart failure.

[0044] In some embodiments, the inflammatory disease or disorder is selected from type 1 diabetes, type 2 diabetes, pancreatitis, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH).

[0045] In some embodiments, the disease or disorder is a kidney disease.

[0046] In some embodiments, the subjects are humans. Brief description of the attached figures

[0047] Figure 1 Western blot analysis of the glycosylated form of the CD164 mucin domain fusion protein. MYDGF-164 and FGF21-164 are expressed in 293F cells. Cell culture supernatant was collected for SDS-PAGE and Western blot analysis. “R” indicates DTT-reduced sample.

[0048] Figure 2This is a Western blot analysis of the native glycosylated protein fused to the CD164 mucin domain. CD164-FSHa or FSA, and FSHb-164 or FSHb), along with their respective heterodimeric subunits, were stably transfected into CHO cells. Non-reduced (NR) and reduced (R) samples were detected by SDS-PAGE and Western blot. The same analysis was performed on FGFBP3-164 expressed in 293F cells.

[0049] Figure 3A and 3B The image shows the Coomassie brilliant blue staining analysis of the purified MYDGF-164 fusion protein after SDS-PAGE. Figure 3A ) or RP-HPLC analysis ( Figure 3B ).

[0050] Figure 4 The MYDGF-164 fusion protein expressed in 293 cells was shown to be in acidic form. The purified MYDGF-164 fusion protein (sample A001) was analyzed by isoelectric focusing (IEF) and according to pI standards in adjacent lanes. The major isoelectric point of the MYDGF-164 fusion protein was below 5.1.

[0051] Figure 5 Monosaccharide composition analysis showed that the GlcNAc:GalNAc:Gal:Man:Fuc ratio in the MYDGF-164 fusion protein was 7:5:11:2:1. The MYDGF-164 fusion protein contains a large amount of galactose, indicating the presence of both O-glycans and N-glycans.

[0052] Figure 6 For β-elimination analysis, the content of O-glycans in the MYDGF-164 fusion protein was much higher than that of N-glycans.

[0053] Figure 7 These are the characteristic peaks of the N-chain glycan derivative of the MYDGF-164 fusion protein after PNGase treatment and 2-AB labeling. Elution times for different peaks are expressed in minutes. The different glycan chains corresponding to the peaks are represented by pictographic symbols for the sugars.

[0054] Figure 8 This study analyzed the content and types of sialic acid in the MYDGF-164 fusion protein using UPLC. The MYDGF-164 fusion protein contains 6.6% Neu5Ac.

[0055] Figure 9 This is a MALDI-TOF mass spectrum of the MYDGF-164 fusion protein. M+: single proton peak. M2+: double proton peak.

[0056] Figure 10A and 10B For the purified MYDGF-164 fusion protein ( Figure 10A ) and standard protein ( Figure 10B SEC-HPLC analysis of the standard proteins (thyroglobulin: 670,000; γ-globulin: 150,000; ovalbumin: 45,000; myoglobin: 17,000; angiotensin: 1,000) was performed. The molecular weights (MWs) of the standard proteins (thyroglobulin: 670,000; γ-globulin: 150,000; ovalbumin: 45,000; myoglobin: 17,000; angiotensin: 1,000) were correlated with elution time, and a nonlinear regression model was used for fitting (R² = 0.99898). The hydrodynamic radius of the MYDGF-164 fusion protein was 98.744 kDa.

[0057] Figure 11 Pharmacokinetic (PK) analysis of purified MYDGF-164 fusion protein administered intravenously to C57BL / 6 mice. Serum concentration of MYDGF-164 fusion protein was analyzed by LC-MS / MS.

[0058] Figure 12 The results showed that co-incubation of MYDGF-164 fusion protein with 5% fetal bovine serum (FBS) promoted HUVEC cell proliferation. The addition of MYDGF-164 to HUVEC cells in 5% FBS resulted in a dose-dependent effect on HUVEC cell proliferation. One-way ANOVA was used: ***P<0.001, **P<0.01, *P<0.05.

[0059] Figure 13 The left image shows that the MYDGF-164 fusion protein promotes cell cycle activity of HUVECs in 1% fetal bovine serum. The proportion of cells at different cell cycles is shown in the right image. One-way ANOVA was used: ***P<0.001, **P<0.01, *P<0.05.

[0060] Figure 14 For scratch repair analysis, it was demonstrated that MYDGF-164 enhances cell migration. Mechanical scratching of monolayer HUVEC cells resulted in scratch repair via cell migration. Scratch repair of monolayer endothelial cells was tested in the presence of different concentrations of MYDGF-164 or 100 ng / mL VEGFA. Images of monolayer HUVEC cells were acquired at 0, 12, and 24 hours. Migration rate was analyzed using Image-Pro-Plus software. Migration rate was defined as area of ​​change / wound area. One-way ANOVA was used: ***P<0.001, **P<0.01, *P<0.05.

[0061] Figure 15This indicates that after 4 hours of incubation with MYDGF-164 or 100 ng / mL VEGFA, HUVEC cells exhibited enhanced tubular formation in a low-growth-factor-rich matrix gel. Endothelial cell casts were defined as circular structures surrounded by endothelial cells, with the number of closed casts representing the total number of endothelial tubules. One-way ANOVA was used: ***P<0.001, **<0.01, *P<0.05.

[0062] Figure 16 MYDGF-164 reduced hydrogen peroxide-induced apoptosis in HUVEC cells. HUVEC cells were pre-incubated with 1 μg / mL MYDGF-164 for 24 hours, followed by treatment with 400 μmol / L hydrogen peroxide. Annexin V-FITC and propidium iodide staining, and flow cytometry were used to detect apoptosis or cell death. One-way ANOVA was used: ***P<0.001, **<0.01, *P<0.05.

[0063] Figure 17A and Figure 17B A schematic diagram of the experimental plan for testing the protective function of MYDGF-164 in a rat model of myocardial ischemia is depicted. The first ischemia experimental plan ( Figure 17A In this study, MYDGF-164 was administered intravenously 5 minutes before and 4 hours after reperfusion, followed by twice-daily intravenous injections for 7 consecutive days. Rats were then sacrificed, and the infarct area was assessed. Tirofiban, a non-peptide antagonist of the GP IIb / IIIa receptor that inhibits platelet aggregation, was used as a positive control. In the second ischemia experimental program ( Figure 17B During reperfusion, MYDGF-164 was administered intravenously, followed by two intravenous injections at 6 and 12 hours after reperfusion. Infarct area was analyzed 24 hours later. In both experimental plans, cardiac troponin I (cTnI), a biomarker of myocardial injury, was analyzed.

[0064] Figure 18A and Figure 18B Reproducible studies showed that MYDGF-164 reduced cardiac troponin I levels, a marker of myocardial infarction, in two independent experiments conducted by two different research organizations. A positive control was the antiplatelet drug tirofiban. In MYDGF-164-treated rats, cTnI release was reduced to levels seen in sham-operated rats. Two-way ANOVA was used: ***p<0.001 (model group vs. sham-operated group), ###p<0.001 (treatment vs. model group).

[0065] Figure 19A -F indicates the therapeutic effect of MYDGF-164 in a rat ischemia model. Figure 19A and 19BThe study showed a reduction in infarct area after MYDGF-164 treatment based on triphenyltetrazolium chloride (TTC) staining, and the infarct area of ​​rat hearts after 7 days was determined by excluding darker staining of heart sections. Figure 19A In an independent rat ischemia model, the danger zone and infarct area of ​​the rat heart 24 hours later were determined by excluding darker staining with triphenyltetrazol chloride (TTC) and Evans blue (Evans blue). Figure 19D -E). Percentage of the dangerous area in the left ventricle (LV). Percentage of the infarcted area in the dangerous area. One-way ANOVA, Tukey multiple comparison test. ****P<0.0001 vs. sham surgery group, ***P<0.001 vs. sham surgery group, ###P<0.001 vs. MI / R model group, ##P<0.01 vs. MI / R model group. Figure 19F Capillaries are shown in the infarct area and at the edge of the infarct area. Figure 19G -H indicates that rats treated with MYDGF-164 have higher capillary density at the periphery of the myocardial infarction area.

[0066] Figure 20 The study showed that MYDGF-164 treatment increased the survival advantage after myocardial infarction in rats with myocardial infarction.

[0067] Figure 21 A schematic diagram of the experimental plan for testing MYDGF-164's protection of subjects from kidney failure is depicted.

[0068] Figure 22 The results showed that MYDGF-164 reduced serum urea nitrogen and creatinine levels in a rat model of adenine-induced renal failure. One-way ANOVA was used: ***P<0.001, **<0.01, *P<0.05.

[0069] Figure 23 A-23G shows that the MYDGF-164 protein resists adenine-induced kidney structural damage. Figure 23 Image A is a histological image of kidney tissue stained with H&E, showing intraluminal crystal deposition (arrow 1), glomerular atrophy (arrow 2), necrotic tubules (arrow 3), casts (arrow 4), and inflammation (arrow 5). Scale bar is 250 μm. Figure 23 In section B, the ratio of kidney weight to body weight, i.e., relative kidney weight, was measured after treatment. N=5. The 5-point lesion score for each kidney tissue injury was displayed in graphical form: renal tubular necrosis ( Figure 23 C) Glomerular atrophy ( Figure 23 D) Inflammation Figure 23 E) Renal tubular dilation ( Figure 23 F), pigmentation ( Figure 23G). 5-point method: 1) Mild, affecting <10%; 2) Mild to moderate, affecting 11-25%; 3) Moderate, affecting 26-50%; 4) Severe, affecting 51-75%; 5) Severe, affecting 76-100%. One-way ANOVA was used: ***P<0.001, **<0.01, *P<0.05.

[0070] Figure 24 A-24D shows the immunohistochemical analysis of kidney tissue. The left side is a representative image, and the proportion of characteristic cells was calculated using Image-pro Plus 6.0. The right side is a bar chart. Figure 24 A shows a paraffin section of kidney tissue stained with KIM-1, the molecule responsible for renal tubular injury. Scale bar: 100 μm. Figure 24 B shows the RECA-1 immunohistochemical (IHC) analysis of paraffin sections of kidney tissue. Black arrows indicate that the luminal structure of perivascular capillaries (PTCs) remains unchanged, while red arrows indicate that the luminal structure of PTCs has collapsed. Scale bar is 100 μm. Figure 24 C shows Ki-67 immunohistochemical staining of paraffin-embedded kidney tissue sections. Scale bar is 250 μm. Figure 24 D shows the TUNEL assay of apoptotic cells in a paraffin section of kidney tissue. Scale bar is 50 μm.

[0071] Figures 25A-25C The addition of MYDGF-164 to HUVEC cells showed a dose-dependent stimulation of cell proliferation. Figure 25A ) and activate MAPK1 / 3 phosphorylation ( Figure 25B Furthermore, MYDGF-164 fusion enhanced the expression of cyclin D1. Figure 25C )

[0072] Figures 26A-26D MYDGF-164 showed superior efficacy compared to nicorandil in promoting HUVEC cell migration in the scratch assay. In this assay, HUVEC cell monolayers were scraped 24 hours before treatment with either MYDGF-164 or nicorandil. Representative photomicrographs taken at 100x magnification (scratch repair distance indicated by black double arrows) Figure 26A and Figure 26C The migration rate of HUVECs was determined after treatment with MYDGF-164 or nicorandil. Figure 26B and Figure 26D Two-way ANOVA and Tukey's multiple comparison test were used. N=3, ***P<0.001, *P<0.05.

[0073] Figures 27A-27BThe results show enhanced tube formation in HUVECs after 4 hours of stimulation with MYDGF-164 compared to treatment with nicorandil. VEGFA was used as a positive control. Representative photomicrographs were taken at 40x magnification. Figure 27A Number of closed tubes per field of view for HUVEC after treatment with MYDGF-164, nicorandil, or VEGFA. Figure 27B One-way ANOVA and Tukey's multiple comparison test were used. N=3, ***P<0.001, *P<0.05.

[0074] Figure 28 The purified FGF21-164 fusion protein is shown as being stably expressed in 293F cells. Left: SDS-PAGE of the purified FGF21-164 fusion protein followed by Coomassie Brilliant Blue staining. Right: Western blot analysis.

[0075] Method: Using SDS-PAGE was performed using a Tetra System (Bio-Rad), followed by Coomassie Brilliant Blue (CBB) staining and densitometric analysis. Molecular weight standards (11-180 kDa, Tanon) were used for analysis. For purity analysis, approximately 10 μg of the fusion protein was dissolved in sample buffer and separated on a 12% SDS-PAGE gel, with densitometric analysis performed using a Tanon 4600SF gel imaging system.

[0076] Figure 29 The data for MALDI-TOF analysis of the purified FGF21-164 fusion protein are shown. In the analysis, the MALDI-TOF mass peak at 42751.5 Da represents the unprotonated FGF21-164 protein, and the peak at 21601.173 Da represents the diprotonated protein.

[0077] Methods: Mass spectra were acquired using a Bruker Autoflex velocity instrument (equipped with a 1000 Hz Smartbeam-II laser) with 2,5-dihydroxybenzoic acid as the matrix. Mass spectra of FGF21-164 were analyzed using Bruker Flexanalysis software version 3.3.80.

[0078] Figure 30A-C shows analytical SEC data for the purified FGF21-164 protein. A. FGF21-164 migrates as a single symmetrical peak in the SEC analysis. B. Retention times of various standard proteins during the SEC analysis. The molecular weight (MWs) of the standard proteins (thyroglobulin: 67,000; γ-globulin: 150,000; ovalbumin: 45,000; myoglobin: 17,000; angiotensin: 1,000) is plotted against their elution times. C. Regression analysis and fitting of retention time against the molecular weight of standard proteins were performed using a nonlinear regression model. The hydrodynamic radius of the FGF-164 fusion protein was calculated to be 87.679 kDa.

[0079] Figure 31A UPLC characterization of N-linked glycan forms from FGF21-164 after PNGase treatment and 2-AB labeling. Elution times for various peaks are expressed in minutes. The glycan forms corresponding to the peaks are represented by pictographs of the glycans. Figure 31B This is a β-elimination analysis showing the O-glycan content in the FGF21-164 fusion protein.

[0080] Methods: O-glycans and N-glycans were released from the sample using modified β-elimination. A water-equilibrated graphitized carbon column (Supelclean) was used. TM ENVI TM Proteins and salts were removed using 2-Carb SPE, and the glycans were eluted with 20% and 40% acetonitrile. The eluent was evaporated under vacuum. The glycans were fluorescently labeled with 2-aminobenzamide (2-AB) and analyzed in a HILIC UPLC (ACQUITYUPLC Glycan BEH Amide Column). Separation of labeled polysaccharides on a plate (1.7 μm, 2.1 mm × 150 mm).

[0081] According to the manufacturer's specifications (Sigma), N-linked glycans are labeled with 2-aminobenzamide after PNGase F treatment. In HILIC UPLC (ACQUITY UPLC Glycan BEH Amide Column), Labeled glycans were separated on a 1.7 μm, 2.1 mm x 150 mm substrate, and the form of N-linked glycans was determined based on comparison with 2-AB labeled glycan standards.

[0082] Figure 32 Pharmacokinetic analysis of the FGF21-164 fusion protein after intravenous injection into C57 / BL6 mice. Serum concentrations of FGF21-164 protein over time were determined by LC-MS.

[0083] Methods: The in vivo half-life of the FGF21-164 fusion protein, YLYTDDAQQTE AHLEI (YLY peptide), a characteristic peptide of FGF21-164, was determined using LC-MS. FGF21-164 protein (22.7 mg / kg) was administered intravenously to C57 / BL6 mice. Serum samples were prepared at different time points after injection and treated with trypsin to release YLY peptide. The concentration of YLY peptide was quantitatively analyzed by LC-MS to track FGF21-164 protein levels.

[0084] Figure 33 FGF21-164 fusion protein stimulates glucose uptake by adipocytes. After incubating differentiated mature 3T3-L1 adipocytes with various concentrations of FGF21-164, the glucose concentration in the conditioned medium was measured.

[0085] Methods: Cultured, mature 3T3-L1 adipocytes were pretreated with high-glucose DMEM containing 0.1% FBS for 24 hours. The medium was then replaced with different concentrations of FGF21-164. The glucose concentration in the medium was measured after 24 hours of culture. FGF21-164 significantly increased glucose uptake by adipocytes (one-way ANOVA and Dunnett's multiple comparison test were used; *p<0.05, ***p<0.001, ****p<0.0001).

[0086] Figure 34. Decreased serum glucose in ob / ob mice

[0087] Figure 34A Even after overnight starvation, ob / ob mice exhibited high baseline glucose levels. A single administration of the FGF21-164 fusion protein to mice reduced serum glucose levels. Figure 34B The high-dose treatment group showed a greater trend of decreasing glucose levels.

[0088] Methods: Male ob / ob mice of the B6 / JGpt-Lepem1Cd25 / Gpt strain were purchased and processed according to the IACUC guidelines of China Pharmaceutical University. Mice were provided free of charge unless testing was required. Mice were randomly assigned to a control group (n=4), a treatment group receiving 6 mg / kg FGF21-164 (low dose, n=5), and a treatment group receiving 12 mg / kg FGF21-164 (high dose, n=5). After overnight fasting, mice were subcutaneously injected with either PBS or the FGF21-164 fusion protein. Blood glucose levels were determined at different time points using a tail vein blood sampler and quantified using a glucometer. One-way ANOVA and Tukey's multiple comparison test were used to assess the difference between the control and FGF21-164.

[0089] Figure 35AThe purified FSH164 fusion protein was analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining. Figure 35B Western blot analysis was performed on the purified FSH164 fusion protein using an antibody against the α subunit of glycoprotein hormone.

[0090] Figure 36 FSH164 incubation stimulated progesterone synthesis in KGN cells in vitro.

[0091] Methods: KGN cells were grown in DMEM / F12 medium containing 10% FBS and 1% P / S, and seeded at 2 × 10⁴ cells per well in 96-well plates. Cells were grown for 24 hours, then switched to low-serum (1% FBS) medium for another 24 hours. Different concentrations of recombinant human FSH or FSH164 were added to the medium, bringing the final volume to 150 μL. After 72 hours, the culture supernatant was collected, and progesterone biosynthesis was measured by ELISA (DRG).

[0092] Figure 37 Compared to FSH, the FSH164 fusion compound has a prolonged half-life in vivo.

[0093] Methods: Female, 6-week-old immature SD rats (180-190 g) were used for pharmacokinetic analysis. rh-FSH (10 μg / kg) or FSH164 (18.5 μg / kg) was administered subcutaneously into the rat peritoneum. Blood was collected via the orbital sinus to prepare serum samples, which were analyzed using a DRG FSH ELISA. Pharmacokinetic parameters were calculated using PK Solver 2.0. A non-compartmental model and linear trapezoidal fitting were applied to analyze the data. Invention Details

[0094] 1. Overview

[0095] This invention extends known half-life extension techniques, such as Fc fusion, albumin fusion, or polyethylene glycol fusion. The methods and compositions described herein are unique, in part because they alter the pKa of the molecule, converting proteins with a basic charge into acidic molecules, thereby enhancing solubility, tissue distribution, and absorption, and improving the bioavailability of the fusion protein. Furthermore, the immunogenicity of the fusion protein is reduced through glycosylation coating, which is advantageous for biotherapeutic agents requiring frequent and / or long-term administration.

[0096] As expected, fusing proteins or peptides into small mucin domains with high glycosylation and sialylation can also enhance the pharmacokinetic properties and bioactivity of biotherapeutic agents. Therefore, this invention provides a novel technological platform for delivering biotherapeutic agents with the desired characteristics for clinical use.

[0097] More specifically, the fusion protein provided by this invention comprises (human) CD164 mucin domains I and / or II and the allogeneic protein of interest. Results show that when the fusion protein is recombined, the glycosylation and sialylation of the mucin domains are preserved, and surprisingly, the half-life of the fusion protein is significantly prolonged compared to those reported in the literature. Therefore, the host fusion protein provided by this invention, as part of a protein engineering platform, helps to optimize the pharmacokinetic (PK) properties of protein therapies.

[0098] This invention is partly based on the understanding that the brush-like structure of the mucin domain (due to O-linking and N-linking glycosylation) can significantly alter the hydrodynamic behavior of fusion proteins with such glycosylated domains, and the observation that novel fusion proteins with highly glycosylated CD164 mucin domains improve pharmacokinetic properties, such as significantly prolonging serum half-life and improving tissue distribution.

[0099] While not wishing to be bound by any particular theory, there are several potential advantages to using the mucin-like domain fusion proteins of this invention. First, the fusion proteins are highly glycosylated and sialylated, thereby reducing their immunogenicity. Second, the sialylation of the fusion proteins may contribute to their favorable pharmacokinetic properties, including distribution and absorption. Isoelectric focusing (IEF) results show that fusion proteins containing mucin-like domains are strongly acidic, a characteristic that can improve their solubility. For example, FGF21 is known to be unstable, forming aggregates at high concentrations (Hecht 2012), which may lead to unintended immune responses. Furthermore, FGF21 exhibits poor tissue distribution, which is improved by fusion with the mucin domain of CD164.

[0100] Compared to Fc fusion strategies that extend half-life, the CD164 mucin domain fusion protein of this invention does not possess the additional effector immune functions of the Fc region (such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)). Therefore, this may be a safer option for fusion proteins that bind to cell surface receptors. Furthermore, while Fc fusion is an effective method for extending half-life to several days, the extended half-life of CD164 mucin domain fusion is better suited for shorter durations. This is particularly useful for treatments where prolonged overstimulation may be harmful.

[0101] Polyglycoling strategies have previously been used to extend the half-life of binding proteins. According to the present invention, the cumbersome polyethylene glycolation chemical process is replaced by a natural amino acid polymer fused to the CD164 mucin-like domain. Therefore, the manufacturing process is simpler, and the fusion of the CD164 mucin domain may be more cost-effective. Furthermore, the mucin domain is more natural than the non-natural polyethylene domain, which may lead to toxicity with prolonged use.

[0102] Therefore, in one aspect, the present invention provides a fusion protein comprising: (1) a polypeptide containing an amino acid sequence having at least 90% homology with SEQ ID NO:1 or 2, and (2) a heteropeptide.

[0103] In this invention, "heterologous polypeptide" refers to a polypeptide or protein that does not contain a polypeptide or protein derived from SEQ ID NO: 1 and / or 2. It can be a protein or polypeptide from the same species (other human proteins / polypeptides) or from a different species.

[0104] The fusion protein contains the heterologous polypeptide comprising an amino acid sequence that is at least 90% homologous to SEQ ID NO:1 or 2, and optional additional sequences, such as linking peptides, which link the heterologous polypeptide and the polypeptide comprising an amino acid sequence that is at least 90% homologous to SEQ ID NO:1 or 2.

[0105] In some embodiments, there is no linking peptide between the heterologous polypeptide and the polypeptide having at least 90% homology to the amino acid sequence of SEQ ID NO:1 or 2.

[0106] In some embodiments, the polypeptide comprising an amino acid sequence having at least 90% homology with SEQ ID NO:1 or 2 is composed of the amino acid sequence of SEQ ID NO:1 or 2. That is, in this embodiment, the fusion protein is composed of a heterologous polypeptide, a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 or 2, and optional sequences (such as linker peptides that may or may not be present).

[0107] The sequence of the heteropeptide (2) and the polypeptide containing an amino acid sequence having at least 90% homology with SEQ ID NO:1 or 2 (1) can be either (1) being the N-terminus of (2) or (1) being the C-terminus of (2).

[0108] In some embodiments, (1) is the N-terminus of (2). In this embodiment, (1) may be SEQ ID NO:2.

[0109] In some embodiments, (1) is the C-terminus of (2). In this embodiment, (1) may be SEQ ID NO:1.

[0110] In some embodiments, the fusion protein may include two or more polypeptides, each polypeptide containing an amino acid sequence that has at least 90% homology with SEQ ID NO:1 or 2.

[0111] For example, in some embodiments, the fusion protein may include SEQ ID NOs:1 and 2. In this embodiment, the heteropeptide may have two peptides flanking it, the N-terminal peptide comprising an amino acid sequence having at least 90% homology with the amino acid sequence of SEQ ID NO:1, and the C-terminal peptide comprising an amino acid sequence having at least 90% homology with the amino acid sequence of SEQ ID NO:2. In another embodiment, the heteropeptide may have three or more peptides flanking it, each peptide comprising an amino acid sequence having at least 90% homology with SEQ ID NO:1 or 2, wherein the N-terminus of the heteropeptide comprises any peptide having at least 90% homology with the amino acid sequence of SEQ ID NO:1, and / or the C-terminus of the heteropeptide comprises any peptide having at least 90% homology with the amino acid sequence of SEQ ID NO:2. For example, there may be two (identical or different) peptides that share at least 90% homology with the amino acid sequence of SEQ ID NO:1, both of which are the N-terminus of a heteropeptide, and a multipeptide that shares at least 90% homology with the amino acid sequence of SEQ ID NO:2, which is the C-terminus of a heteropeptide, and so on.

[0112] Therefore, in some embodiments, the fusion protein further includes (3) a second type polypeptide that has at least 90% homology with the amino acid sequence of SEQ ID NO:1 or 2, wherein (1) and (3) contain different amino acid sequences in SEQ ID NO:1 and 2, respectively.

[0113] In some embodiments, the SEQ ID NO:1 peptide is fused to the N-terminus of the heteropeptide, and the SEQ ID NO:2 peptide is fused to the C-terminus of the heteropeptide.

[0114] In some embodiments, the fusion protein may contain only SEQ ID NOs:1 or 2, not both. For example, the fusion protein may contain one or more (identical or different) polypeptides, each containing an amino acid sequence that has at least 90% homology with SEQ ID NO:1, and all of these polypeptides are N-terminal to a heterologous polypeptide. In some embodiments, the fusion protein may include one or more (identical or different) polypeptides, each containing an amino acid sequence that has at least 90% homology with SEQ ID NO:2, and all of these polypeptides are C-terminal to a heterologous polypeptide.

[0115] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1. These sequence homology percentages may be based on a query sequence (e.g., the polypeptide is different from SEQ ID NO:1), SEQ ID NO:1, or a query sequence that is adjacent to SEQ ID NO:1.

[0116] In some embodiments, the polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO:2. These sequence homology percentages may be based on a query sequence (e.g., the polypeptide is different from SEQ ID NO:2), SEQ ID NO:2, or a query sequence adjacent to SEQ ID NO:2.

[0117] In some embodiments, the heterologous peptide is a peptide for which it is necessary to prolong the serum half-life of an animal (such as a human or non-human mammal). In some embodiments, the heterologous peptide is a peptide with a relatively short serum half-life (e.g., about 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, or 2 hours).

[0118] In some embodiments, the heterologous peptide is a therapeutic peptide.

[0119] The term "therapeutic peptide" as used herein includes the subject of research and development (R&D) of peptides as pharmaceuticals for the treatment of diseases or unhealthy conditions in humans and / or animals. In some embodiments, a therapeutic peptide refers to a peptide or protein therapy that is being or has been evaluated in clinical trials.

[0120] Catalogs of therapeutic peptides in completed or ongoing drug development and / or clinical trials are available from public or proprietary sources. For example, the Peptide Therapy Foundation (PTF) maintains and publishes a dataset of commercially sponsored protein therapies that have entered clinical trials. Other data can be collected from public sources such as clinicaltrials.gov, PubMed, company and regulatory agency websites, and proprietary or commercial databases (such as Thomson Reuters Partnering, Thomson Reuters Integrity, Sagient Research Systems BioMedTracker, etc.).

[0121] In some embodiments, the therapeutic peptide comprises a single peptide chain, such as those with a length not exceeding 500, 450, 400, 350, 300, 250, 200, 150, 100, 80, 50, 40, 30, or 20 amino acids. In some embodiments, the therapeutic peptide comprises two or more peptides linked together by one or more disulfide bonds, such as those with a total length not exceeding 1500, 1000, 800, 700, 600, 500, 450, 400, 350, 300, 250, 200, 150, 100, 80, or 50 amino acids.

[0122] In some embodiments, the therapeutic peptide includes peptides with a single polypeptide chain, such as those with a length not exceeding 500 amino acids, 450 amino acids, 400 amino acids, 350 amino acids, 300 amino acids, 250 amino acids, 200 amino acids, 150 amino acids, 100 amino acids, 80 amino acids, 50 amino acids, 40 amino acids, 30 or 20 amino acids.

[0123] In some embodiments, typical but non-limiting heterologous peptides include fibroblast growth factor 21 (FGF21), follicle-stimulating hormone (FSH), myeloid-derived growth factor (MYDGF), fibroblast growth factor binding protein 3 (FGFBP3), natriuretic peptide B, cholecystokinin, glucagon-like peptide-1 (GLP-1), gonadotropin-releasing hormone, secretagogue, levolactamase, enfovir dipivoxil, glucagon, divaline, sermotide, adrenocorticotropic hormone tetrapeptide, insulin-like growth factor (IGF), parathyroid hormone, or amylin.

[0124] In some embodiments, the serum half-life of the therapeutic peptide (in humans or mice) is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% lower than that of the fusion protein.

[0125] In some embodiments, the fusion protein includes O and / or N chain glycosylation.

[0126] In some embodiments, the fusion protein includes sialylation.

[0127] In some embodiments, the fusion protein further includes a linker peptide located between (1) (a polypeptide containing at least 90% homology to the amino acid sequence of SEQ ID NO:1 or 2) and (2) (a heteropeptide).

[0128] In some embodiments, (1) in the fusion protein is the C-terminus of (2), (1) may be SEQ ID NO:2, and the heteropeptide is MYDGF or a functional fragment thereof.

[0129] In some embodiments, the amino acid sequence of the fusion protein is at least 90% homologous to SEQ ID NO:3, such as SEQ ID NO:3.

[0130] Another aspect of the invention also provides a polynucleotide encoding any one of the fusion proteins described herein.

[0131] In some embodiments, the polynucleotide is expressed in the target host cell after codon optimization.

[0132] In some embodiments, the target host cell is a human cell, a rodent cell (e.g., a mouse cell), or a non-human mammalian cell.

[0133] Another aspect of the present invention provides a vector comprising the polynucleotides of the present invention.

[0134] In some embodiments, the vector is an expression vector, such as a plasmid.

[0135] Another aspect of the present invention provides a host cell comprising the fusion protein of the present invention, the polynucleotide of the present invention, or the vector of the present invention.

[0136] In some embodiments, the host cell is a tissue culture cell.

[0137] In some embodiments, the host cell is a CHO cell or a HEK293 cell or a derivative thereof.

[0138] Another aspect of the present invention provides a method for extending the serum half-life of a protein, comprising fusing the protein into a polypeptide comprising an amino acid sequence having at least 90% homology with SEQ ID NO:1 or 2.

[0139] In some embodiments, the polypeptide consists of the amino acid sequence of SEQ ID NO:1 or 2.

[0140] In some embodiments, the protein is fused at the -N end of SEQ ID NO:2.

[0141] In some embodiments, the protein is fused to the polypeptide via a linker peptide.

[0142] In another aspect, the present invention provides a method for treating a disease, disorder, or unhealthy state in a subject in need, the method comprising administering to the subject a therapeutically effective dose of the fusion protein of the present invention, the polynucleotide of the present invention, or the carrier of the present invention, wherein the disease, disorder, or unhealthy state can be treated with the said heterologous peptide.

[0143] In some embodiments, the disease, disorder, or unhealthy state is selected from a disease group that includes tissue damage, cardiovascular disease, inflammatory disease or disorder, and kidney disease.

[0144] In some embodiments, tissue damage is an acute injury, such as myocardial infarction or stroke.

[0145] In some embodiments, the tissue damage is a chronic injury, such as kidney damage caused by diabetes.

[0146] In some cases, cardiovascular disease was selected from groups including myocardial infarction, arteriosclerosis, hypertension, angina pectoris, hyperlipidemia, and heart failure.

[0147] In some cases, inflammatory diseases or disorders were selected from groups of type 1 diabetes, type 2 diabetes, pancreatitis, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH).

[0148] In some embodiments, the disease or disorder is a kidney disease.

[0149] In some embodiments, the subjects are humans.

[0150] Having briefly described the general aspects of the invention, specific embodiments of certain aspects of the invention are provided below. It should be understood that any embodiment, including those disclosed only in the examples, claims, or a portion of the specification, can be combined with any one or more other embodiments of the invention, unless expressly denied or otherwise improper.

[0151] 2.CD164

[0152] CD164 is also known as sialovisin or endorphin. Its 197-residue human isoform 1 precursor sequence is RefSeqNP_006007, including the N-terminal 23-residue signal peptide (bold), and mucin domains I and II (SEQ ID NOs: 1 and 2, both double-underlined):

[0153] MSRLSRSLLWAATCLGVLCVLSADKNTTQHPNVTTLAPISNVTSAPVTSLPLVTTPAPETCEGRNSCVSCFNVSVVNTTCFWIECKDESYCSHNSTVSDCQVGNTTDFCSVSTATPVPTANSTAKPTVQPSPSTTSKTVTTSGTTNNTVTPTSQPVRKSTFDAASFIGGIVLVLGVQAVIFFLYKFCKSKERNYHTL

[0154] The aforementioned human sequences lacking a signal peptide were searched in the NCBI nr database using BLASTp, yielding numerous homologs (including homologous sequences and paralogous genes) from other species. The least homologous primate homolog in *Aotus nancymaae* showed 88% homology with the query sequence, while the least homologous of the top 100 search results were rodents (*Cast* or *canadensis*), with 69% homology.

[0155] Similarly, SEQ ID NO:2 was searched using BLASTp, which identified 99 results. Homologous entries in higher primates generally showed >98% sequence identity, while those in lower primates (such as *M. leucophaeus*) generally showed approximately 90-94% sequence identity. Rodents, such as rats and mice, typically showed approximately 70% sequence identity in this region.

[0156] Therefore, in some embodiments, the fusion protein of the present invention comprises a mammalian CD164 mucin domain I or II having at least about 70%, 80%, 90%, 95%, 97%, or 99% sequence alignment (SEQ ID NOs: 1 or 2) with human CD164 mucin domain I or II.

[0157] In some embodiments, mammalian CD164 mucin domain I or II is SEQ ID NO: 1 or 2.

[0158] In some embodiments, the fusion protein of the present invention comprises a variant, a mutant, or a synthetic CD164 mucin domain I or II having at least about 90%, 95%, 97%, or 99% sequence identity (SEQ ID Nos: 1 or 2) with human CD164 mucin domain I or II. In some embodiments, the variant, mutant, or synthetic CD164 mucin domain I or II each has the same N and / or O-glycosylation sites as wild-type human CD164 mucin domain I or II.

[0159] 3. Therapeutic peptides and treatable diseases

[0160] In some embodiments, the fusion protein of the present invention comprises a heterologous peptide of a therapeutically active peptide, such as “Future Directions for Peptide Therapy” disclosed by Kaspar and Reichert (Drug Discov. Today, 18:807-817, 2013; and Fosgerau and Hoffmann, Drug Discov. Today, 20(1):122-128, 2015) (both are referenced herein).

[0161] Naturally occurring peptides are generally unsuitable as direct, convenient treatments due to their inherent weaknesses, including poor chemical and physical stability and short circulating plasma half-life. Drugs must address these aspects.

[0162] Therapeutic peptides typically have short half-lives, which severely limits their therapeutic efficacy and necessitates frequent dosing (usually via intravenous injection or other injection routes, requiring hospitalization, or requiring trained patients to self-administer). Therefore, extending the half-life of a drug in circulation would be a significant improvement in patient convenience, compliance, cost, and ultimately, therapeutic efficacy.

[0163] As of 2015, over 60 peptide drugs approved by the U.S. Food and Drug Administration (FDA) were on the market, approximately 140 peptide drugs were in clinical trials, and over 500 therapeutic peptides were in preclinical trials. These figures are expected to grow significantly in the future. Therefore, the fusion protein of this invention provides a universal method for extending the therapeutic half-life of existing and developing therapeutic peptides, including those already approved by the FDA and EMA.

[0164] In some embodiments, the therapeutic peptide is used to treat metabolic diseases, cancer, inflammation, or as a vaccine.

[0165] In some embodiments, the therapeutic peptide is effective in treating a variety of diseases: endocrine disorders, respiratory disorders, bone disorders, urological disorders, ophthalmic disorders, dermatological disorders, central nervous system disorders, pain disorders, gastrointestinal disorders, allergic / immune disorders, infectious diseases, cardiovascular diseases, oncological diseases, or metabolic diseases.

[0166] In some embodiments, the therapeutic peptides are used to treat gastrointestinal disorders such as short bowel syndrome (e.g., linaclotide and tidulutide).

[0167] In some embodiments, the therapeutic peptide is useful for treating respiratory distress syndrome, such as high-risk preterm infants and premature infants (e.g., lucinantane).

[0168] In some embodiments, the therapeutic peptide is used to treat anemia, such as anemia in adult dialysis patients with chronic kidney disease (e.g., polyethylene glycol peptide).

[0169] In some embodiments, therapeutic peptides are useful for treating Cushing's disease, such as in adult patients who cannot undergo pituitary surgery or for whom a cure is not possible (e.g., paretin).

[0170] In some embodiments, the therapeutic peptide is used to treat cancers, such as blood cancers (e.g., carfilzomib).

[0171] In some embodiments, the therapeutic peptide is used to treat erythropoietic protoporphyria (EPP), a rare genetic disorder characterized by a severe response to sunlight (e.g., the photoprotective afanotide, a melanocortin 1 receptor agonist).

[0172] In some embodiments, the therapeutic peptide is used to treat EPP and solar urticaria (e.g., afanotide).

[0173] In some embodiments, therapeutic peptides, such as glatiramer acetate, are useful for treating multiple sclerosis.

[0174] In some embodiments, the therapeutic peptide is propleurelin, octreotide (for the treatment of acromegaly and symptoms in cancer patients), or goserelin (for the treatment of endometriosis and palliative care in advanced prostate and breast cancer).

[0175] In some embodiments, the therapeutic peptide is its antibody or antigen-binding fragment, such as scFv, Fab, Fab', F(ab')2, Fd, disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single-domain antibody, DVD-lg, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0176] In some embodiments, the therapeutic peptide is a natural peptide, such as a protein fragment, degradation product, or a signaling molecule derived from the gut microbiota.

[0177] In some embodiments, the therapeutic peptide is an antibody-drug conjugate (ADC; gemtuzumab-ozogamicin, brentuximab-vedotin, trastuzumab-emtansine).

[0178] In some embodiments, the therapeutic peptide is a peptide-drug conjugate (PDC; such as zoptarelin dox or ubicin, EP100) for the treatment of urothelial carcinoma, endometrial cancer, prostate cancer, breast cancer, and ovarian cancer.

[0179] In some embodiments, the therapeutic peptide targets the GLP receptor, CXCR4, opioid receptor, Ghrelin receptor, GNRH-R, vasopressin, oxytocin receptor, melanocorticoid receptor, or parathyroid hormone receptor.

[0180] In some embodiments, the therapeutic peptides are used to treat type 2 diabetes or obesity, such as GLP-1R agonist peptides (e.g., lixilatide; exenatide / Byetta1 / Bydureon1; liraglutide; abirate (albumin fusion); dulaglutide (Fc fusion); smegglutide (acylated GLP-1 analog); PB 1023 (recombinant GLP-1 analogue fusion with biopolymer; Cpd86; ZPGG-72; ZP3022; MOD-6030; ZP2929; HM12525A; VSR859; NN9926; TTP273 / TTP054; ZYOG1; MAR709; TT401; HM11260C; ITCA); RO6811135; ZP2929; TT401). These therapeutic peptides can also be used to treat cardiovascular diseases, neurodegenerative diseases, and weight management, such as myocardial infarction, Alzheimer's disease, Parkinson's disease, and mild cognitive impairment.

[0181] In some embodiments, the therapeutic peptide is a multifunctional peptide, such as a dual agonist of GLP-1-GIP and GLP-1-GCG. For example, the GLP-1-GCG dual agonist, compared to a GLP-1 agonist alone, exhibits increased energy expenditure induced by GCG, resulting in greater weight loss in overweight patients with type 2 diabetes mellitus (T2DM). Simultaneously, in the GLP-1-CCKB dual agonist, the CCKB (gastrin) agonist effect is superimposed on the GLP-1 effect, enhancing pancreatic β-cell function, which in turn helps reduce / prevent the progression of T2DM.

[0182] In clinical studies, the most common peptide therapeutic target is GLP-1R. Of the 265 peptide therapies that entered clinical trials between 2000 and 2012, 32 (12.1%) were GLP-1R agonists. Meanwhile, all other targets accounted for less than 3%. GLP-1R has been well-validated as a target for type 2 diabetes drugs. Since exenatide's approval in 2005, designing, developing, or delivering peptides has become a significant trend in this product category, enabling them to be administered less frequently than twice-daily exenatide. The endogenous ligand GLP-1 is degraded by dipeptidyl peptidase 4 (DPP4) within 1–2 minutes. Exenatide has a half-life of approximately 2.4 hours, as does liraglutide, which is specifically designed to resist DPP4 and also has a half-life of 2–4 hours. Liraglutide's peptide backbone is modified by adding a lipid (i.e., palmitic acid), thereby increasing its half-life to 13 hours, allowing liraglutide to be administered once daily. Abirate consists of an anti-ddp4 GLP-1 (7-36) amide analog fused to HAS via a tandem repeat sequence. Its half-life is 6-7 days. Dulaglutide comprises an anti-ddp4 GLP-1 (7-36) amide analog fused to the Fc region of IgG4, which is engineered to reduce binding to the Fcg receptor and potential immunogenicity, and to eliminate hapten formation. Its half-life is approximately four days. Smegglutide is an acylated GLP-1 analog with a half-life of 6-7 days.

[0183] In some embodiments, the therapeutic peptide is MYDGF. Myeloid-derived growth factor (MYDGF, also known as C19 or f10) is a paracrine cytokine produced by bone marrow-derived monocytes and macrophages and has been shown to promote cardiac recovery after ischemic myocardial infarction (MI). MYDGF also improves glucose tolerance and lipid metabolism by inducing the production and secretion of glucagon-like peptide-1 (GLP-1) to maintain glucose homeostasis. In diabetic nephropathy (DKD), MYDGF protects podocytes from damage by preserving slit diaphragm protein expression and reducing podocyte apoptosis.

[0184] Although MYDGF can inhibit organ and tissue damage in animal models through continuous intravenous administration or adenovirus overexpression, its clinical application is limited due to its short blood half-life (approximately 15.3 minutes). To reduce renal filtration rate, we developed MYDGF with a longer serum half-life.

[0185] Therefore, MYDGF fusion proteins with CD164 mucin domains can be used to treat tissue damage, cardiovascular disease, inflammatory diseases or disorders, and kidney disease.

[0186] In some embodiments, the therapeutic peptide is FGF21. Fibroblast growth factor 21 (FGF21) is an endocrine molecule belonging to the FGF superfamily that plays a role in maintaining lipid metabolism and energy homeostasis (Hecht 2012). FGF21 is a drug for treating diabetes, pancreatitis, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH).

[0187] In some embodiments, the therapeutic polypeptide is follicle-stimulating hormone (FSH). FSH is a gonadotropin, a glycoprotein polypeptide hormone synthesized and secreted by gonadotropic cells of the anterior pituitary gland.

[0188] In some embodiments, the therapeutic peptide is FGFBP3. Fibroblast growth factor binding protein 3 (FGFBP3) is a secretory chaperone that regulates lipid and glucose metabolism. FGFBP3 is a potential therapy for non-alcoholic fatty liver disease and type 2 diabetes (Tassi et al. 2018).

[0189] It should be noted that the above-described therapeutic peptides are for illustrative purposes only, and many other therapeutic peptides, especially those formulated for intravenous injection but with relatively short circulating half-lives, are within the scope of this invention.

[0190] 4. Polynucleotides and Carriers

[0191] In another aspect, the present invention provides a polynucleotide encoding the fusion protein of the invention described herein. In one embodiment, the polynucleotide encodes any one of SEQ ID NOs:1-8, for example, any one of SEQ ID NOs:3-8.

[0192] In some embodiments, the polynucleotide is a synthetic nucleic acid. In some embodiments, the polynucleotide is a DNA molecule. In some embodiments, the polynucleotide is an RNA molecule (e.g., an mRNA molecule). In some embodiments, the mRNA is capped, polyadenylated, substituted with 5-methylcytidine, substituted with pseudouridine, or a combination thereof.

[0193] In some embodiments, the polynucleotide (e.g., DNA) is operatively linked to a regulatory element (e.g., a promoter) to control the expression of the polynucleotide. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is an organism-specific promoter.

[0194] Suitable promoters are known in the art, including, for example, the pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, retro-transcribed Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, and β-actin promoter.

[0195] In one aspect, the present invention provides a polynucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to the polynucleotide sequence described herein, i.e., a nucleic acid sequence encoding any fusion fragment described herein.

[0196] To determine the homology of two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal alignment purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences to achieve optimal alignment, and non-homologous sequences may be ignored to facilitate alignment). Generally, the length of the reference sequence for alignment purposes should be at least 80% of the reference sequence length, and in some embodiments, at least 90%, 95%, or 100% of the reference sequence length. The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. Homology between two sequences is a function of shared positions, which, considering the number of vacancies and the length of each vacancy, necessitates the introduction of optimal alignment between the two sequences. For the purposes of this disclosure, a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5 can be used to perform sequence alignment and determine the homology between two sequences.

[0197] In some embodiments, the nucleic acid molecule encoding the fusion protein, its derivative, or functional fragment is codon-optimized for expression in a host cell or organism. The host cell may include an established cell line or isolated primary cells. The polynucleotide may be codon-optimized for use in any organism of interest, particularly human immune cells. Codon usage tables are readily available, for example, at www.kazusa.com or JP / codon / , and these tables can be adapted in various ways. See Nakamura et al., Nucl. Acids Res. 28:292, 2000 (included in this reference). Codon optimization of specific sequences for expression in specific host cells using computer algorithms is also desirable, such as Gene F or ge (Aptagen; Jacobus, Pa.).

[0198] An example of codon-optimized sequences is the optimization of polynucleotide coding sequences for expression in eukaryotes (e.g., humans) (i.e., optimization of polynucleotide coding sequences for expression in humans), or optimization of polynucleotide coding sequences for expression in other eukaryotes, animals, or mammals described herein. While this is preferred, it should be understood that other examples are possible, and codon optimization may be performed for host species other than humans, or for known specific organs. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance its expression by replacing at least one codon of the original sequence (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) with a codon that is more frequently or most frequently used in the target host cell, while preserving the native amino acid sequence. Different species exhibit specific biases towards specific codons for specific amino acids. Codon bias (the difference in codon usage among organisms) is generally associated with the translation efficiency of messenger RNA (mRNA), which in turn depends on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The preference of the tRNAs selected in a cell usually reflects the most frequently used codons in peptide synthesis. Therefore, based on codon optimization, genes can be tailored to achieve optimal gene expression in a given organism. Codon usage tables are readily available, for example, at www.kazusa.com or jp / codon / , and these tables can be modified in various ways. See Nakamura, Y., et al., "Codon usage tabulated from the international DNA sequence databases: status f or the year 2000," Nucl. Acids Res. 28:292 (2000). Codon optimization of specific sequences for expression in specific host cells using computer algorithms is also desirable, such as Gene F or ge (Aptagen; Jacobus, Pa.). In some embodiments, one or more codons in the sequence encoding the fusion fragment (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all of the codons) correspond to the most commonly used codons for a particular residue.

[0199] In some embodiments, the polynucleotides or nucleic acids of the present invention are present in a vector (e.g., a viral vector).

[0200] As used herein, the term "vector" generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art.

[0201] In some embodiments, the vector may be a cloning vector or an expression vector. The vector may be a plasmid, bacteriophage, sticky plasmid, etc. The vector may contain one or more regulatory elements to allow the vector to replicate in target cells (e.g., mammalian cells, such as CHO cells, HEK293 cells, etc.).

[0202] In some embodiments, the vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA fragments can be inserted, for example, through standard molecular cloning techniques.

[0203] In some embodiments, the vector is a viral vector in which a virus-derived DNA or RNA sequence is present to package into a virus (e.g., retrovirus, lentivirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, HSV, and adeno-associated virus (AAV)). The viral vector also includes polynucleotides carried by the virus for transfection into host cells.

[0204] In some embodiments, the vector is a lentiviral vector. In some embodiments, the lentiviral vector is a self-inactivating lentiviral vector. See, for example, Zufferey et al., "Self-Inactivating Lentivirus Vect or Safe and Efficient In vivo Gene Delivery." J Virol. 72(12):9873-9880, 1998. (included in this reference).

[0205] In some embodiments, the vector is based on the Sleeping Beauty (SB) transposon, which has been used as a non-viral vector for introducing genes into vertebrate genomes for gene therapy. Because the SB system is composed entirely of DNA, its production and delivery costs are significantly lower compared to viral vectors. SB transposons have been used for T-cell gene modification in human clinical trials.

[0206] In some embodiments, vectors are capable of autonomous replication in the host cells to which they are introduced. In some embodiments, vectors (e.g., mammalian vectors containing non-free genes) are integrated into the host cell's genome upon introduction and replicate along with the host genome. In some embodiments, vectors, referred to herein as "expression vectors," are capable of directing the expression of the gene linked to them. Vectors used to accomplish expression in eukaryotic cells are "eukaryotic expression vectors."

[0207] In some embodiments, the vector is a recombinant expression vector containing the nucleic acid of the present invention, in a form suitable for expression in host cells. The recombinant expression vector may include one or more regulatory elements that are selectable based on the host cell to be expressed and operatively linked to the nucleic acid sequence to be expressed. Here, "operatively linked" means that the target nucleotide sequence is linked to the regulatory element in a manner that allows the expression of the nucleotide sequence (e.g., in the host cell when the vector is introduced into the host cell in an in vitro transcription / translation system).

[0208] The term "regulatory element" includes promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences). See, for example, Goeddel, *GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY* 185, Academic Press, San Diego, Calif. (1990), which describes these regulatory elements. Regulatory elements include those that constitutively express nucleotide sequences directly in a variety of host cell types, as well as those that express nucleotide sequences directly only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters may be expressed directly primarily in the desired tissue, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, pancreas) or specific cell types (e.g., lymphocytes such as T cells or NK cells). Regulatory elements may also be expressed directly in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, and may or may not be tissue- or cell type-specific.

[0209] In some embodiments, the vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, the U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral sarcoma virus (RSV) LTR promoter (RSV enhancer optional), the cytomegalovirus (CMV) promoter (CMV enhancer optional) [see, for example, Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the glycerol phosphokinase (PGK) promoter, and the EF1a promoter.

[0210] The term “regulatory element” also includes enhancer elements, such as WPRE; CMV enhancer; R-U5' fragment in the LTR of HTLV-I (Mol.Cell.Biol., Vol 8(1), p.466-472, 1988); SV40 enhancer; intron sequence between exons 2 and 3 of rabbit b-globin (Proc.Natl.Acad.Sci.USA., Vol.78(3), p.1527-31, 1981).

[0211] Those skilled in the art will understand that the design of expression vectors can depend on factors such as the selection of host cells to be transformed and the desired expression level. Vectors can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides encoded by nucleic acids as described herein.

[0212] In some embodiments, the vector is a lentivirus or AAV vector, which may be selected to target a specific type of cell (e.g., with tissue and / or cell type specific orientation).

[0213] The vector of the present invention can be introduced into target cells or host cells by any of the methods recognized by the prior art, such as transfection, lipid carrier, infection, electroporation, microinjection, parenteral injection, aerosol, gene gun or ballistic particles.

[0214] In some embodiments, the fusion proteins described herein may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (e.g., yeast), plant cells, insect cells, and mammalian cells. Such expression may be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express the peptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S and DG44 cells; and PER. Crucells; and NSO cells. In some embodiments, the fusion proteins described herein may be expressed in yeast. See, for example, US Publication No. US 2006 / 0270045 A1. In some embodiments, specific eukaryotic host cells are selected based on their ability to perform desired post-translational modifications to the mucin domains. For example, in some embodiments, peptides produced by CHO cells have higher levels of sialylation than the same peptides produced in 293 cells.

[0215] Introducing one or more nucleic acids into a desired host cell can be accomplished by any method, including but not limited to calcium phosphate transfection, DEAE-glucan-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc. Non-limiting exemplary methods are described, for example, in Sambrook et al., Molecular Cloning, A Lab or at or at Manual, 3rd ed., Cold Spring Harb or Lab or at or at Press (2001). According to any suitable method, nucleic acids can be transiently or stably transfected into the desired host cell.

[0216] In some embodiments, one or more polypeptides may be produced in animals engineered or transfected with one or more vectors or polynucleotides encoding the fusion protein of the present invention, according to any suitable method.

[0217] In some embodiments, transfection includes chemical transfection via, for example, introducing a vector through calcium phosphate, lipids, or protein complexes. Calcium phosphate, DEAE-glucan, liposomes and lipid complexes (for oral delivery of genes), surfactants, and perfluorinated chemical liquids for aerosol delivery of genes are also mentioned.

[0218] In some embodiments, the lipid carrier is produced by combining plasmid DNA and a lipid solution, leading to the formation of liposomes that can fuse with the cell membranes of various cell types, thereby introducing the carrier DNA into the cytoplasm and nucleus for expression of gene-encoding genes. In some embodiments, folic acid is linked to DNA or a DNA-lipid complex to more efficiently deliver the carrier to cells expressing high levels of the folic acid receptor. Other targeting portions can be similarly used to target the delivery of the carrier to specific cell types targeted by the targeting portion.

[0219] In some embodiments, the vector DNA is internalized via receptor-mediated endocytosis.

[0220] In some embodiments, the vector is a lentiviral vector, and the target cell infection spectrum of the vector is expanded by replacing the gene for the surface glycoprotein with a gene from another viral genome in the vector's packaging cell line (PCL).

[0221] 5. Drug combination

[0222] Another aspect of the invention provides pharmaceutical compositions for treating diseases or conditions (e.g., cancer or inflammatory diseases), or any other disease or indication treated with the therapeutic peptides described herein. The pharmaceutical compositions comprise therapeutically effective amounts of the fusion protein, polynucleotide, or carrier of the invention. The pharmaceutical compositions also include pharmaceutically acceptable carriers or excipients.

[0223] As used herein, "pharmaceutically acceptable carriers or excipients" includes any and all physiologically compatible carriers or excipients such as solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). In some embodiments, the pharmaceutical composition comprises a carrier or excipient of the fusion protein of the present invention for intravenous (iv), subcutaneous (sc), inhalation, or oral administration (e.g., oral delivery of peptides expressed directly in the gastrointestinal tract).

[0224] In another embodiment, the pharmaceutical composition comprises a carrier or excipient of the fusion protein of the present invention for nasal, transdermal, or oral administration. (e.g., via gold nanoparticles (MidTech) and PharmFilm) TM (Combined with Monosol Rx technology for delivery)

[0225] As used herein, "therapeutic effective amount" or "therapeutic effective dose" or "effective amount" means the administration of a sufficient quantity of a substance, compound, material, or cell to produce the desired therapeutic effect. Therefore, the dosage is sufficient to prevent, treat, or improve at least one symptom of a disease or condition, or to completely or partially block the progression / worsening of the disease or condition. The dosage is also below a threshold toxicity level, above which may / will cause the subject to discontinue or terminate treatment.

[0226] The quantity and dosage level of the fusion protein in the pharmaceutical compositions of this invention vary depending on the specific patient's needs, route of administration, the subject's disease type and / or severity, desired therapeutic response, tolerable toxicity, and other factors deemed relevant by the attending physician. That is, the selected dosage level can depend on a variety of pharmacokinetic factors, including the specific composition used, route of administration, patient age, other pharmaceutical compositions used in combination, duration and timing of administration, excretion or elimination rate, sex, weight, condition, general health status, patient's condition and medical history, etc., which are well known in the medical community. Those skilled in the art can empirically determine the effective amount of the invention without unnecessary experimentation. In conjunction with the guidance provided herein, by selecting and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred route of administration among the various fusion proteins of this invention, an effective preventative or therapeutic regimen can be planned that does not itself cause substantial toxicity but is fully effective in treating a specific subject.

[0227] The toxicity and efficacy of the present invention can be determined using standard pharmaceutical procedures in cell cultures or laboratory animals, for example, to determine the LD50 (median lethal dose) and ED50 (median effective dose). The dose ratio of toxic side effects to therapeutic effects is a therapeutic indicator and can be expressed as the LD50 / ED50 ratio. Prophylactic and / or therapeutic agents with a large therapeutic index are preferred. Although prophylactic and / or therapeutic agents with toxic side effects can be used, care should be taken to design a delivery system that targets such drugs to the affected tissue site to minimize potential damage to uninfected cells, thereby reducing side effects.

[0228] In some embodiments, data obtained from cell culture analysis, animal studies, and human studies can be used to formulate dose ranges for prophylactic and / or therapeutic agents for human use. The doses of such agents are preferably within a range of circulating concentrations, including an ED50 with low or no toxicity. The dose may vary within this range depending on the dosage form and route of administration used. For any agent used in the methods of this invention, a preliminary therapeutically effective dose can be estimated from cell culture analysis. Doses can be formulated in animal models to achieve a range of circulating plasma concentrations, including the IC50 (i.e., the concentration of the test compound that achieves half-maximal symptom inhibition) determined in cell culture. This information can be used to more accurately determine the effective dose in humans. For example, plasma levels can be measured by high-performance liquid chromatography.

[0229] In some embodiments, the pharmaceutical composition is formulated for use in individuals such as humans, non-human primates, cattle, horses, pigs, sheep, goats, dogs, cats, or rodents. In some embodiments, the subject is a human. Example

[0230] Example 1: CD164 mucin domain fusion protein and its characterization method

[0231] Optimized recombinant expression was performed on gene codons containing CD164 mucin domain I (aa24-60) and mucin domain II (aa110-162). Typically, mucin domain I is fused to the N-terminus of the selected protein, while mucin domain II is fused to the C-terminus.

[0232] At least the following fusion proteins were constructed: fibroblast growth factor 21 (FGF21), follicle-stimulating hormone (FSH), myeloid-derived growth factor (MYDGF), and fibroblast growth factor-binding protein 3 (FGFBP3), all of which are current or potential protein therapeutics. The sequences of the mucin domains and several representative fusions are as follows:

[0233] CD164 mucin domain I:

[0234] DKNTTQHPNVTTLAPISNVTSAPVTSLPLVTTPAPET (SEQ ID NO:1).

[0235] CD164 muxin protein structure regionⅡ:

[0236] SVSTATPVPTANSTAKPTVQPSPSTTSKTVTTSGTTNNTVTPTSQPVRKS TFD(SEQ ID NO:2).

[0237] MYDGF-164:

[0238] VSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMETYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTELSVSTATPVPTANSTAKPTVQPSPSTTSKTVTTSGTTNNTVTPTSQPVRKSTFD(SEQ ID NO:3)

[0239] CD164-MYDGF fusion protein:

[0240] DKNTTQHPNVTTLAPISNVTSAPVTSLPLVTTPAPETVSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTE(SEO ID NO:4)

[0241] FGF21-164 fusion protein:

[0242] DSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGN KSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYASSVSTATPVPTANSTAKPTVQPSPSTTSKTVTTSGTTNNTVTPTSQPVRKSTFD(SEQ ID NO:5)

[0243] In some embodiments, the FGF21 in the 164 fusion is a wild-type FGF21 sequence. In some embodiments, the FGF21 in the 164 fusion contains L146C and / or A162C mutations.

[0244] FGFBP3-164 fusion protein:

[0245] RREKGAASNVAEPVPGPTGGSSGRFLSPEQHACSWQLLLPAPEAAAGSELALRCQSPDGARHQCAYRGHPERCAAYAARRAHFWKQVLGGLRKKRRPCH DPAPLQARLCAGKKGHGAELRLVPRASPPARPTVAGFAGESKPRARNRGRTRERASGPAAGTPPPQSAPPKENPSERKTNEGKRKAALVPNEERPMGTGPDPDGLDGNAELTETYCAEKWHSLCNFFVNFWNGSVSTATPVPTANSTAKPTVQPSPSTTSKTVTTSGTTNNTVTPTSQPVRKSTFD(SEQ ID NO:6)

[0246] 164-FSHa fusion protein:

[0247] DKNTTQHPNVTTLAPISNVTSAPVTSLPLVTTPAPETAPDVQDCPECTLQ ENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMVTSESTCCVAKSYNVAKS YNRVTVMGGFKVENHTACHCSTCYYHKS(SEQ ID NO:7)

[0248] FSHb-164 fusion protein:

[0249] MKTLOFFFLFCCWKAICCNSCELTNITIAIEKEECRFCISINTTWCAGYCYTRDLVYKDPARPKIQKTCTFKELVYETVRVPGCAHHADSLYTYPVATQCHCGKCDSDSTDCTVRGLGPSYCSFGEMKESVSTATPVPTANSTAKPTVQPSPSTTSKTVTTSGTTNNTVTPTSQPVRKSTFD(SEQ ID NO:8)

[0250] All gene fragments were cloned into the pD2531 expression vector (ATUM) and stably transfected into serum-free Chinese hamster ovary (CHO) cells or 293F cells for recombinant expression of the fusion protein. Recombinant cell lines were screened in glutamine-deficient medium, and subclonal selection was performed in 96-well tissue culture plates using a limiting dilution method. Western blot analysis was performed using polyclonal antibodies (SinoBiologicals or Abcam) to preliminarily identify the expression of the recombinant protein, followed by protein purification and analytical identification.

[0251] protein After separation by SDS-PAGE using the Tetra System (Bio-rad), protein purity was analyzed by Coomassie Brilliant Blue (CBB) staining and density determination. Molecular weight standards (11-180 kDa, Tanon) were used for analysis.

[0252] For purity analysis, approximately 10 μg of the fusion protein was dissolved in sample buffer and analyzed in a 12% SDS-PAGE gel. Density was determined using a Tanon 4600SF gel imaging system.

[0253] Polyacrylamide gel electrophoresis (PAGE) was used, with isoelectric focusing (IEF) performed using a Multiph or II Electrophoresis System (GE Healthcare). IEF standards (Bio-rad) with a pH range of 3–10 and IEF Standard (GE Healthcare) with a pH range of 2.5–6.5 were used as reference pI ranges.

[0254] Samples were desalted before isoelectric focusing. A 10 μg protein sample was isoelectrically focused at 15 °C with an initial focusing step of 700 V for 20 minutes, followed by a linear gradient to 500 V for 20 minutes, and finally a linear gradient to 2000 V for 90 minutes. After isoelectric focusing, proteins in the PAGE gel were observed by silver staining. Gel images were scanned, and pI values ​​were determined by analysis using IMAGEQUANT TLversion 7.0 (GE Healthcare) software.

[0255] High-performance liquid chromatography (HPLC) equipped with an autosampler (Agilent Technologies) was used at AdvanceBioSEC. SEC analysis was performed on an Agilent Technologies column at a flow rate of 0.3 mL / min. Protein standards (Agilent Technologies) were analyzed using 150 mmol / L sodium phosphate buffer (pH 7.0). Fusion proteins were analyzed using 150 mmol / L sodium phosphate buffer (pH 7.0). Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed on a Z or BAX SB300 C8 column (Agilent Technologies) at a flow rate of 1 mL / min using a linear elution gradient from 0% v / v acetonitrile, 0.01% v / v TFA to 60% v / v acetonitrile, 0.085% TFA.

[0256] According to the manufacturer's protocol (Sigma), the MYDGF-164 fusion protein was hydrolyzed with 1 mL of 4M trifluoroacetic acid (TFA), and the released monosaccharides were labeled with 2-AA. Analysis was performed using a Shimadzu Nexera UPLC system equipped with an RF-20Axs fluorescence detector and a reverse-phase column (Phenomenex Hyperclone 5μm ODS). (250×4.60mm). The composition and proportion of various monosaccharides were determined based on monosaccharide standards and area normalization method.

[0257] O-glycans and N-glycans were released from the sample using improved β-elimination. A water-equilibrated carbon column (Supelclean) was used. TM ENVI TM Proteins and salts were removed using 2-Carb SPE. The polysaccharides were eluted with 20% and 40% acetonitrile. The eluent was evaporated under reduced pressure and fluorescently labeled with 2-aminobenzamide (2-AB) in a HILIC UPLC (ACQUITY UPLC-CLYCAN BEH Amide Column). Separation of labeled polysaccharides on a plate (1.7 μm, 2.1 mm × 150 mm).

[0258] According to the manufacturer's protocol (Sigma), the MYDGF-164 fusion protein was treated with PNGase F, followed by labeling the released N-linked glycans with 2-aminobenzamide. The labeled glycans were then processed in HILIC-UPLC (ACQUITY UPLC Glycan BEHAmide Column). Separation was performed on a 1.7 μm, 2.1 mm × 150 mm plate to determine the form of N-linked glycans by comparison with 2-AB labeled glycan standards.

[0259] The content of sialic acid in the MYDGF-164 fusion protein was determined by high performance liquid chromatography (HPLC). Sialic acid released from the hydrolysis of the MYDGF-164 fusion protein was labeled with OPD and analyzed using a C18 reversed-phase column (Phenomenex Hyperclone 5μm C18, 250×4.6mm) via UPLC-FLD or UPLC-FLD-MS (Shimadzu LCMS 2020ESI mass spectrometer coupled with a fluorescence detector). The type and content of sialic acid were determined by comparison with OPD-labeled sialic acid standards.

[0260] Six different extrinsic / endoglycosidases were used to remove glycosylation modifications from MYDGF-164 fusion protein samples, including β-N-acetylglucosidase SN384, neuraminidase AuNeu54, β-galactosidase Am0874, commercially available galactosidase, fucosidases Eo0918, Eo3066, and Eo3141, and PNGase. The reaction mixture consisted of 38 μL of the protein sample dissolved in water, 5 μL of 200 mM PBS buffer (pH 6.5), and 1 μL of each enzyme. The mixture was incubated at 37 °C for 16 h.

[0261] Mass spectra of the MYDGF-164 fusion protein and the glycan-free MYDGF-164 fusion protein were acquired using a MALDI-TOF instrument (Bruker Autoflex 244 velocimeter equipped with a 1000Hz Smartbeam-II laser). Protein ionization was performed using 2456-aza-2-thiothymine as the matrix. Molecular masses were obtained through spectroscopic analysis using BrukerFlexanalysis software version 3.3.80.

[0262] Twelve C57BL / 6 mice were injected intravenously with 17.5 mg / kg of MYDGF-164 protein for pharmacokinetic analysis. Mice were randomly divided into three groups, and blood samples were collected at 0, 30, 240 min, 5, 60, 360 min, and 15, 120, 480 min post-injection. Serum was obtained by centrifugation at 11000 rpm for 5 minutes. Serum MYDGF-164 fusion protein concentration was analyzed by LC-MS / MS, and plasma fusion protein concentration was quantified using MassHunter software (Agilent, USA). Pharmacokinetic parameters were calculated using a non-compartmental model based on Phoenix Winnonlin 7.0 software (Pharsight, USA). Cmax and Tmax were determined by LC-MS / MS.

[0263] Example 2: Glycosylation of mucin domains is unaffected in fusion proteins.

[0264] CD164 mucin domains I (SEQ ID NO:1) and II (SEQ ID NO:2) consist of 37 and 53 amino acids, respectively, with molecular weights of 3.8 kDa and 5.4 kDa in their unglycosylated forms.

[0265] To investigate whether fusion with non-glycosylated proteins affects the glycosylation of the CD164 mucin domain, chimeric proteins were constructed by fusing mucin with the non-glycosylated proteins MYDGF and FGF21, resulting in MYDGF-164 (SEQ ID NO:3) and FGF21-164 (SEQ ID NO:5). The fusion proteins were stably expressed in CHO or 293F cells, and their secretion in conditioned medium was analyzed by SDS-PAGE and Western blot. Figure 1 The predicted molecular weight of the unglycosylated mature peptide MYDGF-164 was 21.1 kDa, and the predicted molecular weight of FGF21-164 was 24.3 kDa. SDS-PAGE and Western spectroscopy analyses showed that MYDGF-164 migrated 48 kDa and FGF21-164 migrated 52 kDa during electrophoresis, indicating that fusion with the CD164 mucin domain resulted in an additional mass of 20-30 kDa.

[0266] In addition, CD164 mucin fusion proteins chimeric with FSH alpha, FSH beta, and FGFBP3 proteins were generated; these proteins are all naturally glycosylated. Figure 2 Compared to non-fusion proteins, the FSH fusion protein also achieved a mass increase of 20 to 30 kDa. Glycosylation of the fusion CD164 mucin region was detected in all cases.

[0267] These data convincingly demonstrate that the fusion of mucin domains with other heterologous proteins has no significant effect on glycosylation.

[0268] Example 3: Characterization of CD164 mucin fusion protein

[0269] To identify chimeric / fusion proteins containing mucin domains, the MYDGF-164 fusion protein was purified using conventional chromatography. The purified MYDGF-164 fusion protein was separated by SDS-PAGE, stained with Coomassie Brilliant Blue, and then analyzed. Figure 3A ).

[0270] After purification, the MYDGF-164 protein migrated to a 48 kDa protein, similar to Western blotting analysis. Figure 1 The purified protein was subjected to N-terminal sequencing, which determined the N-terminal sequence of 5 amino acids. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis also confirmed the homogeneity of the protein. Figure 3B ).

[0271] In RP-HPLC elution, the MYDGF-164 spectrum showed a single absorption peak, indicating that both N-linked glycosylation sites of CD164 mucin domain II were uniformly utilized, meaning either complete glycosylation or neither was observed. Furthermore, Coomassie Brilliant Blue staining of the purified protein revealed a relatively simple banding pattern. These observations suggest that the polysaccharide form of MYDGF-164 is predominantly homogeneous.

[0272] Isoelectric focusing (IEF) analysis showed that the purified MYDGF-164 fusion was a mixture containing multiple acidic isomers, with a pI of 5.22-3.31. Figure 4 Unmodified MYDGF-164 is a basic protein with a pI of 7.93. IEF data strongly suggest that the fusion protein may be converted to an acidic form via sialylation of the glycan. Sialic acid can be added to O- and N-linked glycans.

[0273] N-linked glycans were further analyzed by PNGase treatment and 2-AB labeling, followed by UPLC analysis. Figure 7 The dominant form of the glycan was biantennary, while the triantennary found at 34.078 and 35.181 min was negligible. Therefore, the relatively homogeneous biantennary structure found was consistent with Coomassie Brilliant Blue staining and RP-HPLC data (Figure 3), further demonstrating the highly homogeneous N-linked glycosylation of the CD164 mucin domain.

[0274] Therefore, the presence of N-linked glycans was confirmed. The 2-AB labeling process removed sialic acid, so no elution peaks showed the structure of sialic acid.

[0275] For example, the elution peak at 29.668 min corresponds to a fucosylated doyonnaise containing galactose but no sialic acid. As previously mentioned, the mucin II domain of CD164 contains two putative N-linked glycosylation sites (Doyonnas). Based on the analysis of the N-linked glycans, it is shown that the MY64 fusion is fully modified at all N-glycosylation sites.

[0276] To determine the quality of the purified MYDGF-164 fusion protein, MALDI-TOF mass spectrometry analysis was performed. In the mass spectrum, the purified MYDGF-164 fusion protein exhibited a single charged peak at 42370.668 Daltons and a double charged peak. Figure 9 The predicted mass of the double-charged peak was 42,432 Daltons, higher than the 42,370.668 Daltons of the single-charged peak. This difference is likely due to the heterogeneity of the fusion protein in various glycosylated forms during laser-induced protonation and the subsequent release of protonated proteins in the mass spectrometry. However, the mass of the single-charged peak was approximately 20 kDa higher than the predicted molecular weight of 21,182.6 Daltons for the unglycosylated MYDGF-164, indicating that the fusion protein is highly glycosylated.

[0277] The hydrodynamic properties of MYDGF-164 were characterized using SEC chromatography. Protein elution was characterized by a single symmetrical peak. Figure 10A ), compared with standard protein ( Figure 10B At this stage, the hydrodynamic radius of the MYDGF-164 fusion protein corresponds to a 98.744 kDa globular protein. Due to the brush conformation of the mucin domain, the MYDGF-164 fusion protein is unlikely to fold into a globular structure. However, similar to Fc fusion proteins or PEGylated proteins, the increased hydrodynamic radius of MYDGF-164 is thought to decrease glomerular filtration rate, thereby increasing the half-life of the fusion protein in vivo.

[0278] Example 4: Enhancing the serum half-life of CD164 mucin fusion protein

[0279] Early data suggest that due to its extremely short half-life, MYDGF has limited bioavailability in vivo, and its activity in cardiac repair models requires continuous intravenous infusion (K or f-klingebiel 2015).

[0280] This experiment uses a mouse model to determine whether the half-life of MYDGF-164 fusion is prolonged.

[0281] The protein was purified by intravenous injection, and then pharmacokinetics were studied by LC-MS / MS analysis to quantify the concentration of MYDGF-164 fusion protein in serum at different time points. Figure 11 Pharmacokinetic parameters of the MYDGF-164 fusion protein were calculated based on a non-compartmental model (Table 1).

[0282] Table 1. Summary of MYDGF-164PK data

[0283]

[0284] *T was determined using LC-MS / MS. max and C max .

[0285] Based on the analysis of end-stage pharmacokinetic data, the MYDGF-164 fusion protein can be cleared in mice with a half-life of 3.86 hours.

[0286] In stark contrast, MYDGF without the fusion of the mucin domain has a clearance half-life of only 15.3 minutes in vivo (K or f Klingebiel 2015). The increased half-life of the fusion-generated MYDGF-164 protein is expected to significantly reduce the frequency of its use as a therapeutic agent in disease treatment and improve treatment outcomes.

[0287] Furthermore, the steady-state volume of distribution was 248.15 mL / kg, which is much higher than the blood volume of mice (70 mL / kg), indicating that most of MYDGF-164 entered the tissues and that the fusion of the mucin domain did not affect the tissue distribution.

[0288] In summary, the pharmacokinetic parameters strongly suggest that the CD164 mucin domain can be used for protein engineering to improve half-life without affecting tissue distribution.

[0289] References

[0290] Chan et al., Relationship between novel isoforms, functionally important domains, and subcellular distribution of CD164 / endolyn. JBC 276(3):2139-52, 2001.

[0291] Doyonnas et al., CD164 monoclonal antibodies that block hematopoietic progenitourinary cells or cell adhesion and proliferation interact with the first mucin domain of the CD164 receptor. The Journal of Immunology 165(2):840-51, 2000.

[0292] Fares et al., Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit. PNAS USA 89(10):4304-8, 1992.

[0293] Fares et al. developed a long-acting erythropoietin by fusing the carboxyl-terminal peptide of human chorionic gonadotropin β-subunit to the coding sequence of human erythropoietin. Endocrinology 148(10):5081-7, 2007.

[0294] Forde et al., Endolyn (CD164) modulates the CXCL12-mediated migration of umbilical cord blood CD133+ cells. Blood 109(5):1825-33, 2007.

[0295] Hecht et al., Rationale-based engineering of a potent long-acting FGF21 analog or the treatment of type 2 diabetes. PloS One 7(11):e49345,2012.

[0296] Ihrke et al., Endolyn is a mucin-like type I membrane protein targeted to lysosomes by its cytoplasmic tail. Biochemical Journal. 345(2):287-96, 2000.

[0297] Korf-klingebiel et al., Myeloid-derived growth factor (C19 or f10) mediates cardiac repair following myocardial infarction. Nature Medicine 21(2):140,2015.

[0298] Lee et al., Identification of a role for the sialomucin CD164 in myogenic differentiation by signal sequence trapping in yeast. MCB 21(22):7696-706, 2001.

[0299] Tassi et al., Fibroblast Growth Factor Binding Protein 3 (FGFBP3) affects carbohydrate and lipid metabolism. Scientific Reports 8:15973, 2018.

[0300] All references are inserted where they are cited.

[0301] Example 5: MYDGF-164 fusion protein promotes the proliferation, migration, and tubule formation of human umbilical vein endothelial cells and inhibits apoptosis.

[0302] To characterize the proliferative activity of HUVEC cells, MYDGF-164 was added to HUVEC cells seeded in 96-well plates and stimulated with different concentrations of MYDGF-164 in the presence of 5% or 1% FBS. Figure 12 The results showed that MYDGF-164 promoted HUVEC cell proliferation in a dose-dependent manner in 5% FBS. In 1% FBS, MYDGF-164 had an effect on cell proliferation, but this effect did not increase with increasing dose, indicating that serum-added growth factors have a synergistic effect with MYDGF-164.

[0303] To confirm that MYDGF-164 can promote the proliferation of HUVEC cells, flow cytometry was performed to determine the proportion of cells at different stages of the cell cycle. Figure 13 In 1% FBS medium, after 24 hours, approximately 79% of HUVECS were in the G0 / G1 phase, while with the addition of 1 μg / mL MYDGF-164, approximately 65% ​​of HUVECS were in the G0 / G1 phase. Simultaneously, approximately 14% of cells entered the G2 phase, compared to approximately 5% in untreated cells. The number of cells in the S phase after treatment was approximately 21%, also higher than the 16% in the untreated sample. These results confirm that MYDGF-164 has a significant impact on cell cycle activity.

[0304] One characteristic of endothelial cells is their migration response to angiogenesis stimuli. The migration behavior of HUVEC cells in the presence of MYDGF-164 was measured. Figure 14 In scratch repair assays, endothelial cell monolayers closed via migration. MYDGF-164 promoted HUVEC cell migration, as the resulting scratch closure increased with increasing MYDGF-164 concentration. Figure 14 ).

[0305] The ducts formed by endothelial cells are a key part of angiogenesis, and this article describes their in vitro testing. Figure 15 Traditionally, the ability of HUVEC cells to attach to low-growth factor matrix gels (BD, Bioscience) and form tubular structures is an indicator of endothelial cell angiogenesis potential. Compared to 100 ng / mL VEGFA, different concentrations of MYDGF-164 promoted tubular structure formation. Notably, compared to the same concentration of VEGFA, the closed tubules of the ring-shaped structures stimulated by MYDGF-164 appeared to be more tightly packed and regular.

[0306] Data further demonstrate that the MYDGF-164 fusion protein can reduce apoptosis induced by various stimuli. In this experiment, HUVEC cells were pre-incubated with 1 μg / mL MYDGF-164 for 24 hours, and then treated with hydrogen peroxide (400 μmol / L) for another 24 hours. Figure 16 In the presence of MYDGF-164, cells stained with both PI and Annexin-V decreased to 20.54%, while cells in the untreated sample decreased to 27.85%, indicating a significant reduction in hydrogen peroxide-induced apoptosis. Figure 16 ).

[0307] method:

[0308] Cell lines. Human umbilical vein endothelial cells (HUVECs) were purchased from Promocell (Heidelberg, Germany, primary cells) and cultured in endothelial cell growth medium supplemented with endothelial cell growth supplement (supplement mixture, Promocell) and 5% (v / v) FBS and 1% (vol / vol) streptomycin at 37°C in a 5% CO2 atmosphere. Before the experiment, HUVECs were passaged at least three times in T75 flasks (passed every 3 days).

[0309] Cell proliferation analysis. Human umbilical vein endothelial cells were seeded at a rate of 3000 cells / well in 96-well culture plates and cultured for 24 hours. Cells were then stimulated for 24 hours with 5% or 1% FBS containing different concentrations of MYDGF-164. Cell proliferation was quantified using the CCK-8 assay (Yeasen).

[0310] Flow cytometry analysis. For cell cycle analysis, human umbilical vein endothelial cells were seeded at a density of 2 × 10⁵ cells per well in 6-well plates. Cells were placed in 1% FBS, followed by the addition of solvent or 1 μg / mL MYDGF-164. After 24 hours of treatment, cells were collected, fixed, and stained with a cell cycle analysis kit (Solarbio). Signals were acquired using Novocyte (Agilent BIO), and data were analyzed using NovoExpress software. Apoptosis analysis: Cells were incubated with 1 μg / mL MYDGF-164 for 24 hours. Then, stimulation with H₂O₂ (400 μmol / L) or cisplatin (DDP, 5 μg / mL) was performed for 24 hours. Cells were collected, washed with PBS, stained with an apoptosis detection kit (Yeasen), and apoptotic and necrotic cells were detected using NovoCyte (Agilent BIO). Data were analyzed using NovoExpress software.

[0311] Scratch assay. For scratch recovery analysis, human umbilical vein endothelial cells were seeded at a density of 1 × 10⁵ cells per well in 12-well plates. The supernatant was removed, and a straight line through the cell monolayer was drawn in the center of each well using a 200 μl pipette tip. Endothelial cell growth medium with different concentrations of MYDGF-164 or 100 ng / mL VEGFA was added. Images were taken at 0, 12, and 24 hours. Migration rate was analyzed using Image-Pro-Plus software. Migration rate = area of ​​change / wound area.

[0312] Tube formation assay. Low growth factor matrix gel (BD, Bioscience) was mixed with cell culture medium at a ratio of 1:2 and 100 μL / well was placed in 48-well plates. The plates were then polymerized at 37°C for 1 h. Two × 10⁴ human umbilical vein endothelial cells incubated with different concentrations of MYDGF-164 or 100 ng / mL VEGFA were added to each well. After 4 hours of culture, the tubes were photographed. The closed tubes formed a ring structure, completely surrounded by HUVEC cells. The number of closed tubes was counted.

[0313] Example 6: MYDGF-164 fusion protein reduces the infarct area after myocardial infarction.

[0314] Testing the potential protective function of MYDGF-164 in a rat model of myocardial ischemia. Figure 17A and Figure 17B In the first model, 32 six-week-old male SD rats were randomly divided into a sham-operated group (sham-operated group) and a model group. The model group underwent 45 minutes of myocardial ischemia-reperfusion. The MYDGF-164 treatment group received a tail vein injection of MYDGF-164 5 minutes before reperfusion. MYDGF-164 was administered 4 hours after reperfusion, and then twice daily at 6-hour intervals for the following week. The positive control group received tirofiban (a non-peptide reversible antagonist of platelet glycoprotein (GP) IIb / IIIa receptors and an inhibitor of platelet aggregation). In the second model, MYDGF-164 treatment was similar, except that the heart was assessed 24 hours after the ischemic event to determine the area of ​​danger.

[0315] Cardiac troponin I (cTnI) is a specific protein released by the myocardium during myocardial infarction. The measurement of cardiac troponin I is widely used as a diagnostic indicator for myocardial infarction and acute coronary syndrome. Serum cTnI was collected preoperatively, and at 24 hours, 72 hours, and 8 days postoperatively. Treatment with MYDGF-164 and tirofiban significantly reduced serum cTnI levels, indicating the cardioprotective effect of MYDGF-164 during experimental myocardial infarction. Figure 18A and Figure 18B ).

[0316] Figures 19A-19HMYDGF-164 is shown to reduce cardiac tissue damage caused by ischemia-reperfusion and promote angiogenesis. Triphenyltetrazolium chloride (TTC) staining is the preferred method for assessing postmortem myocardial ischemia. Non-infarcted biopsy tissue stains red, while infarcted areas remain unstained. Figure 19A and 19B Rat hearts were cut into 1 mm sections and stained with TTC. Figure 19A The results showed that, compared with the model group, the infarct area in the Tirofiban and MYDGF-164 groups tended to decrease. Figure 19B ).

[0317] In a second rat ischemia model, Evans blue staining was added for analysis. Unstained ischemic tissue represented areas of permanent tissue damage. Twenty-four hours after the ischemic event, MYDGF-164 treatment significantly reduced both the danger zone and infarct area. Figure 19C , Figure 19D , Figure 19E )

[0318] Figure 19F-19H This demonstrates MYDGF-164-enhanced angiogenesis in the boundary region between infarcted and non-infarcted tissues. Seven days post-ischemia and treatment, H&E-stained sections of cardiac tissue were analyzed to examine capillaries in the infarcted area and its periphery. Figure 19F (Red arrow). The capillary density in the infarct area was similar in the MYDGF-164 group and the tirofiban (positive control) group. Figure 19G MYDGF-164 treatment significantly increased capillary density in the infarct margin area. Figure 19H One-way ANOVA and Tukey's multiple comparison test were used. ***P<0.001 vs. sham surgery group; #P<0.05 vs. model group.

[0319] On day 8, the survival rates of different groups were also calculated. Figure 20 Compared with the model group and the positive control group, the MYDGF-164 group had the highest survival rate. This result is consistent with the overall infarct area data, in which the MYDGF-164 group also had the lowest myocardial infarction area. The results indicate that MYDGF-164 can significantly reduce mortality in patients with myocardial infarction.

[0320] Example 7: MYDGF-164 fusion protein reduces adenine-induced kidney damage

[0321] To investigate the protective effect of MYDGF-164 against kidney injury, Wistar rats were randomly divided into experimental groups. The experimental groups received oral administration of 200 mg / kg / day of adenine for 4 weeks, followed by gavage with PBS or 675 mg / kg of okra, or subcutaneous injection of 0.7 mg / kg / day of MYDGF-164 for 1 week, followed by two weeks of 0.7 mg / kg / 2 days. As a negative control, one group of rats was fed PBS throughout the experiment. During the treatment period, serum samples were collected weekly to measure creatinine and urea levels according to the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute). The experimental protocol is as follows: Figure 21 As shown.

[0322] After treatment, animals were euthanized and kidneys were weighed. Kidney tissue was fixed in 4% paraformaldehyde for 24 hours and then embedded in paraffin. Hematoxylin-eosin (H&E) staining was performed according to standard procedures (Bio-year Tech). The degree of kidney lesions was determined using the 5-point method. For immunohistochemical (IHC) staining, sections were incubated overnight with anti-KI-67 (GB14102: Servicebio), anti-KIM-1 (BA3537; BASTER), and anti-rat endothelial cell antigen-1 (ab9774; Abcam). The corresponding HRP-labeled secondary antibodies were then bound to the sections, followed by incubation with DAB solution and nuclear staining with hematoxylin. TUNEL signals were stained according to the manufacturer's instructions (Beyotime, Beijing, China).

[0323] Serum blood urea nitrogen and creatinine are both serum markers of kidney function. Their accumulation in serum indicates impaired kidney function, such as adenine-induced tissue damage. Figure 22 Serum urea nitrogen and creatinine were measured in each group from week 4 to week 7. Compared with the control group fed only PBS (4 weeks), the model group developed severe renal insufficiency after 4 weeks of adenine administration, as evidenced by significantly elevated serum creatinine and urea nitrogen levels (p<0.001).

[0324] Simultaneously, one week after subcutaneous injection of MYDGF-164 protein, serum creatinine and blood urea nitrogen levels (at week 5) significantly decreased, showing no significant difference compared to the control group. The efficacy of the MYDGF-164 treatment group was comparable to that of the okra gavage group (weeks 5, 6, and 7). One week after treatment, MYDGF-164 significantly reduced serum urea nitrogen levels, while okra did not show a significant reduction, suggesting that MYDGF-164 is more effective than okra in treating kidney injury. Two weeks after medication, serum creatinine (week 6) and urea (week 7) decreased to levels indistinguishable from the control group.

[0325] After the treatment plan was completed, all animals were euthanized. Histopathological examination showed that the kidneys in the model group were pale, and relative kidney weight (mg / g body weight) analysis showed severe kidney hypertrophy. Figure 23 A). Compared with the model group, the relative kidney weight was significantly reduced after treatment with MYDGIF-164 or Abelmoschus manihot (Abelmoschus manihot). Figure 23 B) indicates that MYDGF-164 protein and okra protect the kidneys from adenine damage. A five-point method was used to assess the degree of renal tissue damage. Data showed that after MYDGF-164 treatment, renal tubular necrosis ( Figure 23 C) Glomerular atrophy ( Figure 23 D) Inflammation Figure 23 E) All were reduced compared to the model group, and the degree of reduction was similar to that of the okra treatment group.

[0326] Notably, after MYDGF-164 treatment, the glomerular atrophy score recovered to the same level as the control group. Figure 23 D). However, MYDGF-164 and okra treatment did not improve renal tubular dilation following adenine-induced kidney injury (D). Figure 23 F) and pigmentation ( Figure 23 G).

[0327] In an adenine-induced kidney injury model, persistent epithelial cell death and subsequent proliferation are cellular processes of injury and repair. As part of the tissue repair process in chronic kidney disease, renal tubular interstitial fibrosis is associated with a reduction in peritubular capillaries (PTCs).

[0328] The effects of MYDGF-164 on apoptosis and proliferation of PTCs were observed. The levels of the biomarker kidney injury molecule-1 (KIM-1) were significantly reduced in the MYDGF-164 and Abelmoschus manihot treatment groups. Figure 24 A) indicates that renal tubular damage was effectively repaired after treatment with MYDGF-164 and Abelmoschus manihot.

[0329] To assess the effect of MYDGF-164 on PTCs, the status of PTCs was evaluated by RECA-1 staining. RECA-1 is a rat endothelial cell marker that can visualize vascular structures in histological analysis. In the model group, many PTCs showed abnormal vascular systems (i.e., luminal collapse). Figure 24 B). In contrast, the RECA-1 stained area in the MYDGF-164 group accounted for 2.46% of the total examined tissue area, which was significantly higher than that in the model group and the okra group. Figure 24 B). This result indicates that MYDGF-164 can effectively restore or prevent damage to PTCs after adenine withdrawal in a kidney injury model.

[0330] Cell proliferation is an important process in the recovery process after kidney injury. Kidney tissue sections were stained with Ki-67 to observe cell proliferation. After treatment with MYDGF-164 and okra, the number of Ki-67-positive cells in the kidney tissue increased (…). Figure 24 C). In addition, MYDGF-164 treatment increased cell proliferation in renal tubules and peritubular capillaries, suggesting that MYDGF-164 may promote the proliferation of renal tubular epithelial cells and interstitial capillary epithelial cells.

[0331] Terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling (TUNNEL) is commonly used to identify apoptotic cells in tissues. Following MYDGF-164 treatment, the frequency of apoptotic cells in kidney tissue observed by TUNEL staining was significantly reduced. Figure 24 D) indicates its protective effect against apoptosis / cell death.

[0332] Example 8: MYDGF-164 fusion protein-driven HUVEC proliferation is associated with MAPK and cyclin D1 activation.

[0333] Early results indicated that native MYDGF activates the MAPK1 / 3 pathway, stimulates AKT phosphorylation, and increases cyclin D1 expression in HUVECs. To assess whether the fusion of the CD164 mucin II domain alters MYDGF function, human umbilical vein endothelial cells (PromoCell, Heidelberg, Germany) were cultured in endothelial cell growth medium with endothelial cell growth supplement (Supplement Mix, PromoCell) at 37°C and 5% CO2. Cells were seeded at 3000 cells / well in 96-well plates and cultured for 24 hours, followed by stimulation with different concentrations of MYDGF-164 for 24 or 48 hours. Cell proliferation was quantified using a CCK-8 assay (Yeasen). When adjusting for quality differences, there was no difference in proliferative activity between native MYDGF and the MYDGF-164 fusion protein. Figure 25A In Western spectroscopy analysis of HUVECs treated with either MYDGF (300 ng / mL) or MYDGF-164 (1 μg / mL) fusion protein, the phosphorylation kinetics of MAPK1 / 3 were identical in both treatments. Figure 25B In addition, MYDGF-164 also enhanced the expression of cyclin D1. Figure 25CIn short, the MYDGF-164 fusion is most likely to maintain the cellular signaling function of MYDGF. The antibodies used for Western blot analysis were p-MAPK1 / 3 (Y204 / T202, CST, Cat#4370), total MAPK1 / 3 (CST, Cat#4695), cyclin D1 (CST, Cat#2922), and β-actin (Abways, Cat#AB0035).

[0334] Example 9: Compared with nicorandil, the in vitro MYDGF-164 fusion protein can drive HUVEC migration.

[0335] The antianginal drug nicorandil has been clinically used to treat injuries resulting from coronary angioplasty or reperfusion after thrombolysis. The protective effect of nicorandil is attributed to the opening of adenosine triphosphate-sensitive potassium (KATP) channels, and possibly the effect of nitric oxide produced after hydrolysis. It remains unclear whether nicorandil promotes endothelial cell migration in response to angiogenic stimuli via nitric oxide. We determined the migration behavior of HUVECs in the presence of MYDGF-164 and nicorandil in a scratch repair assay. Closure of the monolayer endothelial cells via migration was enhanced by dose-dependent addition of MYDGF-164. Figure 26A B), while adding nicorandil had no effect on migration. Figure 26C Therefore, MYDGF-164 can enhance endothelial cell migration, which is an alternative tissue repair mechanism not present when nicorandil is used.

[0336] Example 10: Compared with nicorandil, the MYDGF-164 fusion protein is superior in promoting tube formation using HUVECs.

[0337] Nitric oxide has been shown to participate in angiogenesis processes involving endothelial cells. Endothelial cell tube formation is a characteristic of angiogenic potential, and MYDGF-164 promotes tube formation compared to nitric oxide (…). Figure 27A (B) VEGFA was used as a positive control to promote tube formation. MYDGF-164 exhibited dose-dependent activation of tube formation similar to VEGFA. In contrast, endothelial cells treated with the highest concentration of nicorandil showed only mild tube formation.

[0338] Example 11: Purified FGF21-164 fusion protein

[0339] The FGF21-164 fusion protein was stably expressed in 293F cells, and the secreted protein was purified from conditioned medium. The purified protein was analyzed by SDS-PAGE and Western blot analysis. Figure 28For FGF21-164, the predicted molecular weight of the unglycosylated mature peptide is 24.3 kDa. Both SDS-PAGE and Western spectroscopy analyses showed that FGF21-164 migrated at approximately 52 kDa, indicating that fusion with the CD164 mucin domain resulted in an additional mass between 20 and 30 kDa.

[0340] Example 12: MALDI-TOF mass of purified FGF21-164 fusion protein

[0341] To more accurately determine the molecular weight of the FGF21-164 fusion protein, MALDI-TOF analysis was performed on the purified protein. Although the predicted molecular weight for the unglycosylated mature peptide FGF21-164 was 24.3 kDa, the MALDI-TOF data ( Figure 29 The results showed that the mass of the single-charged FGF21-164 protein was 42751.5 Daltons, and the mass of the double-charged protein was 21601.173 Daltons. Similarly, in the MYDGF-164 fusion, the increased mass is likely due to glycosylation of the CD164 mucin domain portion during the fusion.

[0342] Example 13: SEC analysis of purified FGF21-164 fusion protein

[0343] SEC analysis remains a method for determining protein hydrodynamic behavior. The FGF21-164 fusion protein exhibited an apparent molecular weight comparable to an 88 kDa globular protein in phosphate buffer. Figure 30A -C). This observation suggests that the mucin domain of CD164 primarily contributes to the hydrodynamic properties of the FGF21-164 fusion protein. Due to the brush-like conformation of the mucin domain, the FGF21-164 fusion protein is unlikely to fold into a globular structure. However, it is believed that, similar to Fc-fusion proteins or PEGylated proteins, the increased hydrodynamic radius of FGF21-164 should decrease glomerular filtration rate, thereby increasing the half-life of the fusion protein in vivo.

[0344] Example 14: Glycan analysis of purified FGF21-164 fusion protein

[0345] N-linked glycans were further analyzed by PNGase treatment and 2-AB labeling, followed by UPLC analysis. Figure 31A The dominant glycan forms were sialylated biantennary and triantennary glycans eluted at 6.87, 7.39, and 8.24 min. Therefore, the relatively uniform biantennary glycan structure observed was consistent with Coomassie brilliant blue staining data. Figure 28 This further demonstrates that the N-linked glycosylation of the CD164 mucin domain is highly uniform. O-linked glycans were also found. Figure 31B ).

[0346] Example 15: Pharmacokinetic analysis of purified FGF21-164 fusion protein

[0347] The mean serum half-life of FGF21 protein was previously described as 30 minutes. To determine whether fusion with the CD164 mucin domain prolongs the serum half-life of FGF21, the serum concentration of FGF21-164 was quantified using LC-MS after intravenous injection into four C57 / BL6 mice. Figure 32 The pharmacokinetic parameters of FGF21-164 were analyzed using a non-compartmental model (Table 2). The terminal half-life of the FGF21-164 fusion protein was determined to be 2.60 ± 0.335 h, the clearance rate (CL) was 21.9 ± 1.22 mL / h / kg, and the stable volume of distribution (Vss) was 64.2 ± 1.78 mL / kg.

[0348] Table 2. FGF21-164 PK Parameters

[0349]

[0350]

[0351] The concentration of mouse endogenous YLY peptide is approximately 50 pg / mL to 2 ng / mL, which is far below the lower limit of quantification of 4.00 μg / mL.

[0352] Example 15: Glucose uptake stimulated by purified FGF21-164 fusion protein

[0353] FGF21 is known to bind to FGFR1 and Klotho, and to act as an agonist to stimulate glucose uptake in adipocytes. The dose-dependent stimulation of glucose uptake in 3T3-L1 adipocytes by the FGF21-FGF21-164 fusion protein is shown to be... Figure 33 ).

[0354] Example 15: Purified FSH-164 fusion protein

[0355] The FSH-164 fusion protein was stably expressed in CHO cells. The secreted protein was purified from conditioned medium, and the purified protein was analyzed by SDS-PAGE and Western blot analysis (Figure 35). The predicted molecular weight of the mature heterodimer FSH, including glycosylation, was approximately 45 kDa. Both SDS-PAGE and Western blot analyses showed that FSH-164 migrated to approximately 70 kDa, indicating that the fusion with the CD164 mucin domain resulted in an additional mass between 20 and 30 kDa.

[0356] Example 16: The purified FSH-164 fusion protein is active in stimulating progesterone biosynthesis.

[0357] FSH stimulates the synthesis and secretion of progesterone in human granulocytes without causing luteinization. The KGN cell line, a granuloid tumor cell line, can be used to test in vitro FSH function. The purified FSH-164 fusion protein was tested on KGN cells. Figure 36 FSH-164 stimulated progesterone biosynthesis in KGN cells, with an EC50 of 6.3 nM, similar to FSH (EC50 = 5.8) in the same assay.

[0358] Example 17: The purified FSH-164 fusion protein exhibited prolonged PK in vivo.

[0359] Compared to recombinant FSH, the FSH164 fusion protein clearly showed a prolonged in vivo half-life. Figure 37 The half-life of purified FSH164 is approximately 26.7 hours, compared to 10 hours for recombinant FSH (Table 3). Theoretically, this 2.5-fold increase in the FSH half-life is sufficient to support a single-dose treatment regimen per ovulation cycle.

[0360] Table 3. Summary of PK data for FSH164

[0361]

Claims

1. A fusion protein comprising: (1) a polypeptide with the amino acid sequence shown in SEQ ID NO: 2, and (2) a heteropeptide, wherein the heteropeptide is follicle-stimulating hormone (FSH), the follicle-stimulating hormone being FSHβ, wherein (1) is located at the C-terminus of (2); the amino acid sequence of the fusion protein is shown in SEQ ID NO: 8; the fusion protein is O- and / or N-linked glycosylation, the fusion protein is sialylated; and the serum or circulating half-life of the FSH is at least 50% shorter than the half-life of the fusion protein.

2. A polynucleotide encoding the fusion protein of claim 1.

3. A vector comprising the polynucleotide of claim 2.

4. The carrier as described in claim 3, characterized in that, The carrier is an expression carrier.

5. The carrier as described in claim 3, characterized in that, The vector is a plasmid.

6. A host cell comprising the fusion protein of claim 1, the polynucleotide of claim 2, or the vector of any one of claims 3-5.

7. The host cell as described in claim 6, wherein it is a tissue culture cell.

8. The host cell as described in claim 6 or 7, characterized in that, The host cells are either CHO cells or HEK293 cells.

9. A pharmaceutical composition, characterized in that, The invention comprises a therapeutically effective amount of the fusion protein of claim 1, the polynucleotide of claim 2, or the carrier of any one of claims 3-5, and a pharmaceutically acceptable carrier or excipient.

10. The pharmaceutical composition according to claim 9, characterized in that, The drug combination is formulated for subcutaneous injection.

Images