A collagen peptide capable of effectively delaying aging and a preparation method and application thereof

Abstract

The application discloses a ternary fusion polypeptide Col-TF-1 targeting senescent cells, a matching monoclonal antibody mAb SC-1, and an anti-aging composition containing both and application. The fusion polypeptide is composed of a targeting peptide targeting a uPAR receptor on the surface of senescent cells, a collagen active peptide promoting collagen synthesis, and an immunomodulatory peptide activating NK cells through a flexible linker GGGGS, and the amino acid sequence is shown as SEQ ID NO. 1. The matching antibody mAb SC-1 is reconstructed by humanizing the CDR region and designed with a pH-sensitive histidine switch, the affinity is improved by 5 times in a slightly acidic environment, and the ADCC effect is enhanced through a fucose-deficient host cell. Experiments show that the combination of the fusion polypeptide and the antibody can make the clearance rate of senescent cells reach 45.2%, and the collagen density is improved by 83.5%, which is significantly better than the effect of a single drug. The composition provided by the application can be applied to the fields of cosmetics, medical devices, drugs and functional foods, and provides a new strategy for the treatment of aging-related diseases and the development of anti-aging products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering and anti-aging technology, and specifically relates to a collagen peptide capable of effectively delaying aging and a preparation method and application thereof. BACKGROUND

[0002] In the field of biomedical anti-aging, collagen peptides activate the TGF-β / Smad signaling pathway, stimulate fibroblasts to synthesize type I and type III collagen, effectively improve skin elasticity and dermal structure, and become a classic anti-aging ingredient. As shown in patent CN117402929B, small molecular peptide segments (molecular weight <3000 Da) obtained by enzymatic hydrolysis of fish scale collagen can significantly increase type I collagen expression by 35%. However, traditional collagen peptides have significant technical defects: first, they lack the ability to recognize specific markers on the surface of senescent cells (SnCs), and cannot remove senescent cells that secrete IL-6, MMPs and other SASP factors and continuously degrade collagen; second, large molecular peptide segments (>5000 Da) have very low transdermal efficiency. According to a 2023 study in Journal of Controlled Release, the transdermal penetration rate is less than 5%; third, the function is single, only acting on collagen synthesis, and it is difficult to block the formation of pro-aging microenvironment.

[0003] In recent years, "Senolytic therapy" has become a hot spot in anti-aging research. The E16-uPA24 chimeric peptide disclosed in Nature Aging in 2024 can induce senescent cell apoptosis by targeting uPAR receptors, but does not have tissue repair and regeneration function; patent CN117126285A uses a combination of collagen peptides and PTEN antibodies, which has a production cost of $200 / g and needs to be administered separately, limiting clinical application.

[0004] Currently, there is no single molecule that can simultaneously achieve precise targeting and removal of senescent cells and simultaneously promote efficient collagen regeneration. Therefore, it is urgent to develop an innovative molecule with dual functions and supporting technology to break through the limitations of existing anti-aging technology. SUMMARY

[0005] The purpose of the present application is to provide a collagen peptide capable of effectively delaying aging and a preparation method and application thereof.

[0006] Therefore, the present application provides a ternary fusion polypeptide, which is Col-TF-1, wherein the amino acid sequence of Col-TF-1 is shown in SEQ ID NO. 1; and the nucleotide sequence of Col-TF-1 after codon optimization is shown in SEQ ID NO. 2.

[0007] In one aspect, the present application also provides an anti-aging composition comprising the triple fusion polypeptide Col-TF-1 and the monoclonal antibody mAb SC-1, and a pharmaceutically or cosmetically acceptable carrier.

[0008] Preferably, the monoclonal antibody mAb SC-1 of the present application specifically binds to the uPAR receptor on the surface of senescent cells, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 4; the histidine at position 310 of the CH2 domain of the monoclonal antibody mAb SC-1 forms a pH-sensitive affinity modulation module; and the fucose modification of the Fc fragment of the monoclonal antibody mAb SC-1 is deleted by knocking out the FUT8 gene of the host cell.

[0009] Preferably, the concentration of the triple fusion polypeptide Col-TF-1 of the present application is 0.1-2%, and the concentration of the monoclonal antibody mAb SC-1 is 0.1-1 mg / g, and the dosage form includes cream, essence, wound repair dressing, inhalation powder, or enteric-coated capsule.

[0010] In one aspect, the present application also provides the use of the triple fusion polypeptide Col-TF-1 in the preparation of an anti-aging composition.

[0011] The present application has the following advantages: first, the fusion structure is novel, and the target peptide, collagen peptide and immune-regulating peptide are fused for the first time, which solves the problem that traditional collagen peptides cannot remove senescent cells; second, the antibody mAb SC-1 has pH-sensitive properties, and the affinity can be increased by 5 times in the slightly acidic environment of senescent tissues; third, the synergistic effect is obvious, and the removal rate of senescent cells is increased to 45.2% when the fusion peptide is combined with the antibody, which is much higher than 22.7% when the peptide is used alone; fourth, the process is innovative, and the enzymatic efficiency is increased by 40% under red light irradiation (630 nm), and the production cycle is shortened to 1 / 3 of the traditional method; fifth, the safety advantage is outstanding, and the rabbit skin irritation test proves that it has no irritation, and the irritation index is 0.21, which is better than 2.85 of tretinoin.

[0012] The fusion peptide and antibody of the present application have wide application prospects and can be applied in multiple fields: in the field of cosmetics, it can be used in anti-aging essence, facial mask and other products, and the concentration range is 0.1-2%; in the field of medical devices, it can be made into wound repair dressing and used in combination with red light irradiation equipment; in the field of drug development, it can be used in preparations for treating pulmonary / liver fibrosis, such as inhalation powder; and in the field of functional food, it can be made into oral anti-aging capsules with enteric-coated. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1SDS-PAGE detection results of the ternary fusion polypeptide Col-TF-1.

[0014] Figure 2 SDS-PAGE detection results of the monoclonal antibody (mAb SC-1), wherein 1 is the mAb SC-1.

[0015] Figure 3 SEC-HPLC detection results of the monoclonal antibody (mAb SC-1). DETAILED DESCRIPTION

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0017] Unless otherwise specified, the reagents, methods, and equipment employed in the present application are the conventional reagents, methods, and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0018] Example 1: Design of the ternary fusion polypeptide Col-TF-1

[0019] In the field of research and treatment of aging-related diseases, how to achieve precise action on senescent cells and promote tissue repair has always been a key problem. There are specific receptors on the surface of senescent cells, such as uPAR receptors, and if these receptors can be specifically combined, precise targeting of senescent cells can be achieved; at the same time, promoting collagen synthesis to maintain the structure and function of tissues, and activating the immune system to remove senescent cells are important directions in anti-aging research. Based on this, the present application designs a ternary fusion polypeptide Col-TF-1 with multiple functions to solve the problems existing in the prior art.

[0020] 1. Structure of the ternary fusion polypeptide Col-TF-1

[0021] The ternary fusion polypeptide Col-TF-1 designed by the present application has the general structure of: targeting peptide-linker-collagen active peptide-linker-immunomodulatory peptide. This structure realizes the synergistic effect of multiple biological functions by reasonably combining different functional peptide segments. Details of each component are shown in Table 1, and the amino acid sequence of the Col-TF-1 designed in this way is shown in SEQ ID NO. 1.

[0022] Table 1 Composition and function of the fusion polypeptide Col-TF-1

[0023]

[0024] 2. Function implementation of each part

[0025] (1) Precise targeting: Targeting peptide uPA24 can specifically recognize and bind to the uPAR receptor on the surface of senescent cells, thereby achieving precise positioning of senescent cells and laying the foundation for subsequent effects on senescent cells. This specific binding reduces the impact on normal cells, improving the accuracy and safety of the effect.

[0026] (2) Collagen regeneration: Collagen active peptide CP3 can stimulate the proliferation of fibroblasts, which are the main cells that synthesize collagen. The proliferation of fibroblasts can promote collagen synthesis, helping to maintain the structure and function of skin, connective tissue, etc., and improve the problem of collagen loss caused by aging.

[0027] (3) Immune activation: Immune regulatory peptide 16E activates the mGluR5 receptor on the surface of NK cells, enhancing the activity of NK cells. NK cells are an important component of the immune system that eliminates abnormal cells (such as senescent cells), and the enhancement of their activity helps the body more effectively identify and eliminate senescent cells.

[0028] (4) Synergistic anti-aging: The three functional peptide segments are connected by a linker, and the linker GGGGS has good flexibility, which can maintain the flexibility of each domain, allowing the functions of the three peptide segments to be synergistically exerted. In the process of eliminating senescent cells, it promotes tissue repair and achieves a multi-dimensional anti-aging effect.

[0029] 3. Collagen active peptide (CP3) is obtained through bioinformatics screening, the specific process is as follows:

[0030] (1) Screening: First, from the fish scale collagen peptide library, with high cell activity as the screening standard, the peptide segment with potential function of promoting collagen synthesis is preliminarily screened. The fish scale collagen peptide library contains a variety of collagen peptides with different sequences, providing rich materials for screening.

[0031] (2) Analysis of molecular docking technology: The molecular docking technology is used to analyze the binding energy of the preliminarily screened peptide segment and the key receptor DDR2 of collagen synthesis. Molecular docking technology is a method of simulating the interaction between molecules, which can predict the affinity of the peptide segment and the receptor.

[0032] (3) Optimization to obtain the optimal sequence: Through the analysis of the binding energy, the 14-peptide sequence with the optimal binding energy (-10.2 kcal / mol) to DDR2 is screened, which is the CP3 peptide segment. The lower the binding energy, the more stable the binding of the peptide segment and the receptor, and the stronger the activity, thereby ensuring that the CP3 peptide segment can effectively stimulate collagen synthesis.

[0033] In summary, the three fusion polypeptide Col-TF-1 designed by the application realizes precise targeting of senescent cells, promotes collagen regeneration, and activates the immune system through rational structural design and the synergistic effect of each functional peptide segment, thereby providing a new idea and method for anti-aging research and treatment of related diseases.

[0034] Example 2: Preparation of the three fusion polypeptide Col-TF-1

[0035] 1. Plasmid construction

[0036] (1) Sequence design: Col-TF-1 contains 2 GGGGS connecting peptides, and a 6xHis tag is added at the C-terminus. The coding sequence is optimized for E. coli codons, as shown in SEQ ID NO. 2.

[0037] (2) Vector construction: The upstream and downstream primers contain BamH I / Xho I enzyme digestion sites. After PCR amplification, they are ligated with the pET-28a(+) vector, transformed into E. coli BL21(DE3), and positive clones are selected (sequence verified to be correct).

[0038] 2. Fermentation of co-expression

[0039] (1) Seed culture: a single colony was inoculated into TB medium (50mL, kanamycin 50ug / mL + chloramphenicol 34ug / mL), and cultured at 30℃, 220rpm until OD 600 =1.0, and then inoculated into a 5L fermenter (3L TB medium) at a 2% inoculation amount.

[0040] (2) Induction of expression: culture at 30℃ until OD 600 =0.6, add 0.1mM IPTG, maintain at 30℃ for 4h, then cool to 25℃, start 630nm red light irradiation (2J / cm 2 , 10min), continue to induce for 16h.

[0041] 3. Purification process

[0042] (1) Bacterial treatment: centrifuge at 8000rpm to collect the bacterial cells (wet weight 23g / L), ultrasonic breakage (300W, 3s / 3s, 100 times), centrifuge at 12000rpm to obtain the supernatant (soluble protein concentration 1.3mg / mL).

[0043] (2) Two-step chromatography purification

[0044] Nickel column affinity: HisTrap HP column (5mL), equilibration buffer (20mM Tris, 500mM NaCl, 10mM imidazole, pH8.0), 50mM imidazole to wash the impurities, 500mM imidazole for elution (peak concentration 3.8mg / mL, purity 82%).

[0045] Molecular sieve: Superdex 75 column (PBS equilibration), 500 μL sample loading, 0.5 mL / min collection of main peak (96% purity, SDS-PAGE showed Figure 1 ) a single band at about 7 kDa, consistent with the theoretical value).

[0046] 4. Freeze-drying

[0047] Purified liquid -80℃ pre-freezing 24h, vacuum freeze-drying (cold trap -50℃, vacuum degree <10 Pa, 48h), white powder 38.2mg (yield about 88%), stored at -80℃ for standby.

[0048] 5. Summary

[0049] By codon optimization and sequence design, the soluble expression of the target polypeptide (containing GGGGS connecting peptide and 16E repeat sequence) is realized. The segmented temperature control (30℃→25℃) combined with the molecular chaperone co-expression improves the protein folding efficiency, and the red light irradiation enhances the enzymatic activity. Two-step chromatography purification obtains high-purity product (96%), and the freeze-drying yield is 88%, and the process is stable and can be scaled up. It provides a good foundation for subsequent scale application.

[0050] Example 3: Design and preparation of monoclonal antibody (mAb SC-1)

[0051] The present inventors analyzed the monoclonal antibodies (containing monoclonal antibodies 1-10 screened by the present inventors using hybridoma cell technology) that have been developed to specifically recognize the surface marker uPAR receptor of senescent cells, and on this basis, they were modified and engineered to further improve their affinity and specificity, thereby achieving enhanced clearance of senescent cells.

[0052] I. Antibody molecule design and engineering

[0053] (I) Functional domain directed modification

[0054] 1. CDR region humanization reconstruction

[0055] According to the CDR sequence, Rosetta software was used to simulate the conformation of CDR-H3 loop region, and mouse CDR region was grafted into human IgG1 framework region (FR). In order to maintain antigen binding activity, key contact residues were specifically retained, heavy chain Y95, L100, light chain W32, Q91.

[0056] To achieve the response to the tumor microenvironment, a pH-sensitive histidine switch was introduced. A histidine (His) was inserted at position 310 of the CH2 domain of the heavy chain (IgG1 numbering system), and the Fc conformational change triggered by the protonation of His at the pH 6.5 of the tumor microenvironment was utilized. Crystal structure prediction showed that the displacement of the β2-β3 loop region was 0.8 nm, and accordingly, an affinity-enhancing module was designed.

[0057] The optimized monoclonal antibody was named mAb SC-1, and the amino acid sequences of the variable region of the heavy chain and the variable region of the light chain were shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

[0058] 2. Glycosylation engineering

[0059] To optimize the ADCC effect, the FUT8 gene of CHO-K1 cells was knocked out using CRISPR-Cas9 technology, targeting exon 6, causing an 8-bp deletion, thereby constructing a fucose-deficient host cell line. Through genotype verification, qPCR detection showed that the expression of FUT8 decreased by 98%, and lectin blotting confirmed that fucose modification was completely absent.

[0060] (II) Expression vector construction

[0061] After codon optimization of the heavy chain / light chain gene sequence in E. coli, the sequence was inserted into the pTT5 vector, which contains a CMV promoter and a hygromycin / pturomycin dual resistance gene. Sequencing verification showed that the sequence was 100% homologous to the designed sequence, and the open reading frame was correct. ORF verification was performed by Nde I / Xho I double enzyme digestion, and the verification was correct.

[0062] Four, engineering cell line construction and large-scale production

[0063] (I) Screening of stable expression strains

[0064] The target clone (SC-1-7) was transfected into fucose-deficient CHO-K1 cells, and the electric conversion parameters were 1800V, 20ms, and 2mm electric conversion cup. After 21 days of screening with puromycin (8μg / mL), the cells were cloned using the limiting dilution method, and a monoclonal cell line was obtained, in which >99% of the cells expressed anti-uPAR receptor antibody.

[0065] (II) Bioreactor culture

[0066] The culture medium was selected as serum-free medium SFM4CHO, with the addition of 4mM L-glutamine, and the inoculation density was 2×10 6 cells / mL.

[0067] Process control, temperature was controlled at 37°C for day 0-4, and adjusted to 36°C from day 5; pH was automatically adjusted by CO2 / NaOH, maintained at 7.0±0.1; dissolved oxygen was adjusted by pure oxygen / nitrogen, maintained at 30% air saturation.

[0068] Culture results were good, peak cell density reached 12x10 6 cells / mL, cell viability >95%, antibody expression was 1.8g / L (Bradford method), which was 50% higher than conventional CHO cells.

[0069] Five, antibody purification and quality control

[0070] (1) Three-step purification process

[0071] 1. Capture stage (Protein A affinity chromatography)

[0072] MabSelect Sure chromatography column (5mL, dynamic binding capacity 40mg / mL) was used. The process was as follows: first equilibrated with equilibration buffer (100mM NaH2PO4, pH7.0), then sample was loaded at a flow rate of 1mL / min, followed by 10 column volumes of equilibration buffer, and finally eluted with 0.1M glycine-HCl (pH3.0), and the elution peak with A280>1.0 was collected. The yield was 85%, and the purity was >95% (SDS-PAGE results as shown in Figure 2 ).

[0073] 2. Purification stage (cation exchange chromatography)

[0074] SP Sepharose FF chromatography column (5mL, binding capacity 60mg / mL at pH5.5) was used. The conditions were as follows: equilibration buffer (20mM NaAc, pH5.5), linear gradient 0-1M NaCl elution (10CV), main peak was collected, and SEC-HPLC detection showed that the purity was >98%. Figure 3 ).

[0075] 3. Virus safety treatment

[0076] 0.22μm sterilization filter (Millipore Express SHC) was performed, and 60℃ / 10h pasteurization was performed. Mycoplasma detection was not detected by PCR method, and endotoxin was <0.05EU / μg.

[0077] Table 2 Summary of monoclonal antibody (mAb SC-1) detection results

[0078]

[0079] Six, experimental results analysis

[0080] 1. Targeting mechanism verification

[0081] His-switch design through pH-dependent conformational change, MD simulation shows the RMSD change of CH2 domain The binding free energy (ΔG) of antibody and uPAR receptor in acidic environment is reduced from -8.2 kcal / mol to -9.8 kcal / mol, which is consistent with the theoretical expectation of the enhancement of electrostatic interaction.

[0082] Fucose knockout eliminates the steric hindrance of FcγRIIIa binding, and the crystal structure shows the distance between fucose and receptor Asn297 sugar chain The degranulation rate (CD107a+) of ADCC effector cells (NK cells) is increased by 2.3 times, and the secretion of effector molecules IFN-γ is increased by 1.8 times.

[0083] 2. Screening system efficiency improvement

[0084] Flow cytometry sorting combined with single cell ELISA, the positive clone screening accuracy is improved from 65% of traditional method to 92%, the key is to exclude non-specific binding "background clones", about 20% of ELISA positive wells are excluded due to flow cytometry negative.

[0085] 3. Production process engineering optimization

[0086] Temperature step control (37℃→36℃) reduces cell metabolic rate, glucose consumption rate from 0.8 mM / day to 0.5 mM / day, prolongs the exponential growth phase by 24 h, and directly improves antibody production by 30%.

[0087] Seven, summary

[0088] 1. Mechanism-oriented molecular modification

[0089] His-switch utilizes the pH gradient of tumor microenvironment to achieve dynamic regulation of affinity, and fucose knockout enhances effector function through glycosylation engineering, both based on structural biology and glycoimmunology principles.

[0090] 2. Integration of high-efficiency screening technology

[0091] Combining phenotypic screening with flow cytometry and molecular interaction analysis with BIAcore, a dual verification system from cell level to molecular level is established to ensure clone specificity and functional activity.

[0092] 3. Process control engineering

[0093] Optimization of bioreactor culture parameters (temperature, dissolved oxygen) followed the cell metabolic kinetics model, which broke through the antibody expression level of 1.8 g / L and reached the industrial production level.

[0094] Process verification showed that the coefficient of variation of key quality attributes (CQA) between batches was <3% (n=5), which laid a solid foundation for subsequent preclinical studies. It is recommended that future research focus on the synergistic mechanism of antibodies and fusion polypeptides, and further optimize the combination therapy regimen through in vivo pharmacokinetics and tissue distribution experiments.

[0095] Example 4: In vitro clearance of senescent cells experiment

[0096] I. Experimental materials and methods

[0097] 1. Cell model construction

[0098] Cell source: human foreskin fibroblasts (HFF-1) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and placed in a 37°C, 5% CO2 incubator.

[0099] Senescence induction: logarithmic growth phase cells were adjusted to a density of 1×10 5 cells / well were seeded in 6-well plates and cultured for 24 h to 80% confluence. Single 10Gy irradiation was performed using an X-ray irradiator (dose rate 1 Gy / min), and the cells were cultured for another 72 h after irradiation. The proportion of senescent cells was verified by SA-β-gal staining.

[0100] SA-β-gal staining steps: cells were washed with PBS twice, fixed with 4% paraformaldehyde for 10 min, and washed with PBS three times. Staining solution (containing 1 mg / mL X-gal, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, pH 6.0) was incubated at 37°C for 16 h (avoiding light). Under a light microscope, 10 fields of view (200x) were randomly selected, and the proportion of SA-β-gal positive cells (blue granular cells) was counted. The positive rate after induction was 87.3±2.1% (n=3), which met the experimental requirements.

[0101] 2. Experimental grouping and treatment, grouping design (n=6, 6 replicate wells per group), as shown in Table 3:

[0102] Table 3 Experimental grouping and treatment

[0103]

[0104] The maximum non-toxic concentration of peptide / antibody on HFF-1 cells was determined by CCK-8 method (polypeptide ≤ 2 mg / mL, antibody ≤ 20 μg / mL), and finally 1 mg / mL peptide and 10 μg / mL antibody were selected as working concentrations.

[0105] 3. Detection index and method

[0106] SA-β-gal positive rate: After 72 h of treatment, 200 cells were randomly counted in each group according to the above staining procedure, and the positive rate was calculated.

[0107] Apoptosis rate: Annexin V-FITC / PI apoptosis detection kit was used, and the steps were as follows: collect cells, wash with PBS for 2 times, resuspend with 100 μL binding buffer. Add 5 μL Annexin V-FITC and 5 μL PI, incubate at room temperature for 15 min in the dark. Flow cytometry detection, calculate the proportion of early apoptosis (Annexin V+ / PI-) + late apoptosis (Annexin V+ / PI+) cells.

[0108] IL-6 level: The cell culture supernatant was collected, and human IL-6 ELISA kit was used according to the instruction, detection wavelength was 450 nm, standard curve range was 15.6-1000 pg / mL, and sensitivity was <7 pg / mL.

[0109] 4. Statistical analysis

[0110] The data was expressed as "mean ± standard deviation (Mean ± SD)", and single factor analysis of variance (One-Way ANOVA) was performed by GraphPad Prism 9, Tukey post-hoc test was used for comparison between groups, and *P<0.05, **P<0.01 indicated significant difference.

[0111] II. Experimental results are shown in Table 4.

[0112] 1. Senescent cell clearance efficiency

[0113] The SA-β-gal positive rate of the control group (group A) was 85.2±3.1%, and the traditional collagen peptide (group B) was only slightly reduced to 79.5±2.8% (P>0.05 vs group A), indicating that the traditional peptide had no significant clearance effect.

[0114] When the fusion peptide Col-TF-1 (group C) and the antibody mAb SC-1 (group D) were used alone, the positive rates were reduced to 52.3±4.2% and 48.6±3.7% respectively (*P<0.05 vs group A), indicating that both could partially clear senescent cells.

[0115] The positive rate of the combination group (group E) was further reduced to 30.1 ± 2.9% (**P < 0.01 vs C / D group), and the clearance efficiency was increased by about 40% compared with single drug, showing significant synergistic effect.

[0116] 2. Apoptosis induction ability

[0117] The apoptosis rate of the control group was 3.2 ± 0.8%, and that of group B increased to 5.1 ± 1.2% (P > 0.05). The apoptosis rates of groups C and D were 22.7 ± 3.5% and 25.3 ± 2.9% respectively (*P < 0.05 vs group A). The apoptosis rate of the combination group (group E) was as high as 45.2 ± 4.1% (**P < 0.01 vs C / D group), suggesting that the combination can remove senescent cells by enhancing the apoptosis pathway.

[0118] 3. Inflammatory factor level

[0119] The IL-6 secreted by senescent cells was 1250 ± 85 pg / mL in group A, and decreased to 1102 ± 76 pg / mL in group B (P > 0.05). The IL-6 in groups C and D decreased to 623 ± 45 pg / mL and 587 ± 52 pg / mL respectively (*P < 0.05 vs group A), and that in the combination group (group E) further decreased to 298 ± 31 pg / mL (**P < 0.01 vs C / D group), indicating that the combination can effectively alleviate the senescence-associated secretory phenotype (SASP).

[0120] Table 4 Experimental results (n = 6)

[0121]

[0122]

[0123] *P < 0.05, **P < 0.01 compared with group A (ANOVA, F values were SA-β-gal: 128.5, P < 0.0001; apoptosis rate: 327.4, P < 0.0001; IL-6: 215.6, P < 0.0001).

[0124] **P < 0.01 compared with C / D group (Tukey test).

[0125] Three, Summary

[0126] The experiment confirmed that the combination of fusion polypeptide Col-TF-1 and monoclonal antibody mAb SC-1 can significantly enhance the ability of senescent cell clearance through the X-ray-induced HFF-1 senescent cell model. The mechanism may be related to the synergistic induction of apoptosis and inhibition of inflammatory factor secretion. Although the single drug group (C / D group) can partially remove senescent cells, the SA-β-gal positive rate, apoptosis rate and IL-6 level of the combination group (E group) are significantly better than those of the single drug (P<0.01), indicating that there is a synergistic effect between the two in terms of target recognition (antibody) and effector function (polypeptide). The results provide in vitro experimental evidence for the development of "dual functional molecules" for the treatment of senescence-related diseases.

[0127] Example 5: Anti-aging animal experiment of skin

[0128] I. Experimental materials and methods

[0129] 1. Animal model and grouping

[0130] Animal source: 18-month-old female C57BL / 6 mice (n=32) were raised in a constant temperature (22±2°C), constant humidity (55±5%) environment, 12h light / dark cycle, free access to food and water. The experimental groups are shown in Table 5:

[0131] Table 5 Experimental grouping (n=8 / group)

[0132]

[0133] Cream preparation: weigh 70g of vaseline and 30g of lanolin, heat to melt, cool to 40°C, then add drugs (Col-TF-1 powder or antibody solution), stir evenly to room temperature, store at 4°C for standby. Antibodies need to be sterilized by 0.22μm filter membrane, and the cream preparation process should be sterile.

[0134] 2. Dosing operation

[0135] Hair removal treatment: 24h before the start of the experiment, use hair removal cream (Veet) to remove the hair on the back of the mouse in an area of 2cm×2cm, avoiding damage to the skin.

[0136] Application method: every day at the same time, use a micropipette to suck 50μL of cream and evenly apply it to the depilated area, gently massage until absorbed, and continuously treat for 8 weeks.

[0137] 3. Detection index and method

[0138] Epidermis thickness: After the experiment, the dorsal skin tissue (including the application area) was taken, fixed with 4% paraformaldehyde for 24 h, paraffin-embedded, sectioned (5 pm thickness), and HE stained. Five non-overlapping fields (200x) were selected under an optical microscope (Olympus BX53), and the vertical distance from the basal layer to the stratum corneum was measured using ImageJ software, and the average value was taken.

[0139] Collagen density: Masson trichrome staining was performed on adjacent tissue sections as follows: deparaffinized to water, stained with Weigert iron hematoxylin dye for 5 min, and then washed with running water. Stained with acid magenta dye for 5 min, and then differentiated with 1% phosphomolybdic acid for 2 min. Stained with aniline blue dye for 5 min, dehydrated with 95% ethanol, and then mounted with neutral resin. Five fields (200x) were selected, and the percentage of blue collagen fiber area to total field area was calculated using ImageJ software.

[0140] Skin elasticity: A skin elasticity tester was used for detection, with the following parameter settings: sample suction depth of 0.5 mm, measurement time interval of 1 s, and repeated measurement of each sample for 3 times, and the average value was taken.

[0141] Trans-epidermal water loss (TEWL): A TEWL instrument was used for detection, and the mice were allowed to equilibrate at room temperature for 30 min before measurement. Each sample was measured 3 times, and the average value was taken.

[0142] 4. Statistical analysis

[0143] The data are expressed as "mean ± standard deviation (Mean ± SD)", and one-way ANOVA was performed using GraphPad Prism 9. Tukey's post-hoc test was used for comparison between groups, and *P < 0.05, **P < 0.01 indicate significant differences.

[0144] II. Experimental results are shown in Table 6.

[0145] 1. Improved epidermal structure: The epidermis thickness of the control group was 22.3 ± 1.5 pm, and the tretinoin group (35.2 ± 2.1 pm) and experimental group 1 (38.5 ± 1.8 pm) were significantly thicker (*P < 0.05). Experimental group 2 (45.7 ± 2.3 pm) was further increased by 18.7% compared to experimental group 1 (**P < 0.01), indicating that the combination can enhance the regenerative ability of the epidermis.

[0146] 2. Enhanced collagen synthesis: The collagen density of the control group was 41.2 ± 3.8%, and the tretinoin group was increased to 58.7 ± 4.2% (*P < 0.05). Experimental group 1 reached 62.3 ± 3.5% (*P < 0.05), and experimental group 2 reached 75.6 ± 4.1% (**P < 0.01 vs experimental group 1), which was increased by 83.5% compared to the control group, indicating that the combination significantly promotes the synthesis and deposition of type I collagen.

[0147] 3. Skin elasticity and barrier function: Skin elasticity was 55.3 ± 4.2% in the control group, significantly improved in the tretinoin group (68.7 ± 3.8%) and experimental group 1 (72.5 ± 4.1%) (*P < 0.05), and improved by 16.1% in experimental group 2 (84.2 ± 3.7%) (**P < 0.01) compared with experimental group 1, close to the level of young mice (about 85% in literature). Trans-epidermal water loss (TEWL) reflects barrier function, which was 18.7 ± 1.2 g / m 2 / h in the control group, reduced to 7.3 ± 0.6 g / m 2 / h in experimental group 2 (**P < 0.01 vs experimental group 1), 60.9% lower than the control group, indicating that the combination can effectively repair barrier damage caused by aging.

[0148] 4. Comparison with the positive control group: Experimental group 2 was superior to the tretinoin group in terms of the improvement in collagen density, skin elasticity and TEWL (e.g., collagen density increased by 28.8% compared with the tretinoin group), suggesting that the anti-aging effect of Col-TF-1 / mAb combination may be more significant.

[0149] Table 6 Experimental results

[0150]

[0151]

[0152] Compared with the control group: *P < 0.05, **P < 0.01 (ANOVA, F values were 185.6, P < 0.0001 for epidermal thickness; 152.3, P < 0.0001 for collagen density; 217.4, P < 0.0001 for skin elasticity; and 245.8, P < 0.0001 for TEWL, respectively). Compared with experimental group 1: **P < 0.01 (Tukey test).

[0153] Three, Summary

[0154] This experiment confirmed that Col-TF-1 and mAb SC-1 compound cream can significantly improve the skin aging phenotype, and its effect is better than that of single drug and commercial tretinoin cream. The specific performance is as follows:

[0155] 1. Synergistically promote epidermal thickening and collagen synthesis: The epidermal thickness and collagen density of the combination group were increased by 18.7% and 21.4% respectively compared with the single drug group, which may be related to the targeting delivery of polypeptides to senescent cells by antibodies and the enhancement of their collagen synthesis activity.

[0156] 2. Improve skin mechanical properties and barrier function: Skin elasticity is improved to 84.2%, and TEWL is reduced to 7.3 g / m 2 / h, suggesting that the combination can simultaneously repair structural damage and dysfunction.

[0157] 3. Mechanism of action: Based on the in vitro experimental results, the combination may act through a dual mechanism of "antibody recognizing senescent cell surface antigen + polypeptide inducing collagen synthesis / clearing senescent cells".

[0158] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A ternary fusion polypeptide, characterized in that, The ternary fusion polypeptide is Col-TF-1, wherein the amino acid sequence of Col-TF-1 is shown as SEQ ID NO.

1.

2. An anti-aging composition, characterized in that, The composition comprises the ternary fusion polypeptide Col-TF-1 of claim 1 and the monoclonal antibody mAb SC-1, and a pharmaceutically or cosmetically acceptable carrier; the monoclonal antibody mAb SC-1 specifically binds to the uPAR receptor on the surface of senescent cells, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 3 and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4; the heavy chain CH2 domain of the monoclonal antibody mAb SC-1 comprises a histidine at position 310 to form a pH-sensitive affinity adjustment module; and the Fc segment of the monoclonal antibody mAb SC-1 is deficient in fucose modification, which is achieved by knocking out the FUT8 gene of the host cell.

3. The composition of claim 2, wherein, The concentration of the ternary fusion polypeptide Col-TF-1 is 0.1-2%, and the concentration of the monoclonal antibody mAb SC-1 is 0.1-1 mg / g, and the dosage form is cream, essence, wound repair dressing, inhalation powder, or enteric capsule.

4. Use of the ternary fusion polypeptide Col-TF-1 of claim 1 in the preparation of an anti-aging drug.

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