Peptides and lysosomal targeting chimeras targeting IL17RA and their application in the treatment of psoriasis

Abstract

This invention discloses a polypeptide targeting IL-17RA and a lysosomal targeting chimera and their application in the treatment of psoriasis, relating to the biomedical field. The amino acid sequence of the polypeptide targeting IL-17RA has at least 80% identity with the sequence shown in SEQ ID NO:1 or 2. It has a high affinity for IL-17RA protein and can effectively block the binding of IL-17RA protein and IL-17A protein. The lysosomal targeting chimera constructed using this polypeptide molecule has excellent therapeutic effects and good biocompatibility for psoriasis, and has good application prospects in the treatment or adjuvant treatment of psoriasis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to a polypeptide and lysosomal targeting chimera that target IL17RA and its application in the treatment of psoriasis. Background Technology

[0002] Psoriasis is a common polygenic inherited skin disease, often manifesting as localized or widespread scaly erythematous patches or plaques. The pathogenesis of psoriasis is not yet fully understood, but it is believed to be caused by the combined effects of genetic and environmental factors.

[0003] There are several types of psoriasis, the most common being plaque psoriasis, followed by psoriatic arthritis, pustular psoriasis, and erythrodermic psoriasis. Symptoms of psoriasis are diverse, primarily including red plaques on the skin, usually covered with silvery-white scales; itching, burning, or pain; dry, cracked skin; and even bleeding. Some patients may also experience joint pain, swelling, and abnormalities in their fingernails and toenails.

[0004] Developing drugs that can effectively treat or improve psoriasis is one of the urgent problems that need to be solved today.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a polypeptide and lysosomal targeting chimera that target IL17RA and its application in the treatment of psoriasis.

[0007] This invention is implemented as follows:

[0008] In a first aspect, embodiments of the present invention provide a polypeptide whose amino acid sequence has at least 80% identity with the sequence shown in SEQ ID NO:1 or 2.

[0009] Secondly, embodiments of the present invention provide a lysosome-targeting chimera, comprising: an aptamer targeting the IL-17RA protein; the aptamer targeting the IL-17RA protein comprises the polypeptide described in the foregoing embodiments.

[0010] Thirdly, embodiments of the present invention provide a composition comprising: raw materials for preparing the lysosome-targeting chimera described in the foregoing embodiments.

[0011] Fourthly, embodiments of the present invention provide a method for preparing a lysosome-targeting chimera as described in the foregoing embodiments, comprising: preparing the chimera using the composition described in the foregoing embodiments.

[0012] Fifthly, embodiments of the present invention provide the use of the polypeptides as described in the foregoing embodiments or the lysosomal targeting chimeras as described in the foregoing embodiments in the preparation of products for the treatment or adjunctive treatment of psoriasis or for improving related clinical symptoms caused by psoriasis.

[0013] The present invention has the following beneficial effects:

[0014] This invention provides a novel polypeptide targeting IL-17RA, which has a high affinity for the IL-17RA protein and can effectively block the binding of IL-17RA and IL-17A proteins. The lysosomal targeting chimera constructed using this polypeptide molecule has excellent therapeutic effects and good biocompatibility for psoriasis, and has good application prospects in the treatment or adjuvant treatment of psoriasis. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a schematic diagram of the phage screening process;

[0017] Figure 2 Schematic diagram of the structure of the lysosome-targeting chimera TdlTac-17RA;

[0018] Figure 3 The results of molecular docking between CZG-7 and IL-17RA protein;

[0019] Figure 4 Western blot results for TdlTac-17RA-targeted degradation of IL-17RA protein;

[0020] Figure 5 ELISA results were used to verify the blocking ability of CZG-7;

[0021] Figure 6 The therapeutic effect of CZG-7 on psoriatic mice;

[0022] Figure 7 HE staining results of the back skin of CZG-7 mice after treatment with psoriasis;

[0023] Figure 8 HE staining results of livers in CZG-7 treated psoriasis mice;

[0024] Figure 9HE staining results of kidneys in CZG-7 treated psoriasis mice;

[0025] Figure 10 HE staining results of the back skin of psoriatic mice after treatment with TdlTac-17RA. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0027] Current research indicates that multiple immune signaling pathways are involved in the occurrence and exacerbation of psoriasis. Helper T (Th)1 cells and Th17 cells are considered to play important roles in the pathogenesis of psoriasis. Studies have found that the proliferation of Th1, Th17, and Th22 cells in psoriasis patients leads to the production of a large number of pro-inflammatory mediators, including tumor necrosis factor-α (TNF-α), interferon, and TNF-α. IFN-c and interleukin (IL)-6, interleukin (IL)-17, and interleukin (IL)-23, these pro-inflammatory mediators cause psoriatic lesions. Among them, the interleukin 17A (IL-17A) pathway is a key axis in the pathogenesis of psoriasis, stimulating the production of antimicrobial peptides, attracting inflammatory cell infiltration, and exacerbating the inflammatory response in psoriasis. Currently, biotherapy for psoriasis has achieved certain clinical efficacy. The UK Psoriasis Guidelines 2020 cover biologics already marketed or soon to be marketed in the UK, including TNF-α inhibitors, IL-12 / 23 (IL-12 / 23p40) inhibitors, IL-23 (IL-23p19) inhibitors, IL-17A inhibitors, and IL-17RA inhibitors. In psoriasis, the main receptor for IL-17A is IL-17RA. IL-17RA-mediated signaling molecules, such as TNF receptor-associated factor 6 (TRAF6), activate the NF-κB signaling pathway by binding to IL-17RA, ultimately triggering immune cell activation and inflammatory responses.

[0028] This application provides a novel polypeptide that has a high affinity for IL-17 RA protein, which can be used to construct a lysosomal targeting chimera to target and degrade IL-17 RA protein, thereby inhibiting the activation of the IL-17 pathway and suppressing the inflammatory response to treat psoriasis.

[0029] On the one hand, embodiments of the present invention provide a polypeptide whose amino acid sequence has at least 80% identity with the sequence shown in SEQ ID NO:1 or 2.

[0030] The "at least 80% identity" refers to sequence identity between any one or any two of the following: 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, and 100%. Identity refers to the proportion of two sequences where the nucleotide or amino acid residues at the same site are completely identical.

[0031] In some embodiments, the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1 or 2.

[0032] On the other hand, embodiments of the present invention provide a lysosome-targeting chimera, comprising: an aptamer targeting the IL-17RA protein; the aptamer targeting the IL-17RA protein comprises the polypeptide described in any of the foregoing embodiments.

[0033] In some embodiments, the lysosome-targeting chimera further includes an aptamer of a lysosome-targeting receptor linked to the aptamer targeting the IL-17RA protein.

[0034] In some embodiments, the molar ratio of the aptamer targeting the IL-17RA protein to the aptamer targeting the lysosomal receptor is 1-6:1-2. Specifically, this molar ratio can be any one or any two of the following: 1:1, 1:2, 2:1, 3:1, 4:1, 5:1, 6:1.

[0035] In some embodiments, the lysosomal receptor aptamer includes an aptamer targeting IGFIIR.

[0036] In some embodiments, the aptamer for the lysosomal targeting receptor comprises retinoic acid. Optionally, the retinoic acid is ATRA.

[0037] In some embodiments, the aptamer targeting the IL-17RA protein is directly linked to the aptamer targeting the lysosomal receptor or linked through a connection system.

[0038] In some embodiments, the connection system includes a biotin-avidin connection system.

[0039] In some embodiments, the connection system includes biotin, a linker, and avidin; the aptamer targeting the IL-17RA protein and the aptamer targeting the lysosomal receptor are respectively linked to biotin via the linker, and the biotin is capable of non-covalently binding to the avidin.

[0040] In some embodiments, the avidin includes any one or more of avidin and streptavidin.

[0041] In some embodiments, the connector includes a PEG.

[0042] In some embodiments, the molecular weight of the PEG is 1k to 10k, specifically any one or any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10k.

[0043] On the other hand, embodiments of the present invention provide a composition comprising: raw materials for preparing the lysosome-targeting chimera described in any of the foregoing embodiments.

[0044] In some embodiments, the raw materials include: the aptamer targeting the IL-17RA protein as described in any of the foregoing embodiments and the aptamer targeting the lysosomal receptor as described in any of the foregoing embodiments.

[0045] In some embodiments, the raw material further includes the connection system described in any of the foregoing embodiments.

[0046] On the other hand, embodiments of the present invention provide a method for preparing a lysosome-targeting chimera as described in any of the foregoing embodiments, which includes: preparing the chimera using the composition described in any of the foregoing embodiments.

[0047] In some embodiments, the preparation includes the following steps:

[0048] The aptamers targeting IL-17RA protein and the aptamers targeting the lysosomal receptor were coupled with biotin via linkers to obtain biotin-coupled aptamers targeting IL-17RA protein and biotin-coupled aptamers targeting the lysosomal receptor.

[0049] The biotin-conjugated aptamer targeting IL-17RA protein, the biotin-conjugated lysosomal receptor aptamer, and avidin are mixed to obtain a lysosomal targeting chimera.

[0050] The molar ratio of the biotin-conjugated aptamer targeting the IL-17RA protein, the biotin-conjugated lysosomal receptor-targeting aptamer, and avidin is 1~2:1~6:1~2. Specifically, this molar ratio can be any one or any two of the following: 1:1:1, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 2:3:1, and 1:3:2.

[0051] Furthermore, embodiments of the present invention also provide the use of the polypeptides as described in any of the foregoing embodiments or the lysosomal targeting chimeras as described in any of the foregoing embodiments in the preparation of products for the treatment or adjunctive treatment of psoriasis or for improving related clinical symptoms caused by psoriasis.

[0052] In some embodiments, the improvement of related clinical symptoms caused by psoriasis includes: improving any one or more of hepatocellular damage, renal cell damage, cell edema, and skin lesions caused by psoriasis.

[0053] In some embodiments, the product includes reagents or kits.

[0054] The term "treatment" in the text includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, achieving a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0055] For cancer, "treatment" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination thereof.

[0056] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0057] Example 1: Obtaining Target Peptides through Phage Screening

[0058] The procedure for obtaining target peptides through phage screening is as follows, which can be referred to... Figure 1 .

[0059] (1) Take two 96-well plates and designate them as well plates 1 and 2, respectively. Add 135 μl of 5 mg / ml BSA solution and 15 μl of 1 μg / μl IL-17 RA protein solution to each well of well plate 1. Then add 15 μL of 1 μg / μL IL-17 RA protein solution to each well of well plate 2. Wrap both well plates with a moistened paper towel and mix overnight at 4°C on a 3D shaker.

[0060] (2) The next day, discard the supernatant from well 1 and add 2 volumes of IL-17A (1 μg / μL solution). Wrap the well plate with a damp paper towel and mix in a 3D shaker at 4°C for 2 hours. Discard the supernatant, add 200 μL of TBST to each well, gently shake the well plate in a cross pattern, discard the TBST, and repeat three times.

[0061] (3) Take 1×10 11 One phage was placed into the well of a No. 1 plate containing protein, and mixed and shaken on a 3D shaker at room temperature for 1 hour.

[0062] (4) Discard the liquid in well 2, add 200 μL TBST to each well, gently shake the plate in a cross shape to discard the TBST, and repeat three times. Add the supernatant from well 1 to well 2. Mix and shake on a 3D shaker at room temperature for 1 h. Discard the supernatant, add 200 μL TBST to each well, gently shake the plate in a cross shape to discard the TBST, and repeat three times.

[0063] (5) Prepare two solutions in advance: 0.2 m clycine-HCl (pH=2.2) and 1 M Tris-HCl (pH=9.1), and filter them using a 0.22 μm filter membrane. Add 150 μL of 0.2 m clycine-HCl (pH=2.2) solution to well 2 and mix well on a 3D shaker at room temperature for 30 min. Add 150 μL of 1 M Tris-HCl (pH=9.1) solution to neutralize, resuspend, and collect the phages at the bottom.

[0064] (6) Add the phage solution from (5) to the liquid culture medium of ER2738 Escherichia coli and incubate overnight at 37°C and 200 rpm.

[0065] (7) The next day, centrifuge the solution at 5000 rpm for 20 min to remove E. coli. Take the supernatant into another conical flask, add 90 mL of NaCl-TEG8000 solution, shake to mix, and let stand at room temperature for 1 h.

[0066] (8) The purpose of centrifuging at 12000 rpm for 15 min in a centrifuge is to collect the phage. Discard the supernatant, resuspend it in TBS solution, collect it in a 2 mL centrifuge tube, and centrifuge at 8000 rpm for 10 min to remove E. coli again. Take the supernatant into a new centrifuge tube, add 300 μL NaCl-TEG8000 solution, mix well, and incubate on ice for 1 h.

[0067] (9) Then centrifuge at 13000 rpm for 10 min, discard the supernatant, add TBS to resuspend. Add 50% glycerol at a 1:1 volume ratio, and finally store in a -20℃ freezer.

[0068] Repeat the above steps 3-5 times, and finally collect the phage solution for sequencing to obtain its amino acid sequence QLRPLVR (SEQ ID NO:1). Sequence optimization was performed through molecular docking using computer simulations, and the final selected sequence was named CZG-7, with the specific amino acid sequence QLRPLFR (SEQ ID NO:2).

[0069] Example 2: Preparation method of lysosomal targeted chimera TdlTac-17RA

[0070] (1) Small retinoic acid molecules targeting IGFII R are conjugated with biotin:

[0071] One mmol of retinoic acid targeting IGFIIR was linked to one mmol of biotin-conjugated PEG 5k (Biotin-PEG 5k-NH3) via an amide bond through its own carboxyl group, thus synthesizing a biotin-conjugated aptamer targeting IGFIIR (V, Biotin-PEG 5k-Va).

[0072] (2) Biotin conjugation method for peptide aptamers targeting membrane proteins:

[0073] One mmol aptamer targeting the IL-17RA protein (peptide CZG-7) was linked to one mmol biotin-conjugated PEG 5k (Biotin-PEG 5k-COOH) via an amide bond through the N-terminal amino group, thus synthesizing a biotin-conjugated peptide ligand targeting a membrane protein (P, Biotin-PEG 5k-CZG-7).

[0074] (3) The preparation method of lysosomal targeting chimera that targets IL-17RA protein degradation includes:

[0075] The lysosome-targeting chimeric TdlTac-17RA was synthesized by mixing a biotin-conjugated aptamer targeting IGFIIR, a biotin-conjugated polypeptide ligand targeting a membrane protein, and streptavidin in a molar ratio of 1:3:1 and linking them through the biotin affinity system.

[0076] The structure of the lysosomal targeting chimeric TdlTac-17RA is as follows: Figure 2 As shown.

[0077] Molecular docking techniques were used to predict the binding posture of TdlTac-17RA to its receptor and to analyze affinity. AutoDock was used to perform molecular docking of TdlTac-17RA with IL-17RA / IL-17RC proteins to determine the binding site of TdlTac-17RA on the IL-17RA protein structure. The docking results were evaluated by analyzing the free function of TdlTac-17RA and IL-17RA proteins to determine the most likely binding site of TdlTac-17RA. AutoDock, a rapid energy assessment method combining calculated affinity potential grids and Lamarck genetic algorithms, can find the ligand binding site. The docking mode of TdlTac-17RA and IL-17RA proteins is shown below. Figure 3As shown, global docking of TdlTac-17RA and IL-17RA proteins is performed, ensuring the receptor protein is contained within a grid frame. The goal is to align the center of the grid frame with the center of the protein, allowing IL-17RA to maintain rigidity and the CZG-7 peptide to remain flexible. The number of independent docking runs for each simulation is set to 100, and all other docking parameters are set to their default values.

[0078] Example 3: Western blot analysis of the dual-targeting degradation ability of lysosomal-targeting chimeras for cell membrane proteins.

[0079] 1. Digest HaCat cells cultured in T25 flasks with EDTA-trypsin, transfer to 15ml centrifuge tubes, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-antibiotic mixture, and count cells using a cell counting chamber. Take a six-well plate, spread an appropriate amount of cell suspension in each well (2 × 10⁶ cells / well). 6 Collect a few cells, gently shake the well plate to distribute the cells evenly, and incubate at 37°C in a 5% CO2 incubator for 24 hours. After 24 hours, when the cells have adhered to the plate, begin drug administration.

[0080] 2. Take four 0.5 ml centrifuge tubes. Add 10 μL of 20 μM biotin-conjugated retinoic acid to each tube, followed by 30 μL of 20 μM biotin-conjugated CZG-7. Incubate at room temperature for 5 min. Add 10 μL of 20 μM s-avidin and immediately vortex to mix. Incubate at room temperature for 30 min to obtain the lysosome-targeting chimeric TdlTac-17RA. Add culture medium (without serum and antibiotics) to each centrifuge tube to a total volume of 1 ml. Discard the culture medium in the well plate, wash with PBS, then discard the PBS. Add the drug system sequentially. For the control group, add 1 ml of culture medium (without serum and antibiotics) directly. Gently shake the well plate to distribute the culture medium evenly. Incubate at 37°C and 5% CO2 for 10 h. Then add 1 ml of DMEM high-glucose medium (containing 10% FBS and 1% antibiotics) to each well and incubate for another 24 h. Collect cells and extract cell proteins.

[0081] 3. Discard the culture medium in the well plate, rinse with pre-cooled PBS, discard the PBS, and dissociate the cells from the bottom of the well plate using a cell scraper or cell dissociation buffer containing EDTA but not trypsin. Transfer the cells to 1.5 ml centrifuge tubes, centrifuge at 8000 rpm for 2 min to collect the cells, discard the supernatant, centrifuge at 8000 rpm for 30 sec, gently aspirate the supernatant with a 200 μL pipette, add cell lysis buffer (RIPA:PMSF = 100:1) according to the cell volume, vortex to disperse the cell pellet, place on ice for lysis, vortex once every 10 minutes, repeat 3 times. Centrifuge at 12000 rpm for 10 min. Then prepare the working solution, A solution: B solution = 50:1, take 4 new 1.5 ml centrifuge tubes, add 27 μL of water, 600 μL of working solution and 3 μL of supernatant (after centrifugation) to each centrifuge tube, vortex until homogeneous, and incubate at 37°C for 30 min. Take a new 96-well plate, add 180 μL of each sample to the plate, with three replicates per sample. Measure the absorbance at 562 nm. Substitute the absorbance into the pre-prepared standard curve to calculate the concentration and loading volume. After centrifugation, transfer the remaining supernatant to a new 1.5 ml centrifuge tube. Based on the supernatant volume, add 5x protein loading buffer, vortex to mix, and incubate at 100°C for 10 min. Then perform protein electrophoresis.

[0082] 4. Prepare separating gel and stacking gel, load samples, perform electrophoresis, transfer membrane, block, incubate with primary and secondary antibodies, and expose the bands.

[0083] Figure 4 This demonstrates that the lysosomal targeting chimera TdlTac-17RA achieves targeted degradation of the HaCat cell membrane protein IL-17RA.

[0084] Example 4: ELISA verification of blocking ability

[0085] The specific steps for quantitatively analyzing the blocking ability of CZG-7 using ELISA are as follows:

[0086] 1. Solution preparation

[0087] Reagent buffer: 1% BSA; Washing buffer: PBS + 0.05% Tween 20; Blocking buffer: 2% BSA.

[0088] 2. Coating

[0089] First, prepare the IL-17RA working solution by diluting the IL-17RA stock solution (1 μg / μL) to 1 μg / mL with coating buffer. Add 100 μL of working solution to each well, and set up several control groups without coating. Seal the plate and incubate overnight at 4°C on a small shaker.

[0090] 3. Cleaning

[0091] Discard the liquid in the well plate, add 300 μL of washing buffer to each well, shake on a 3D shaker for 1 min, then remove the supernatant, invert the well plate onto paper, pat gently and wait for it to dry, repeat 3 times.

[0092] 4. Enclosed

[0093] Add 300 μL of bocking buffer to each well, seal with a vacuum membrane, and incubate at 37°C for 1.5 h.

[0094] 5. Cleaning

[0095] Discard the liquid in the well plate, add 300 μL of washing buffer to each well, shake on a 3D shaker for 1 minute, then remove the supernatant, invert the well plate onto paper, pat gently and wait for it to dry, repeat 3 times.

[0096] 6. Sample loading

[0097] The monoclonal antibody and CZG-7 were diluted with 1% BSA to prepare a monoclonal antibody concentration of 1 μM and a CZG-7 concentration of 10 μM. 100 μL of sample solution was added to each well, and Reagent buffer was added to the NO-coating wells. The plates were then sealed.

[0098] 7. Combining

[0099] First, prepare the working solution of Biotin-IL-17A. Dilute Biotin-IL-17A (50 μg / mL) with Reagent buffer to 0.1 μg / mL. Set up experimental groups and NO-coating groups. Add 50 μL of Biotin-IL-17A working solution to each well of the experimental group and 50 μL of Reagent buffer to each well of the NO-coating group. Gently tap to mix, seal the plate, and incubate at 37°C for 1 h.

[0100] 8. Cleaning

[0101] Discard the liquid in the well plate, add 300 μL of washing buffer to each well, shake on a 3D shaker for 1 min, then remove the supernatant, invert the well plate onto paper, pat gently and wait for it to dry, repeat 3 times.

[0102] 9. Add Streptavidin-HRP

[0103] First, prepare the Streptavidin-HRP working solution by diluting the Streptavidin-HRP stock solution (50 μg / mL) to 0.1 μg / mL with substrate buffer. Add 100 μL to each well, seal the plate, and incubate at 37°C in the dark for 1 h.

[0104] 10. Cleaning

[0105] Discard the liquid in the well plate, add 300 μL of washing buffer to each well, shake on a 3D shaker for 1 min, then remove the supernatant, invert the well plate onto paper, pat gently and wait for it to dry, repeat 3 times.

[0106] 11. Substrate reaction

[0107] Add 100 μL TMB to each well, seal the plate, and incubate at 37°C in the dark for 20 min.

[0108] 12. Termination

[0109] Add 50 μL of stop solution to each well and gently shake the plate to mix thoroughly (blue → yellow).

[0110] 13. Measure absorbance

[0111] The absorbance at 450 nm-630 nm using an ELISA reader is the final result.

[0112] Figure 5 In this study, PBS served as the negative control group, and monoclonal antibody served as the positive control group. By comparing the two, CZG-7 was found to have a good blocking effect.

[0113] Example 5: In vivo experiments to verify therapeutic efficacy

[0114] Experimental process

[0115] 1. Construction of a mouse model of psoriasis

[0116] Six-week-old female Balb / c mice were purchased and, after one week of acclimatization, were divided into four groups of six mice each: PBS group, CON group, CZG-7 group, and MTX (methotrexate) group. The experimental procedures are as follows:

[0117] Normal group: fed normally, without any treatment;

[0118] PBS group: The backs of mice in this group were treated with hair removal, 5% imiquimod cream was applied daily and their backs were photographed. When the skin on the back of the mice became wrinkled, 100 μL of PBS was injected into each mouse intraperitoneally, and multiple injections were required.

[0119] CZG-7 group: The backs of mice in this group were shaved, 5% imiquimod cream was applied daily and their backs were photographed. When skin folds appeared on the backs of the mice, the dosage was 10 mg / kg, 125 μl was injected intraperitoneally. After that, psoriasis induction and drug treatment were performed once a day for one week.

[0120] MTX group: The backs of mice in this group were treated with hair removal, and 5% imiquimod cream was applied daily and photographed. When skin folds appeared on the backs of the mice, the dosage was 1 mg / kg, administered via intraperitoneal injection of 100 μl. For the next week, the injection was given every two days, and psoriasis was induced daily.

[0121] After administration, mice were sacrificed, and their livers, kidneys, and back skin were collected for HE staining to verify the biosafety of CZG-7 and its therapeutic effect on psoriasis. The results are as follows: Figure 6-9 As shown.

[0122] Figures 6-9 The images show, in order, the HE staining results of the dorsal skin, liver, and kidneys of psoriatic mice after treatment. Figure 6 and Figure 7 As shown, the PBS group exhibited significant keratinization of the stratum corneum on the back, with thinning of the granular layer in the epidermis, revealing obvious psoriasis symptoms; Figure 8 and Figure 9 As shown, in the PBS control group, liver and kidney cells of mice exhibited fragmented necrosis, unclear structure, and cellular edema. In contrast, cells in the CZG-7 and MTX experimental groups were neatly arranged, with uniform cytoplasmic staining and clear nuclei, verifying that CZG-7 has no cytotoxicity and possesses extremely high biocompatibility. All indicators showed significant improvement after treatment with CZG-7 and MTX.

[0123] Based on this, we purchased 6-week-old female Balb / c mice, and after feeding them for a week to allow them to adapt to their growth environment, we divided the mice into two groups of 6 mice each: the PBS group and the TdlTac-17RA group.

[0124] Normal group: fed normally, without any treatment;

[0125] PBS group: The backs of mice in this group were treated with hair removal, 5% imiquimod cream was applied daily and their backs were photographed. When the skin on the back of the mice became wrinkled, 100 μL of PBS was injected into each mouse intraperitoneally, and multiple injections were required.

[0126] TdlTac-17RA group: The backs of mice in this group were treated with hair removal, 5% imiquimod cream was applied daily and photographs were taken of their backs. When skin folds appeared on the backs of the mice, they were treated by intraperitoneal injection at a concentration of 10 mg / kg, 125 μl each time. For the next week, psoriasis induction and treatment were performed once a day.

[0127] Figure 10 The effect of TdlTac-17RA treatment on the dorsal skin of psoriatic mice is shown in the figure. After a period of TdlTac-17RA treatment, the psoriasis symptoms of the mice were significantly reduced, verifying that TdlTac-17RA has a therapeutic effect on psoriatic mice.

[0128] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

2.

2. A lysosome-targeting chimera, characterized in that, It includes: An aptamer targeting the IL-17RA protein; the aptamer targeting the IL-17RA protein includes the polypeptide of claim 1.

3. The lysosome-targeting chimera of claim 2, wherein, The lysosomal targeting chimera further includes an aptamer for a lysosomal targeting receptor linked to the aptamer targeting the IL-17RA protein.

4. The lysosome-targeting chimera of claim 3, wherein, The molar ratio of the aptamer targeting the IL-17RA protein to the aptamer targeting the lysosomal receptor is 1~6:1~2.

5. The lysosome-targeting chimera according to claim 3, characterized in that, The lysosomal receptor aptamers include aptamers targeting IGFIIR.

6. The lysosome-targeting chimera according to claim 3, characterized in that, The aptamers for the lysosomal targeting receptor include retinoic acid.

7. The lysosome-targeting chimera according to claim 3, characterized in that, The aptamer targeting the IL-17RA protein is directly linked to the aptamer targeting the lysosomal receptor or linked through a connection system.

8. The lysosome-targeting chimera according to claim 7, characterized in that, The connection system includes a biotin-avidin connection system.

9. The lysosome-targeting chimera according to claim 8, characterized in that, The avidin includes any one or more of ovalbumin and streptavidin.

10. The lysosome-targeting chimera according to claim 8, characterized in that, The connection system includes: biotin, a linker, and avidin; the aptamer targeting the IL-17RA protein and the aptamer targeting the lysosomal receptor are respectively linked to biotin via the linker, and the biotin is capable of non-covalently binding to the avidin.

11. The lysosome-targeting chimera according to claim 10, characterized in that, The linker includes PEG.

12. The lysosome-targeting chimera according to claim 11, characterized in that, The molecular weight of the PEG is 1k~10k.

13. A composition, characterized in that, It includes: raw materials for preparing the lysosome-targeting chimera according to any one of claims 2 to 12; The raw materials include: the aptamer targeting the IL-17RA protein as described in claim 2 and the aptamer targeting the lysosomal receptor as described in claim 5 or 6.

14. The composition according to claim 13, characterized in that... The raw materials also include: the connection system described in any one of claims 8 to 12.

15. The method for preparing the lysosomal targeting chimera according to any one of claims 2 to 12, characterized in that, It includes preparation using the composition of claim 13 or 14.

16. The preparation method according to claim 15, characterized in that, The preparation includes: The aptamers targeting IL-17RA protein and the aptamers targeting the lysosomal receptor were coupled with biotin via linkers to obtain biotin-coupled aptamers targeting IL-17RA protein and biotin-coupled aptamers targeting the lysosomal receptor. The biotin-conjugated aptamer targeting IL-17RA protein, the biotin-conjugated lysosomal receptor aptamer, and avidin are mixed to obtain a lysosomal targeting chimera. The molar ratio of the biotin-conjugated aptamer targeting the IL-17RA protein, the biotin-conjugated lysosomal receptor-targeting aptamer, and avidin is 1~2:1~6:1~2.

17. The use of the polypeptide of claim 1 or the lysosomal targeting chimera of any one of claims 2 to 12 in the preparation of products for targeted treatment of psoriasis or related clinical symptoms caused by psoriasis; The relevant clinical symptoms caused by psoriasis are any one or more of the following: liver cell damage, kidney cell damage, and skin damage caused by psoriasis.

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