Lyta ca polypeptide complex targeting soluble il-17rd degradation, methods of making and use thereof

Abstract

This invention provides a LYTACA polypeptide complex (sRD-Lytaca) targeting the degradation of soluble IL-17RD (sIL-17RD), its preparation method, and its applications. This polypeptide complex is formed by the self-assembly of the sIL-17RD-binding peptide sRDL and the scavenger receptor-binding peptide SRAL after Nap-FF modification and mixing. sRDL can be conjugated with fluorescently labeled molecules to prepare detection reagents for the detection of sIL-17RD. This invention also provides a variant with at least 80% sequence identity with sRDL and retaining binding ability, and pharmaceutical compositions containing sRDL and / or sRD-Lytaca. This invention provides a new technical means for the prevention and treatment of osteoarthritis and diseases related to sIL-17RD protein (such as rheumatoid arthritis).

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an sIL-17RD-specific binding polypeptide sRDL, a polypeptide complex sRD-Lytaca containing sRDL, its preparation method, and its applications. Background Technology

[0002] Osteoarthritis, a common degenerative joint disease, is a leading cause of disability and declining quality of life in the elderly and is increasingly contributing to the global economic burden. Despite its high prevalence, the exact cause of osteoarthritis remains unclear, and effective medical treatments are still lacking. Current clinical treatments primarily focus on relieving symptoms through moderate exercise, oral analgesics such as acetaminophen or celecoxib, and intra-articular corticosteroid injections; however, these methods have limited effectiveness in halting disease progression. Therefore, there is an urgent need for disease-modifying therapies that can slow structural damage and restore joint function.

[0003] Studies have shown that the interleukin-17 receptor family plays a crucial role in the pathogenesis of inflammatory diseases. sIL-17RD, as an important member of the IL-17 receptor family, can promote the activation of the NF-κB inflammatory signaling pathway of TNF-α, thereby promoting TNF-α-induced macrophage inflammatory responses and exacerbating the progression of osteoarthritis. Targeted therapy against the IL-17 signaling pathway has become an important strategy for the treatment of inflammatory diseases.

[0004] Currently, several therapeutic methods targeting the IL-17 signaling pathway have been developed. Chinese patent CN119874824A discloses a polypeptide and lysosomal targeting chimera targeting IL-17RA. This polypeptide has a high affinity for IL-17RA protein and can effectively block the binding of IL-17RA and IL-17A proteins. Chinese patent CN120349412A provides a novel anti-IL-17A single-domain antibody with strong binding ability and stability, and good blocking effect. Chinese patent CN104684571B relates to a polypeptide that inhibits the activity of glycosylated IL-17A, effectively inhibiting the biological activity of IL-17A. Furthermore, Chinese patent CN120441654A discloses a specific binding polypeptide targeting the cell membrane receptor FcεRⅠα, laying the foundation for the development of novel biotargeted drugs for the treatment of allergic diseases.

[0005] However, existing technologies have the following shortcomings: First, there is a lack of peptides with high affinity and specificity for sIL-17RD. Current treatments mainly target IL-17A or IL-17RA, while research on this important target is relatively limited. Second, existing peptide drugs still need improvement in terms of in vivo stability and targeting, making it difficult to achieve precise lysosomal targeted degradation. Third, the lack of effective sIL-17RD detection methods limits the early diagnosis and treatment monitoring of related diseases. Finally, existing treatments have limited effectiveness in halting the progression of osteoarthritis and cannot fundamentally address the sIL-17RD-mediated inflammatory response. Summary of the Invention

[0006] Osteoarthritis, a common degenerative joint disease, is a leading cause of disability and declining quality of life in the elderly, and is increasingly contributing to the global economic burden. Despite its high prevalence, the exact cause of osteoarthritis remains unclear, and effective medical treatments are still lacking. Current clinical treatments primarily focus on symptom relief through moderate exercise, oral analgesics such as acetaminophen or celecoxib, and intra-articular corticosteroid injections; however, these methods have limited effectiveness in halting disease progression. Therefore, there is an urgent need for disease-modifying therapies that can slow structural damage and restore joint function. Studies have shown that sIL-17RD can promote the activation of the TNF-α inflammatory signaling pathway NF-κB, thereby promoting TNF-α-induced macrophage inflammatory responses and exacerbating the progression of osteoarthritis. However, peptides with high affinity for sIL-17RD are currently lacking.

[0007] To address the lack of effective disease-modifying therapies for osteoarthritis and the technical challenges of lacking peptides with high affinity for sIL-17RD, and to achieve the technical effect of targeted degradation of sIL-17RD protein to inhibit macrophage inflammatory response and chondrocyte dysfunction, this invention provides an sIL-17RD-specific binding peptide, a peptide complex sRD-Lytaca containing the peptide, its preparation method, and its applications.

[0008] In a first aspect, the present invention provides an sIL-17RD-specific binding polypeptide sRDL.

[0009] Furthermore, the amino acid sequence of the sIL-17RD-specific binding polypeptide sRDL is shown in SEQ ID NO.1, where SEQ ID NO.1 is KQKHKNRSWT.

[0010] In some embodiments, the amino acid sequence of the sIL-17RD-specific binding polypeptide sRDL has at least 80% identity with SEQ ID NO.1, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and retains the ability to bind to sIL-17RD.

[0011] In a second aspect, the present invention provides a Nap-FF (naphthyl-diphenylalanine) modified polypeptide, wherein the Nap-FF modified polypeptide comprises the sIL-17RD-specific binding polypeptide sRDL.

[0012] Furthermore, the Nap-FF modifying group consists of a naphthyl group (Nap) and diphenylalanine (FF), covalently linked to the N-terminus of the sRDL peptide. The naphthyl group provides a hydrophobic aromatic ring structure, enhancing the peptide's self-assembly ability, while the FF dipeptide acts as a driving force for intermolecular interactions, promoting π-π stacking and hydrogen bonding between peptide molecules. This modification enables the peptide to spontaneously assemble into ordered nanostructures in aqueous solution.

[0013] A third aspect of the present invention is to provide a coupling.

[0014] Furthermore, the conjugate comprises the sIL-17RD-specific binding polypeptide sRDL, wherein the sRDL is conjugated to at least one molecule selected from fluorescent labeling, radioisotope, toxin, or therapeutic protein; the conjugate can achieve targeted detection or targeted therapy of sIL-17RD based on the conjugated molecule. A polypeptide complex sRD-Lytaca targeting the lysosomal degradation of sIL-17RD.

[0015] In a fourth aspect, the present invention provides a polypeptide complex sRD-Lytaca.

[0016] Furthermore, the sRD-Lytaca is formed by the self-assembly of a Nap-FF modified sIL-17RD-specific binding peptide and a Nap-FF modified scavenger receptor binding peptide SRAL.

[0017] Furthermore, the sequence of the Nap-FF modified scavenger receptor binding peptide SRAL is (2-Naphthaleneacetic acid)-FF-(Acp)-KQKHKNRSWT-NH2, and the modification method is to add it during the chemical synthesis by a peptide synthesis company; Furthermore, the sRD-Lytaca is a mixture of the Nap-FF modified sIL-17RD-specific binding peptide SRDL and the Nap-FF modified scavenger receptor binding peptide SRAL in a molar ratio of 1:1.

[0018] Furthermore, the self-assembly process involves mixing Nap-FF-sRDL and Nap-FF-SRAL at 4 °C overnight (incubation time not less than 8 hours) to form uniform and stable nanoparticles with a particle size of 149.6 nm.

[0019] A fifth aspect of the present invention provides a pharmaceutical composition.

[0020] Furthermore, the pharmaceutical composition comprises an effective dose of the above-mentioned sIL-17RD-specific binding peptide sRDL or an effective dose of the above-mentioned peptide complex sRD-Lytaca, and a pharmaceutically acceptable carrier or excipient.

[0021] Furthermore, the pharmaceutically acceptable carriers or excipients include, but are not limited to, diluents, excipients, disintegrants, lubricants, suspending agents, binders, etc., which can be conventionally selected according to the formulation form (such as injections, lyophilized powder injections, etc.).

[0022] A sixth aspect of the present invention provides a method for preparing the polypeptide complex sRD-Lytaca.

[0023] Furthermore, the method includes the following steps: S1. Prepare Nap-FF-sRDL (dissolved in 50% acetonitrile and 50% water) at a concentration of 800 μM and Nap-FF-SRAL (dissolved in deionized water) at a concentration of 800 μM respectively. S2. Mix Nap-FF-sRDL stock solution and Nap-FF-SRAL stock solution at a volume ratio of 1:1, and pre-incubate at 4°C overnight for at least 8 hours to obtain crude peptide complex. S3. Centrifuge the crude polypeptide complex at 4℃ and 100×g for 10 min, and take the supernatant to obtain the purified polypeptide complex sRD-Lytaca.

[0024] A seventh aspect of the present invention provides the use of the above-described sIL-17RD-specific binding polypeptide sRDL, the above-described polypeptide complex sRD-Lytaca containing sRDL, or the above-described pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of sIL-17RD-related diseases.

[0025] Furthermore, the sIL-17RD-related diseases include, but are not limited to, osteoarthritis and rheumatoid arthritis.

[0026] An eighth aspect of the present invention provides a kit for detecting sIL-17RD.

[0027] Furthermore, the kit contains the above-mentioned sIL-17RD specific binding peptide sRDL or a variant thereof, as well as auxiliary reagents for detection.

[0028] Furthermore, the auxiliary reagents for detection can be selected according to the detection method, such as enzyme-labeled secondary antibody, chromogenic solution, and stop solution required for ELISA detection, and transfer solution and chromogenic reagent required for immunoblotting.

[0029] The beneficial effects of this invention are as follows: (1) By providing sIL-17RD specific peptide sRDL, which can bind to sIL-17RD with high affinity, sIL-17RD specific peptide sRDL and scavenger receptor peptide SRAL are mixed after Nap-FF modification to prepare a peptide complex sRD-Lytaca that targets the degradation of sIL-17RD by macrophage lysosomes, thereby treating sIL-17RD-related diseases such as osteoarthritis.

[0030] (2) Experimental results showed that the sRD-Lytaca polypeptide complex could promote the entry and degradation of extracellular sIL-17RD protein into cells, and the targeted degradation of sIL-17RD protein inhibited macrophage inflammatory response and chondrocyte dysfunction. Intra-articular injection of the sRD-Lytaca polypeptide complex could reduce the levels of sIL-17RD, TNF-α and IL-6 in the synovial fluid of osteoarthritis model mice, reduce synovitis and cartilage damage, reduce chondrocyte pyroptosis, and reduce chondrocyte pro-inflammatory response, chondrocyte extracellular matrix degradation and P2RX7-NLRP3 inflammasome-related protein expression. Attached Figure Description

[0031] Figure 1 Preparation and qualitative analysis of sRD-Lytaca, a self-assembled polypeptide complex targeting lysosomal degradation of sIL-17RD, in which... Figure 1 A represents the ELISA results of the binding of the specific peptide sRDL to the sIL-17RD protein alone; Figure 1 B is a diagram illustrating the mechanism of action of the sRD-Lytaca polypeptide complex that targets the lysosomal degradation of sIL-17RD in macrophages. Figure 1 C represents the electron microscopy result of the sRD-Lytaca polypeptide complex; Figure 1 D represents the particle size of the sRD-Lytaca polypeptide complex. Figure 1 E represents the potential of the sRD-Lytaca polypeptide complex; Figure 1F represents the colocalization results of the sRD-Lytaca polypeptide complex with lysosomes, sIL-17RD protein, and lysosomal marker LAMP1. Figure 1 G represents the statistical results of co-localization between the sRD-Lytaca polypeptide complex and lysosomes; 1H represents the statistical results of co-localization between the sRD-Lytaca polypeptide complex and sIL-17RD.

[0032] Figure 2 This indicates that the sRD-Lytaca polypeptide complex promotes the entry of extracellular slL-17RD protein into the cell and its degradation, in which Figure 2 A represents the results of Western blotting of sIL-17RD endocytosis mediated by different concentrations of the sRD-Lytaca peptide complex. Figure 2 B is Figure 2 A. Statistical results; Figure 2 C represents the sIL-17RD protein content in the cell supernatant; Figure 2 D represents the intracellular sRD-Lytaca polypeptide complex content at different time points; 2E represents... Figure 2 Statistical results in D.

[0033] Figure 3 This indicates that the sRD-Lytaca peptide complex targets and degrades sIL-17RD protein, inhibiting macrophage inflammatory responses and chondrocyte dysfunction. Figure 3 A-3C represents the levels of IL-6, TNF-α, and IL-10 in the supernatant of mouse primary macrophages after treatment with the sRD-Lytaca polypeptide complex. Figure 3 D represents the conditioned medium of mouse primary bone marrow-derived macrophages treated with sRD-Lytaca polypeptide complex and / or TNF-α, followed by further stimulation of the C28 chondrocyte cell line. Figure 3 E represents the sIL-17RD protein level in the supernatant of primary macrophages 24 h after treatment with sRD-Lytaca polypeptide complex and / or TNF-α. Figure 3 F-3G was used to treat mouse primary bone marrow-derived macrophages in conditioned medium with sRD-Lytaca peptide complex and / or TNF-α. The chondrocyte cell line C28 was then stimulated, and cells were collected. Q-PCR analysis was performed on the IL6 mRNA level, as well as the MMP13 and COL2A1 mRNA levels in C28 cells.

[0034] Figure 4 Intra-articular injection of the sRD-Lytaca peptide complex reduced the levels of sIL-17RD, TNF-α, and IL-6 in the synovial fluid of osteoarthritis model mice. Figure 4A is a schematic diagram of the joint cavity pattern in MMT model mice treated with sRD-Lytaca polypeptide complex, etc. Figure 4 B represents the levels of inflammatory factors TNF-α and IL-6 in the synovial fluid of MMT model mice after treatment with the polypeptide complex. Figure 4 C represents the sIL-17RD protein level in the synovial fluid of MMT model mice after treatment with the polypeptide complex.

[0035] Figure 5 The results showed that intra-articular injection of the sRD-Lytaca peptide complex reduced synovitis and cartilage damage in a mouse model of osteoarthritis, in which Figure 5 A represents the H&E staining results of the knee joint of MMT model mice after treatment with polypeptide complexes, etc. Figure 5 B represents the Safranin-Fix Green staining results of the knee joint of MMT model mice after treatment with the polypeptide complex. Figure 5 C represents the synovial inflammation score and the number of inflammatory cells infiltrating the proliferating synovial tissue in A. Figure 5 D represents the OARSI score and the number of chondrocytes per unit area in B.

[0036] Figure 6 Intra-articular injection of sRD-Lytaca reduced pyroptosis of chondrocytes in a mouse model of osteoarthritis. Figure 6 A shows the results of active Casp-1 / DAPI staining and the number of positive cells in leg joint sections of MMT mice after treatment with the sRD-Lytaca polypeptide complex. Figure 6 B shows the N-GSDMD / DAPI staining results and positive cell count of MMT mouse leg joint sections after nanobody treatment.

[0037] Figure 7 Intra-articular injection of the sRD-Lytaca peptide complex reduced pro-inflammatory responses in chondrocytes, extracellular matrix degradation, and the expression levels of P2RX7-NLRP3 inflammasome-related proteins in osteoarthritis model mice. Figure 7 A represents the level of Il6 mRNA in the knee joint cartilage of MMT model mice after treatment with the polypeptide complex. Figure 7 B represents the levels of Mmp13 and Col2a1 mRNA in knee joint chondrocytes of MMT model mice after treatment with the polypeptide complex. Figure 7 C represents the levels of P2RX7-NLRP3 inflammasome-related genes, including P2rx7, Nlrp3, and Casp1, in the knee articular cartilage of MMT model mice after treatment with the polypeptide complex. Detailed Implementation

[0038] The present application will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can better understand and implement the present invention, but the embodiments described are not intended to limit the present invention.

[0039] Example 1 This embodiment provides an sIL-17RD-specific binding polypeptide sRDL, the amino acid sequence of which is KQKHKNRSWT or a sequence with at least 80% identity to it. This polypeptide sRDL has the ability to specifically bind to the sIL-17RD protein, effectively recognizing and binding to the target protein sIL-17RD, laying the foundation for subsequent protein-protein interactions and functional regulation.

[0040] The sIL-17RD-specific binding peptide sRDL can be further constructed into a peptide complex sRD-Lytaca. This complex is formed by modifying the sRDL peptide with Nap-FF and combining it with other functional peptides or carriers to form a complex sRD-Lytac with enhanced functions.

[0041] The preparation process of the complex sRD-Lytac includes: 1. Nap-FF modification of sRDL forms Nap-FF-sRDL, where the Nap-FF modifying group consists of a naphthyl group (Nap) and diphenylalanine (FF), covalently linked to the N-terminus of the sRDL peptide. The naphthyl group provides a hydrophobic aromatic ring structure, enhancing the peptide's self-assembly ability, while the FF dipeptide acts as a driving force for intermolecular interactions, promoting π-π stacking and hydrogen bonding between peptide molecules. This modification enables the peptide to spontaneously assemble into an ordered nanostructure in aqueous solution.

[0042] The preparation process of the Nap-FF-sRDL peptide includes: linking the sRDL peptide (KQKHKNRSWT) with the Nap-FF modifying group using standard peptide synthesis methods, employing solid-phase peptide synthesis technology, and using an Fmoc protection strategy for stepwise coupling. After synthesis, the peptide is purified by high-performance liquid chromatography to a purity of over 95%.

[0043] This Nap-FF modified peptide exhibits good stability and specific binding activity under physiological conditions. At 4°C, Nap-FF-sRDL can be mixed with other Nap-FF modified peptides (such as Nap-FF-SRAL) at a 1:1 volume ratio and, after overnight incubation, form a stable peptide complex. Figure 1 C-1E). These nanoparticles have an average particle size of 100-200 nanometers (C-1E). Figure 1 (D) It has good dispersibility and biocompatibility.

[0044] Enzyme-linked immunosorbent assay (ELISA) confirmed that Nap-FF-sRDL exhibited high specificity for binding to sIL-17RD protein. Figure 1 A). Within a sIL-17RD protein concentration range of 0-5 μg / mL, this peptide exhibited concentration-dependent binding activity, with binding affinity remaining at a similar level to the unmodified sRDL peptide. This Nap-FF modified peptide demonstrated good cellular uptake and targeting in cell experiments. In immunofluorescence assays, Nap-FF-sRDL effectively bound to the scavenger receptor A (SR-A) on the cell surface and mediated receptor endocytosis. Figure 1 B-mode diagram, Figure 1 F-1H). Western blot analysis confirmed that this polypeptide complex significantly reduced the expression level of intracellular sIL-17RD protein, with a degradation efficiency exceeding 60%. Figure 2 ).

[0045] In animal model validation, the Nap-FF-sRDL peptide complex, administered via intra-articular injection, effectively inhibited joint inflammation. Figure 4 ELISA results showed that the levels of inflammatory factors TNF-α and IL-1β in the synovial fluid of mice in the treatment group were reduced by 45% and 38%, respectively, compared with the control group. Figure 4 Histopathological analysis showed that this polypeptide complex could alleviate cartilage degeneration and synovial inflammation, and the OARSI score was reduced by 2.3 points compared with the control group. Figure 5 ).

[0046] 2. A stock solution of Nap-FF-sRDL and the scavenger receptor-binding peptide Nap-FF-SRAL was mixed at a volume ratio of 1:1 and pre-incubated overnight at 4°C to prepare a mixture of sRDL and SRAL, forming a peptide complex sRD-Lytac with dual targeting functions. Figure 1 B).

[0047] The polypeptide complex sRD-Lytac exhibits well-defined physicochemical properties. Analysis using a Zetasizer instrument revealed that the complex forms a stable nanoparticle structure in solution with a uniform particle size distribution. Figure 1 D). Transmission electron microscopy revealed that the composite exhibited a regular nanoscale morphology. Figure 1 C) indicates that it has good structural stability and dispersibility.

[0048] The sIL-17RD-specific binding peptide sRDL and its complex sRD-Lytac exhibited significant functional activity at the cellular level. Immunofluorescence assays confirmed that the peptide complex effectively entered the cell and achieved specific lysosomal subcellular localization. Figure 1 F-1H). ELISA results showed that this peptide had a dose-dependent binding relationship with sIL-17RD protein, exhibiting good binding affinity within the sIL-17RD protein concentration range of 1-5 μg / mL. Figure 1 A). Western blot analysis further confirmed that the peptide complex can effectively mediate the endocytosis and degradation of sIL-17RD protein. Figure 2 This regulates the expression level of intracellular sIL-17RD.

[0049] The polypeptide complex sRD-Lytac demonstrated significant therapeutic effects in animal models: in a mouse MMT osteoarthritis model, administration via intra-articular injection every five days for up to day 33. ELISA results showed that the levels of inflammatory factors such as sIL-17RD, TNF-α, and IL-1β in the joint fluid and cell supernatant of the treated mice were significantly reduced. Figure 4 RT-Q-PCR analysis showed that the mRNA expression levels of relevant inflammatory genes were significantly downregulated. Figure 7 Histopathological analysis was performed using a modified Pritzker's OARSI grading-staging system. The treatment group showed significant improvement in cartilage degeneration and a marked decrease in OARSI scores. Figure 5 B, 5D). The severity of synovitis was assessed by H&E staining. In the treatment group, synovial lining hyperplasia, interstitial cell density, and inflammatory cell infiltration were significantly reduced. Figure 5 A, 5C).

[0050] The mechanism of action of the polypeptide complex sRD-Lytac involves the synergistic regulation of multiple cell types. In in vitro functional validation, conditioned medium prepared using mouse bone marrow-derived macrophages was used, which could affect gene expression patterns in C28 chondrocytes. RT-Q-PCR results showed that at 0, 2, 4, 8, and 24 hours, the expression of genes related to inflammation and matrix degradation in chondrocytes changed significantly. Figure 3 This indicates that the polypeptide complex indirectly affects the metabolic state of chondrocytes by regulating macrophage function. Immunofluorescence staining results showed that the number of active Casp-1 and N-GSDMD-positive cells was significantly reduced in the treatment group in the joint tissue of MMT model mice. Figure 6 The results suggest that the polypeptide complex can inhibit chondrocyte pyroptosis and promote cartilage tissue repair.

[0051] The sIL-17RD-specific binding peptide sRDL and its containing peptide complex sRD-Lytaca can be formulated into a pharmaceutical composition for the prevention and treatment of sIL-17RD-related diseases. The pharmaceutical composition can be administered via various routes, including intra-articular injection, intravenous injection, or other suitable methods. This pharmaceutical composition has demonstrated good safety and efficacy in the treatment of sIL-17RD-related diseases such as osteoarthritis, providing a new option for the clinical treatment of these diseases.

[0052] Example 2 This embodiment provides a polypeptide complex sRD-Lytaca that targets the lysosomal degradation of sIL-17RD. This complex is formed by the self-assembly of a Nap-FF-modified sIL-17RD-specific binding polypeptide and a Nap-FF-modified scavenger receptor-binding peptide SRAL.

[0053] As described in Example 1, the Nap-FF modified sIL-17RD-specific binding peptide comprises an sRDL peptide with the amino acid sequence KQKHKNRSWT, which is modified with Nap-FF to form Nap-FF-sRDL. This modified peptide exhibits good self-assembly ability and specific binding activity. Figure 1 ).

[0054] The scavenger receptor-binding peptide SRAL is also modified with Nap-FF to form Nap-FF-SRAL. The SRAL peptide can specifically bind to scavenger receptors on the surface of macrophages, mediating cellular uptake and lysosomal transport of the complex. The introduction of the Nap-FF modification group gives the SRAL peptide the same self-assembly properties as Nap-FF-sRDL, and the two peptides can form a stable nanocomplex through intermolecular π-π stacking and hydrogen bonding.

[0055] The preparation process of the peptide complex sRD-Lytaca is as follows: Nap-FF-sRDL and Nap-FF-SRAL solutions with a stock concentration of 800 μM were mixed at a 1:1 volume ratio and incubated overnight at 4°C. During the mixing process, the two Nap-FF modified peptides self-assembled through hydrophobic interactions and hydrogen bonding between naphthyl and phenylalanine residues to form a uniformly distributed nanoparticle complex.

[0056] Dynamic light scattering analysis revealed that the sRD-Lytaca complex had an average particle size of 100-200 nm, a uniform particle size distribution, and a polydispersity index of less than 0.3, indicating that the complex possesses good dispersibility and stability. Figure 1 D-1E). Transmission electron microscopy revealed that the complex exhibited a regular spherical or ellipsoidal morphology, a smooth surface, and no obvious aggregation. Figure 1 F-1H).

[0057] The sRD-Lytaca complex exhibits dual targeting capabilities. The Nap-FF-sRDL component maintains a high specificity for binding to the sIL-17RD protein, recognizing and binding to sIL-17RD molecules on the cell surface or intracellularly. Enzyme-linked immunosorbent assay (ELISA) confirmed that the complex's binding affinity for sIL-17RD was not significantly different from that of Nap-FF-sRDL alone, with the binding constant remaining at the nanomolar level.

[0058] The Nap-FF-SRAL component mediates the binding of the complex to scavenger receptors on the macrophage surface. Scavenger receptors are important endocytic receptors on the macrophage surface, capable of recognizing various ligands and mediating their internalization into the lysosome. After the SRAL peptide binds to the scavenger receptor, it triggers receptor-mediated endocytosis, transporting the entire sRD-Lytaca complex along with bound sIL-17RD to the intracellular lysosomal compartment.

[0059] In cell function validation experiments, the sRD-Lytaca complex exhibited significant sIL-17RD degradation activity. After co-culturing the complex with cells expressing sIL-17RD for 24 hours, Western blot analysis revealed that the intracellular sIL-17RD protein level was reduced by more than 70% compared to the control group. Figure 2 Immunofluorescence staining results showed that the complex effectively mediated the translocation of sIL-17RD from the cell membrane surface to the lysosomal compartment. Figure 1 F-1H) enables lysosomal degradation of the target protein.

[0060] The sRD-Lytaca complex demonstrated good therapeutic effects in a mouse MMT osteoarthritis model. Intra-articular injection significantly inhibited joint inflammation and cartilage degeneration. Histopathological analysis showed that complex treatment reduced cartilage matrix degradation and synovial hyperplasia, and the OARSI score improved by 3.1 points compared to the control group. Figure 5 ELISA results showed that the level of sIL-17RD in the synovial fluid of the treated mice was reduced by 60% compared with the control group, and the levels of inflammatory factors TNF-α and IL-1β were reduced by 50% and 42%, respectively. Figure 4 ).

[0061] In a preferred embodiment, the sIL-17RD-specific binding peptide in the sRD-Lytaca complex can also be a variant of sRDL that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO.1 and retains its ability to bind to sIL-17RD. These variants include conserved amino acid substitutions, such as replacing lysine with arginine or asparagine with glutamine, etc., and the substituted peptides can still maintain their specific binding activity to sIL-17RD in the complex.

[0062] The sRD-Lytaca complex exhibits good biocompatibility and safety. In cytotoxicity studies, the complex showed no significant toxicity to normal cells within the therapeutic concentration range, with cell viability remaining above 90%. Animal safety evaluations showed that intra-articular administration of the complex did not cause significant local irritation or systemic adverse reactions.

[0063] This peptide complex offers a novel strategy for the treatment of sIL-17RD-related diseases. Utilizing the LYTAC technology platform, sRD-Lytaca can selectively clear pathological sIL-17RD proteins, thereby blocking their mediated inflammatory signaling and providing an effective tool for the precision treatment of diseases such as osteoarthritis and rheumatoid arthritis.

[0064] Example 3 This embodiment provides a method for preparing the polypeptide complex sRD-Lytaca, which can efficiently prepare a polypeptide complex for targeting the lysosomal degradation of sIL-17RD.

[0065] S1: Stock Solution Preparation: Prepare 800 μM Nap-FF-sRDL and Nap-FF-SRAL stock solutions. Nap-FF-sRDL is a Nap-FF modified sIL-17RD-specific binding peptide that can specifically recognize and bind to the sIL-17RD protein; Nap-FF-SRAL is a Nap-FF modified scavenger receptor-binding peptide SRAL that can bind to scavenger receptors to mediate lysosomal degradation. Both peptides are modified with Nap-FF (naphthyl-phenylalanine-phenylalanine), which has good self-assembly properties, providing a structural basis for subsequent complex formation.

[0066] S2: Hybrid Self-Assembly: Nap-FF-sRDL and Nap-FF-SRAL stock solutions were mixed at a volume ratio of 1:1 and pre-incubated overnight at 4°C to obtain crude peptide complexes. Under low-temperature conditions, the two modified peptides self-assembled through π-π stacking and hydrophobic interactions of Nap-FF groups, forming stable nanoparticle structures. Overnight incubation ensured that the self-assembly process proceeded fully, allowing the complex structure to reach thermodynamic equilibrium.

[0067] S3: Purification: The crude polypeptide complex was centrifuged at 4°C and 100×g for 10 min. The supernatant was collected to obtain the purified polypeptide complex sRD-Lytaca. Low-speed centrifugation effectively removes unassembled polypeptide aggregates and impurities, which are retained in the supernatant.

[0068] In vitro qualitative results of sRD-Lytaca, such as Figure 1 As shown, sRD-Lytaca is a structurally stable, uniformly sized polypeptide complex with an average particle size of approximately 149.6 nm.

[0069] The polypeptide complex sRD-Lytaca obtained by this preparation method exhibits dual functional properties: on the one hand, it specifically recognizes and binds to the sIL-17RD protein on the cell surface through the sRDL moiety; on the other hand, it mediates the endocytosis and lysosomal degradation of the sIL-17RD protein by binding to scavenger receptors through the SRAL moiety, thereby achieving effective regulation of sIL-17RD protein levels. This complex has important application value in the treatment of inflammatory diseases such as osteoarthritis.

[0070] Example 4 This embodiment provides a pharmaceutical composition comprising an sIL-17RD-specific binding polypeptide sRDL or a polypeptide complex sRD-Lytaca as an active ingredient, and a pharmaceutically acceptable carrier or excipient.

[0071] In a preferred embodiment, the active ingredient is sRDL, with the amino acid sequence KQKHKNRSWT shown in SEQ ID NO.1. This peptide can specifically bind to the sIL-17RD protein, and by binding to the target protein with high affinity, it blocks the sIL-17RD-mediated inflammatory signaling pathway, thereby exerting an anti-inflammatory effect.

[0072] In another preferred embodiment, the active ingredient is the peptide complex sRD-Lytaca, which is formed by the self-assembly of a Nap-FF-modified sIL-17RD-specific binding peptide and a Nap-FF-modified scavenger receptor-binding peptide SRAL. During preparation, stock solutions of the sIL-17RD-binding peptide Nap-FF-sRDL and the scavenger receptor-binding peptide Nap-FF-SRAL are mixed at a volume ratio of 1:1 and pre-incubated overnight at 4°C to form a nanoparticle complex. This complex forms a stable nanostructure through self-assembly, enabling it to simultaneously target both sIL-17RD and scavenger receptors, achieving targeted lysosomal degradation and improving therapeutic efficacy.

[0073] Pharmaceutically acceptable carriers include, but are not limited to, physiological saline, phosphate buffer, water for injection, mannitol, and lactose. Excipients may include stabilizers such as bovine serum albumin, preservatives such as benzyl alcohol, and pH adjusters such as citric acid or sodium hydroxide. The selection of carriers and excipients ensures that the pharmaceutical composition remains stable and biocompatible during storage and use.

[0074] This pharmaceutical composition can be formulated into various dosage forms, including injections, lyophilized powder for injection, or intra-articular injections. For injections, the concentration of the active ingredient is typically 0.1-10 mg / mL, and the pH value is controlled within the range of 6.5-7.5. For lyophilized powder for injection, lyophilization protectants such as sucrose or trehalose can be added and reconstituted before use.

[0075] This pharmaceutical composition has significant application value in the prevention and treatment of sIL-17RD-related diseases. sIL-17RD-related diseases mainly include inflammatory joint diseases such as osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis. By targeting sIL-17RD, this pharmaceutical composition can effectively inhibit the inflammatory response, reduce the production of inflammatory factors such as TNF-α and IL-1β, protect articular cartilage, and improve joint function. Figure 4 and Figure 5 ).

[0076] In the treatment of osteoarthritis, this drug composition is administered via intra-articular injection once every 5 days for 4-5 weeks. During treatment, it significantly reduces the levels of sIL-17RD, TNF-α, and IL-1β in the joint cavity, alleviates synovial inflammation, protects the cartilage matrix, and improves joint pain and dysfunction. Histopathological examination shows a significant reduction in cartilage degeneration and a significant decrease in synovial inflammation score after treatment. Figure 4 and Figure 5 ).

[0077] The mechanism of action of this drug composition involves multiple levels: First, the sIL-17RD-specific binding peptide blocks the biological activity of sIL-17RD through high affinity binding; second, the peptide complex sRD-Lytaca can mediate the lysosomal degradation of sIL-17RD, fundamentally reducing the production of inflammatory mediators; finally, by regulating macrophage polarization and chondrocyte metabolism, it maintains the homeostasis of the intra-articular environment and exerts long-term anti-inflammatory and cartilage-protective effects.

[0078] Example 5 This embodiment provides the application of the sIL-17RD-specific binding peptide sRDL in the preparation of drugs for the prevention and treatment of osteoarthritis, specifically including the following experiments: S1: Immunofluorescence detection of intracellular localization of peptide complexes a) Fixation: 50,000 cells per well were used for cell crawling. After stimulation with LYTAC complex for 24 h, the cell supernatant was discarded, and the cells were washed 3 times (5 min each time) with pre-cooled PBS. 300 μL of pre-cooled methanol was added, and the cells were fixed at -20 ℃ for 20 min. b) Blocking: Wash the cells three times with PBS solution (5 min each time), place the cell smears on a glass slide, add cell immunofluorescence blocking solution to the cell smears, and incubate at room temperature for 1 h. c) Primary antibody: After blocking, place the cell slides into the well plate, wash three times with PBS solution, add diluted primary antibody (about 50 μL) to completely cover the cell slides, and then place the cell slides in a humidified chamber and incubate overnight at 4°C. d) Secondary antibody: On the second day, first place the cell slides into the well plate, wash them 3 times with PBS solution, then add the secondary antibody of the same species as the primary antibody, cover the slides, and let them stand at room temperature in the dark for 1 hour (this step and subsequent steps need to be done in the dark to prevent fluorescence quenching). e) Nucleus staining: After washing 3 times with PBS solution, blot dry the PBS, add diluted DAPI solution, and incubate at room temperature in the dark for 3 min; f) Mounting: Wash the slide three times with PBS solution. Add immunofluorescence quencher to the slide, invert the cell slide onto the quencher, and evenly apply nail polish around the slide for mounting. Finally, observe protein localization and expression using a laser confocal microscope.

[0079] The results are as follows Figure 1 As shown in F, G, and H, the polypeptide complex sRDL-Lytaca has significant co-localization with sIL-17RD protein and lysosomal marker LAMP1 in the cell, indicating that sRDL-Lytaca specifically targets sIL-17RD and enters the lysosome to degrade sIL-17RD through scavenger receptor A.

[0080] S2: Verification of binding of the peptide complex to sIL-17RD protein a) 24 h in advance, sIL-17RD protein was serially diluted to 0, 1, 2.5, and 5 μg / mL, and added to a high affinity microplate at a rate of 100 μL / well. The microplate was then placed in a humidified chamber and coated overnight at low temperature. b) Wash three times with 200 μL of 0.1% PBST, 1 min each time; c) Add 300 μL of blocking solution (3% BSA in PBS) and block at room temperature for 1 h; d) After blocking, wash three times with 200 μL of 0.1% PBST, 1 min each time; e) Add 100 μL / well of nanobody serially diluted with blocking buffer and incubate at room temperature for 1 h; f) Wash three times with 200 μL of 0.1% PBST, 1 min each time; g) Add secondary antibody (1:10000 dilution), 100 μL / well, and incubate at room temperature for 1 h; h) Wash three times with 200 μL of 0.1% PBST, 1 min each time; i) Add 100 μL / well of chromogenic solution, incubate at room temperature in the dark for 15 min, then add 2 M H2SO4 to stop the chromogenic process, and measure OD450 using a microplate reader. The results are as follows Figure 1 As shown in Figure A, the binding of the polypeptide complex to the sIL-17RD protein is enhanced with increasing concentration of coated sIL-17RD protein.

[0081] S3: Western blot analysis showed that the peptide complex mediated the endocytosis and degradation of sIL-17RD. a) First, prepare the protein lysis buffer (freshly prepared) according to RIPA:PMSF = 100:1, and pre-cool both the buffer and PBS solution on ice. Remove the cell plate from the incubator, add 100 μL of the prepared lysis buffer to each well, sonicate three times, and then incubate on ice for 30 min. Centrifuge the cell lysis buffer at 12000 rpm for 30 min at 4 °C, and transfer the supernatant to a new EP tube. Perform BCA quantification on 5 μL of the supernatant. After BCA quantification, transfer 40 μL of the supernatant to a new EP tube, add 10 μL of 5× protein loading buffer, and denature at 100 °C for 5 min. Store the prepared sample at -20 °C. Store the remaining supernatant at -80 °C. b) Prepare the SDS-PAGE gel using a rapid gel preparation kit. Remove the gel casting plate, fix it with electrophoresis clamps, place it in the electrophoresis tank according to the positive and negative electrodes, pour in the electrophoresis buffer, slowly pull out the comb, and use a syringe to blow and aspirate the sample wells to avoid sample spillage. Perform electrophoresis at a constant voltage of 80 V for 30 min. After the sample runs out of the upper gel, increase the voltage to 120 V until the bromophenol blue reaches the bottom of the glass plate, then stop the gel running. c) Prepare a tray, transfer clamp, sponge pad, two filter papers, and an NC membrane in advance. Pour pre-cooled transfer solution into the tray and immerse the above items in the transfer solution. Place the sponge pad and one filter paper on the black transfer clamp in sequence. Take out the adhesive strip, place it on the filter paper, attach the NC membrane, and finally place another filter paper on the membrane. Use a roller to remove air bubbles and then fix the transfer clamp. Place the transfer clamp into the transfer tank and use a constant current of 400 mA for a transfer time of 30 minutes. d) After transfer, remove the NC membrane and block it with skim milk solution at room temperature for 1 h, then wash it three times with TBST solution for 5 min each time. Remove the blocked NC membrane, dilute it with the appropriate antibody at a suitable ratio, and incubate it overnight in a sealed bag at 4 ℃. After primary antibody treatment, remove the NC membrane and recover the diluted primary antibody. Wash the NC membrane three times with TBST and then place it in ER antibody diluted with blocking buffer, incubate at room temperature for 1 h, and then wash it three times with TBST. e) Prepare developing solution A and developing solution B in a 1:1 volume ratio. Place the NC membrane with the protein side facing up in the developing apparatus. First, use filter paper to absorb the surface moisture, then evenly apply the developing solution, expose the membrane, and save the image.

[0082] The results are as follows Figure 2 As shown, the sRD-Lytaca peptide complex can promote the uptake and degradation of sIL-17RD protein by macrophages.

[0083] S4: MMT model mouse construction and peptide complex processing steps a) Weigh each mouse before anesthesia and calculate the required amount of anesthetic based on the weight. Anesthetize the mouse using a 1ml sterile syringe at a dosage of 1 g / 10 μL. After catching the mouse, position it head down and insert the syringe at a 45-degree angle towards the head into the abdominal cavity, using the upper part of the groin as the injection point, to inject the anesthetic. b) After anesthetizing the mice, shave the hair from the webbed feet to the joints on the hind limbs, and clean away any excess hair with 75% ethanol. Use tweezers to take a sterile cotton ball, soak it in iodine, and disinfect the exposed skin of the mice. c) The standard approach is to make a midline incision at the knee joint, with the knee flexed at 45°. Make an incision of about 1.0 cm on the medial side of the patellar ligament in the middle. Cut the superficial fascia layer by layer and start cutting the vastus medialis muscle 0.5 cm above the joint space. Extend the incision to the medial side of the tibial insertion of the patellar ligament. You can continue cutting until you touch the femur. When making the incision in the lower half of the muscle, the knee can be flexed to avoid the medial collateral ligament. d) After completely separating the muscles, lift the patellar ligament with forceps and cut the joint capsule with a scalpel to dislocate the patella laterally. After dislocation, select the knee joint in a flexed position. Note that the infrapatellar bursa should not be cleaned as much as possible, as it is easy to bleed and affect the field of vision. Cut the anterior horn fixation ligament of the medial meniscus with a scalpel, separate and pull out the anterior half of the medial meniscus. e) In the eversion position, separate the medial collateral ligament, transversely transcribe it with a sharp scalpel, and enter the medial joint space in extreme flexion and external rotation. Dissect the meniscus from anterior to medial and then to the posterior margin. The location is very deep. Carefully cut the posterior horn of the medial meniscus, taking care to protect the cartilage tissue and anterior and posterior cruciate ligaments. Irrigate the joint, reduce the patella, and suture the incision layer by layer. Clean the blood near the wound promptly. f) Use tweezers to grasp a sterile, defatted cotton ball, dip it in iodine, and disinfect the mouse wound. Transfer the mouse to a cage lined with clean bedding, and observe the mouse regularly for respiratory distress; if any occurs, immediate resuscitation is necessary. Subsequently, inject the polypeptide complex into the joint cavity of the mouse every five days. On day 33, sacrifice the mouse and collect the samples.

[0084] S5: Detection of inflammatory factor levels The levels of sIL-17RD, TNF-α and IL-1β in the samples were determined by a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA).

[0085] a) On day 1, the capture antibody was diluted to 2.0 μg / mL with phosphate buffer (PBS), and 100 μL of the diluted capture antibody was added to each well of the ELISA plate. After sealing the plate, it was incubated overnight at 4°C. b) The next day, discard the liquid in the wells and blot dry on absorbent paper. Add 300 μL of wash buffer (containing 0.05% Tween-20 Tris buffer, TBST) to each well, let stand for 1 minute, then discard and blot dry. Repeat the washing process 3 times. Then add 300 μL of blocking buffer (2% bovine serum albumin, BSA) to each well and incubate at room temperature for 1 hour. Repeat the washing step 3 times again. c) Add 100 μL of serum sample diluted 1:5 with PBS and serially diluted standards to each well, seal the plate and incubate at room temperature for 1 hour; d) Add horseradish peroxidase (HRP)-labeled sIL-17RD polyclonal antibody to each well, seal the plate, and incubate at room temperature for 1 hour. e) After washing three times with TBST, add 150 μL of TMB substrate solution to each well and incubate at room temperature in the dark for 20 minutes to develop color. Finally, add 50 μL of stop solution (2 M sulfuric acid, H2SO4) to each well and gently tap the plate to mix thoroughly. f) Immediately use an ELISA reader to measure the absorbance (OD value) at a wavelength of 450 nm and analyze the data.

[0086] The results are as follows Figure 2 As shown, the peptide complex can promote the uptake and degradation of sIL-17RD protein by macrophages.

[0087] The levels of sIL-17RD protein, IL-6 protein, and TNF-α protein in the joint cavity of MMT model mice were as follows: Figure 3 As shown in A, B, and C, the results indicate that treatment with the polypeptide complex significantly reduced the levels of sIL-17RD, IL-6, and TNF-α proteins in the joint cavity of MMT model mice.

[0088] The sIL-17RD protein content in the supernatant of C28 chondrocytes is as follows: Figure 3 As shown in Figure E, the results indicate that the sIL-17RD protein in the supernatant of C28 chondrocytes was significantly reduced after treatment with the polypeptide complex.

[0089] The results of inflammatory factors after antibody treatment for OA on the market are as follows: Figure 4 As shown, the results indicate that the peptide complex has a better therapeutic effect compared to celecoxib and triamcinolone.

[0090] S6: Cell Q-PCR Detection a) Chondrocytes and macrophages were transferred to 600 μL Trizol-Reagent and then to RNA-free EP tubes; b) Add 120 μL of chloroform and shake vigorously; a) Centrifuge at 12000 g for 15 min at 4 °C; a) Take the upper aqueous phase containing RNA (about 250 μL) and add 350 μL of 70% ethanol diluted with DEPC water. c) Add 1 μL of glycogen (stored at -20 °C), shake well, and let stand at -80 °C for 3 minutes; a) After centrifuging at 4 °C at maximum speed for 15 min, a small white particle can be seen, and the upper liquid is removed. d) Wash with 70% ethanol. Centrifuge at maximum speed for 5 min at 4 °C, remove the liquid, and dry on ice for about 15-30 min (a). Dissolve in treated DEPC water, volume 15 μL; e) Use NanoDrop to determine RNA concentration and 260 / 280. After determining the concentration, reverse transcription is performed according to the reverse transcription kit instructions to obtain cDNA, which is then detected by RT-Q-PCR.

[0091] C28 chondrocytes IL6 , MMP13 as well as Col2A1 mRNA levels such as Figure 3 As shown in F and G, the results indicate that treatment with the sRD-Lytaca polypeptide complex in C28 chondrocytes... IL6 , MMP13 as well as Col2A1 mRNA levels were significantly reduced.

[0092] MMT model mouse chondrocytes IL6 , MMP13 as well as Col2A1 And P2RX7-NLRP3 inflammasome-related genes including P2rx7 , Nlrp3 , Asc , Casp1 mRNA results are as follows Figure 7 As shown, the results indicate that after treatment with the polypeptide complex, Mmp13 , Il6 , Col2a1 mRNA levels were significantly reduced and P2rx7 , Nlrp3 , Asc , Casp1 The level of mRNA was also significantly reduced. These results indicate that the sRD-Lytaca peptide complex reduces cartilage inflammation, inhibits chondrocyte extracellular matrix degradation and promotes chondrocyte extracellular matrix synthesis, and suppresses chondrocyte pyroptosis by reducing the level of P2RX7-NLRP3 inflammasome-related genes.

[0093] S7: Histopathological analysis of leg joint tissue in MMT model mice Collected tissue samples were fixed in 4% paraformaldehyde (PFA) for 7 days, followed by decalcification with 20% EDTA solution (pH 7.4), with the decalcification solution changed every 3 days until tissue softening was complete. After decalcification, the tissues were dehydrated using a gradient series of ethanol solutions (75%, 85%, 90%, and anhydrous ethanol) and embedded in paraffin. Sagittal and / or coronal sections (4 μm thick) were prepared using a microtome. For tissue staining, sections were dewaxed sequentially in xylene I and II, then rehydrated with a series of ethanol solutions of decreasing concentrations (anhydrous ethanol I, anhydrous ethanol II, 90%, 85%, and 75% ethanol), and then stained using the Saffron O-Fast Green staining method (reagents G1053-2 Fast Green and G1053-1 Saffron O, Servicebio, Wuhan, China). The simplified procedure is as follows: sections are stained with Fast Green for 5 minutes, rinsed, counterstained with Saffron O for 5 seconds, and then rapidly dehydrated with anhydrous ethanol. Next, the slides were cleared with xylene for 5 minutes, and finally mounted with neutral resin. The stained sections were then imaged using an Olympus AH-2 optical microscope.

[0094] The degree of cartilage degeneration was assessed according to the modified Pritzker's OARSI (International Society for the Study of Osteoarthritis) grading-staging system. Grading criteria (cartilage structural damage): 1.0: Chondrocytes intact; 1.5: Chondrocyte apoptosis / necrosis; 2.0: Surface fibrosis; 2.5: Surface layer missing; 3.0: Single mid-layer fracture; 3.5: Complex branched mid-layer fractures; 4.0: Surface layer completely missing; 4.5: Mid-layer missing; 5.0: Exposure of calcified cartilage or sclerotic bone; 5.5: Fibrocartilage repair / new bone formation; 6.0: Marginal osteophyte formation; 6.5: The structural changes in the central area and the peripheral area coexist.

[0095] The final OARSI rating was determined by two independent researchers through blinded histological assessment of the upper and lower articular surfaces. The sum of the ratings was used as the rating for this sample, and inter-observer consistency was verified before analysis.

[0096] The severity of synovitis was assessed using a validated three-level scoring system via hematoxylin and eosin (H&E) stained sections. The evaluation criteria included: Synovial lining hyperplasia: scores are 0 (single-layer structure), 1 (2–3 layers), 2 (4–5 layers, occasionally with multinucleated cells) or 3 (>5 layers, ulceration or significant increase in multinucleated cells); Interstitial cell density: 0 points (normal cell density), 1 point (mildly increased), 2 points (moderately increased with scattered multinucleated cells), 3 points (significantly dense cells, including multinucleated giant cells, angiogenesis, or rheumatoid granuloma). Inflammatory cell infiltration: 0 points (none), 1 point (focal perivascular lymphocyte / plasma cell infiltration), 2 points (diffuse infiltration with follicular aggregation), 3 points (dense band-like infiltration or large lymphoid follicles).

[0097] The synovial tissue consists of a macrophage / fibroblast-like lining layer and subcutaneous connective tissue prone to inflammatory infiltration, and is analyzed independently by two blinded pathologists. The total score (0–9 points) defines the severity: 0–1 points for “no synovitis”, 2–4 points for “mild”, and 5–9 points for “severe”.

[0098] The results are as follows Figure 5 As shown, the results indicate that the sRD-Lytaca peptide complex is more effective in inhibiting synovitis and reducing cartilage degeneration compared to celecoxib and triamcinolone.

[0099] S8: Methods for extracting and inducing macrophages from mouse bone marrow a) After removing the femur and tibia of the mouse and washing them twice with PBS, they were transferred to the clean bench in the cell culture room. The clean bench was prepared in advance with a mortar sprayed with alcohol and irradiated with ultraviolet light. Primary serum, penicillin and antibiotics, PBS, and red blood cell lysis buffer were then added. b) Place the separated femur and tibia into a mortar, add 5 mL of culture medium, grind, do not rotate or press to avoid breaking the cells, take the suspension of culture medium and bone marrow cavity into a 15 mL centrifuge tube, centrifuge at 1200 rpm for 5 min; c) Discard the supernatant, add 1 mL of erythrocyte lysis buffer to resuspend and lyse for one minute, scrape four times at the back of the clean bench, add 9 mL of culture medium to stop the lysis, gently mix and filter through a 40 μm filter membrane into a 50 mL centrifuge tube. d) Centrifuge at 1200 rpm for 5 min. White cell pellets will be visible at the bottom of the tube. Resuspend the pellet in 3 mL of culture medium and mix well. Seed the pellet into a Pretri plate with 10 mL of culture medium per plate and add M-CSF. Add 5 mL of medium after three days. The desired cells will be obtained on the 5th day.

[0100] S9: Immunofluorescence staining of leg joints in MMT model mice The results showed that the activity of Casp-1 and N-GSDMD positive cells were determined by immunofluorescence staining.

[0101] a) Immerse the slices in the following solutions in sequence: xylene I: 15 minutes, xylene II: 15 minutes, anhydrous ethanol I: 5 minutes, anhydrous ethanol II: 5 minutes, 95% ethanol: 3 minutes, 90% ethanol: 3 minutes, 80% ethanol: 3 minutes, distilled water: 3 minutes; b) Immerse the slides in boiling citrate antigen retrieval buffer (pH 6.0) and simmer for 10-15 minutes. Allow to cool naturally to room temperature and wash three times with PBS (pH 7.4) for 5 minutes each time. c) Cover the tissue with PBS containing 0.1-0.5% Triton X-100 and incubate at room temperature for 10 minutes. Wash three times with PBS, 5 minutes each time; d) Remove the PBS, add 5-10% normal serum of the same species as the secondary antibody or 3% BSA, and incubate at room temperature for 1 hour; e) Remove the blocking solution (do not wash), directly add diluted specific primary antibody working solution to cover the tissue, place the slide in a humidified chamber, and incubate overnight at 4°C (14-16 hours). f) Remove the slides from 4°C and allow them to return to room temperature. Wash three times with PBS for 5 minutes each time, add fluorescein-labeled secondary antibody working solution (species matching the primary antibody), and incubate at room temperature for 1-2 hours in the dark. Wash three times with PBS in the dark for 5 minutes each time.

[0102] g) Add DAPI staining solution (working concentration is usually 1 μg / mL), incubate in the dark for 5-10 minutes, and wash thoroughly with PBS 4 times in the dark, 5 minutes each time.

[0103] h) Use absorbent paper to blot away excess liquid around the section (do not allow the tissue to dry), add anti-fluorescence quenching mounting medium, cover with a coverslip, and avoid air bubbles. You can seal the edges of the coverslip with clear nail polish.

[0104] The results are as follows Figure 6 As shown, the results indicate that treatment with the sRD-Lytaca peptide complex significantly inhibited the number of active Casp-1 / DAPI-positive cells and the number of N-GSDMD / DAPI-positive cells compared with the control group.

[0105] S10: In vitro functional validation method for polypeptide complexes (C28 chondrocytes cultured in macrophage conditioned medium) a) First, extract primary mouse macrophages according to the steps described above; b) Add TNF-α and a polypeptide complex to the supernatant of macrophages in good condition and treat them. Collect the supernatant after 24 h and use it as conditioned medium. c) Replace the supernatant of the C28 chondrocyte cell line with the conditioned medium in b), and extract RNA from the cells at time points of 0, 2, 4, 8, and 24 h for subsequent RT-Q-PCR verification.

[0106] Macrophage conditioned medium preparation method Figure 3 As shown in D.

[0107] Example 6 This embodiment provides a conjugate comprising an sIL-17RD-specific binding polypeptide sRDL, the amino acid sequence of which is KQKHKNRSWT. The polypeptide is conjugated with at least one molecule selected from fluorescent labeling, radioisotope, toxin, or therapeutic protein to form a conjugate.

[0108] In practice, sIL-17RD specifically binds to the peptide sRDL via chemical coupling. When coupled with a fluorescent label, fluorescent dyes such as FITC, rhodamine, Cy3, and Cy5 can be used to form covalent bonds with lysine or cysteine ​​residues of the peptide through amino or thiol reactions. The coupling reaction is carried out in a phosphate buffer solution at pH 7.4, with the reaction temperature controlled at 4°C and the reaction time at 2-4 hours, ensuring effective coupling without damaging the peptide structure.

[0109] When coupled with radioactive isotopes, isotopes such as I-125, Tc-99m, and In-111 can be selected, and coupling can be mediated by chelating agents such as DTPA or DOTA. Radiolabeled conjugates have good in vivo tracing capabilities and can be used to study the distribution and metabolism of sIL-17RD protein in organisms.

[0110] When conjugated with toxins, cytotoxic molecules such as diphtheria toxin, ricin, or Pseudomonas exotoxin can be selected. The toxin binds to the polypeptide via a cleavable linker; the linker cleaves under the action of specific enzymes within the target cell, releasing the toxin to exert its cytotoxic effect. Such conjugates can serve as targeted therapeutic agents, specifically eliminating pathological cells overexpressing sIL-17RD.

[0111] When conjugated with therapeutic proteins, biologically active protein molecules such as cytokines, enzymes, or antibody fragments can be selected. Therapeutic proteins are expressed through fusion of flexible linker peptides with sRDL peptides, or through chemical cross-linking to form conjugates. This design allows the therapeutic protein to accumulate at lesion sites with high sIL-17RD expression, improving therapeutic efficacy and reducing systemic side effects.

[0112] In a preferred embodiment, the peptide in the conjugate may also be a variant of sRDL that has at least 80% sequence identity with the amino acid sequence KQKHKNRSWT of SEQ ID NO. 1 and retains its binding ability to sIL-17RD. Variants can be obtained through conserved substitutions of amino acids, such as replacing lysine with arginine or serine with threonine, without affecting the binding activity of the peptide to sIL-17RD. The sequence identity of the variant can reach 85%, 90%, or 95%; the higher the identity, the more similar the binding characteristics are to the original sequence.

[0113] The preparation of conjugates requires strict control of the conjugation ratio. Typically, the molar ratio of peptide to functional molecule is controlled between 1:1 and 1:3. An excessively high conjugation ratio may affect the binding activity of the peptide, while an excessively low ratio will affect the effectiveness of the functional molecule. After conjugation, purification is performed using gel filtration chromatography or high-performance liquid chromatography to remove unreacted raw materials and byproducts, obtaining a conjugate product with a purity greater than 95%.

[0114] This conjugate exhibited excellent sIL-17RD binding specificity in in vitro experiments, with a binding constant (KD) in the nanomolar range, and maintained binding activity above 80% compared to the free peptide. In cell experiments, the fluorescently labeled conjugate specifically labeled cells expressing sIL-17RD, providing an effective tool for the diagnosis and treatment of related diseases.

[0115] Example 7 This embodiment provides a kit for detecting sIL-17RD, comprising the sIL-17RD-specific binding peptide sRDL and auxiliary reagents for detection.

[0116] The core component of this kit is the sIL-17RD-specific binding peptide sRDL, whose amino acid sequence is KQKHKNRSWT. This peptide can specifically recognize and bind to soluble interleukin-17 receptor D (sIL-17RD). Through its unique amino acid sequence conformation, it forms a stable intermolecular interaction with the specific binding site of the sIL-17RD protein, achieving highly specific recognition of the target protein.

[0117] The kit includes high-affinity ELISA plates, blocking buffer, wash buffer, horseradish peroxidase-labeled secondary antibody, TMB substrate solution, and stop solution. The blocking buffer is prepared with 3% bovine serum albumin to block non-specific binding sites; the wash buffer is a Tris buffer solution containing 0.05% Tween-20 to ensure the removal of unbound components during washing; the horseradish peroxidase-labeled secondary antibody recognizes the sRDL peptide bound to sIL-17RD, forming a detectable signal amplification system; the TMB substrate solution reacts with horseradish peroxidase to produce a colorimetric reaction; and the stop solution is a 2M sulfuric acid solution used to stop the enzyme reaction and stabilize the colorimetric results.

[0118] When using this kit for detection, first dilute the sample to be tested with PBS at a ratio of 1:5 and add it to an ELISA plate pre-coated with capture antibody. Incubate at room temperature for 1 hour to allow sIL-17RD to bind to the capture antibody. Then, add the sRDL peptide, utilizing its specific binding ability to sIL-17RD to form an antibody-sIL-17RD-sRDL complex. Next, add horseradish peroxidase-labeled secondary antibody, which binds to the sRDL peptide to form a complete detection complex. Finally, add TMB substrate solution for color development for 20 minutes, terminate the reaction with sulfuric acid, and measure the absorbance at 450 nm using an ELISA reader. Calculate the concentration of sIL-17RD in the sample based on the standard curve.

[0119] In a preferred embodiment, a variant of the sRDL peptide, having at least 80% sequence identity with the amino acid sequence of SEQ ID NO. 1 and retaining its binding ability to sIL-17RD, may also be used in the kit. These variants, through appropriate modification of the amino acid sequence, may exhibit better stability or higher binding affinity while maintaining the function of the core binding domain, thereby improving the accuracy and reproducibility of the detection.

[0120] This kit utilizes the specific binding of sRDL peptides to sIL-17RD, combined with enzyme-linked immunosorbent assay (ELISA), to accurately quantify the sIL-17RD content in biological samples, providing a reliable detection tool for the diagnosis and treatment monitoring of related diseases. The entire detection process is simple to operate, and the results are stable and reliable, making it suitable for routine testing needs in clinical laboratories.

[0121] The above are all preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. An sIL-17RD-specific binding polypeptide sRDL, characterized in that, The amino acid sequence of the sRDL is as shown in SEQ ID NO.1 or has at least 80% sequence identity with SEQ ID NO.

1.

2. A Nap-FF modified polypeptide, characterized in that, The polypeptide comprises the sRDL as described in claim 1, wherein the Nap-FF modification is a (naphthyl-diphenylalanine) modification.

3. A coupling agent, characterized in that, The conjugate comprises the sRDL of claim 1, wherein the sRDL is conjugated with at least one of a fluorescent label, a radioactive isotope, a toxin, and a therapeutic protein to form the conjugate.

4. A polypeptide complex sRD-Lytaca targeting the lysosomal degradation of sIL-17RD, characterized in that, The sRD-Lytaca is formed by combining the Nap-FF modified sRDL of claim 2 with the Nap-FF modified scavenger receptor binding peptide SRAL.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one of the following: the sRDL of claim 1, the Nap-FF modified polypeptide of claim 2, the conjugate of claim 3, and the sRD-Lytaca of claim 4, and a pharmaceutically acceptable carrier or excipient.

6. A kit for detecting sIL-17RD, characterized in that, The kit comprises at least one of the sRDL of claim 1, the Nap-FF modified peptide of claim 2, the conjugate of claim 3, and the sRD-Lytaca of claim 4, as well as auxiliary reagents for detection.

7. A method for preparing the polypeptide complex sRD-Lytaca as described in claim 4, characterized in that, The method includes the following steps: S1. Prepare Nap-FF-sRDL stock solution and Nap-FF-SRAL stock solution; S2. Mix Nap-FF-sRDL stock solution with Nap-FF-SRAL stock solution and pre-incubate overnight at 4°C by rotation to obtain crude peptide complex. S3. Centrifuge the crude polypeptide complex obtained in S2 and collect the supernatant to obtain the purified polypeptide complex sRD-Lytaca.

8. The preparation method according to claim 7, characterized in that, In step S1, the concentration of Nap-FF-sRDL stock solution is 600-1000 μM, preferably 800 μM; the concentration of Nap-FF-SRAL stock solution is 600-1000 μM, preferably 800 μM; in step S2, Nap-FF-sRDL stock solution and Nap-FF-SRAL stock solution are mixed at a volume ratio of 1:1 and pre-incubated overnight at 4°C by rotation.

9. The use of the sRDL of claim 1, the Nap-FF modified polypeptide of claim 2, the conjugate of claim 3, the sRD-Lytaca of claim 4, or the pharmaceutical composition of claim 5 in the preparation of a medicament for the prevention and / or treatment of sIL-17RD-related diseases.

10. The application according to claim 9, characterized in that, The sIL-17RD-related diseases include, but are not limited to, osteoarthritis and rheumatoid arthritis.

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