Neutralizing nanobodies against soluble il-17rd, methods of making and uses thereof

Abstract

This invention discloses a neutralizing nanobody targeting soluble interleukin-17 receptor D (sIL-17RD), its preparation method, and its applications. The heavy chain variable region (VHH) of this nanobody contains a heavy chain complementarity-determining region 3 (CDR-H3) that specifically binds to sIL-17RD, with a length of 10 amino acids and containing 2–6 lysine residues. This invention provides four nanobodies: CQ5, CQ13, CQ11, and CQ6, corresponding to well-defined CDR-H1, CDR-H2, and CDR-H3 sequence combinations. This nanobody can specifically bind to and neutralize the biological activity of sIL-17RD, reducing chondrocyte death, promoting proliferation, and maintaining extracellular matrix homeostasis by inhibiting the P2rx7-NLRP3 inflammasome-mediated chondrocyte pyroptosis pathway. This invention also protects the nucleic acid encoding this nanobody, the recombinant expression vector, the host cell, the pharmaceutical composition, and the preparation method. The nanobodies described can be used to prepare drugs for the prevention and treatment of inflammatory osteoarthritis and other osteoarthritis. They can inhibit synovitis, reverse cartilage and subchondral bone degeneration, and relieve pain, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a neutralizing nanobody against soluble interleukin-17 receptor D (sIL-17RD), its preparation method, and its application in the treatment and diagnosis of inflammatory osteoarthritis (especially osteoarthritis). Background Technology

[0002] The interleukin-17 (IL-17) family is an important class of pro-inflammatory cytokines that plays a crucial role in the pathogenesis of various inflammatory and autoimmune diseases. Interleukin-17 receptor D (IL-17RD), as an important receptor member of the IL-17 family, has significant pathophysiological implications in its soluble form (sIL-17RD) in inflammation-related diseases such as osteoarthritis. Studies have shown that sIL-17RD can participate in the regulation of inflammatory signaling pathways, activating the P2rx7-NLRP3 inflammasome-mediated chondrocyte pyroptosis pathway, leading to increased chondrocyte death, disruption of extracellular matrix homeostasis, and ultimately, articular cartilage degeneration and increased pain.

[0003] In recent years, the development of antibody drugs targeting members of the IL-17 family has received widespread attention. Nanobodies, as a novel type of antibody molecule, possess advantages such as small molecular weight, good stability, and ease of expression and modification, showing promising application prospects in the treatment of inflammatory diseases. Currently, several nanobodies targeting IL-17A have been developed for the treatment of autoimmune diseases.

[0004] Chinese patent CN120399065B discloses an anti-IL-17A nanobody with high affinity and blocking ability, showing promise for treating autoimmune diseases. Chinese patent CN116769027B discloses another anti-IL-17 nanobody, obtained through phage library screening, capable of specifically recognizing and binding to interleukin-17, applicable to interleukin-17 detection and blood purification. Chinese patent CN117843778B describes a novel nanobody 1-G4 targeting members of the interleukin family; this antibody is easily expressed and exhibits good affinity and blocking effect. Chinese patent CN120554506A provides a novel anti-IL-17A nanobody exhibiting high IL-17A affinity and neutralizing ability. Chinese patent CN118206654B discloses a novel antibody for disease treatment, capable of specifically binding to IL-17A, suitable for detecting IL-17A or treating diseases caused by elevated IL-17A.

[0005] However, the existing technologies mentioned above mainly focus on antibody development targeting IL-17A, while research on specific antibodies against members of the IL-17 receptor family, particularly soluble interleukin-17 receptor D (sIL-17RD), is relatively lacking. sIL-17RD, as a unique target, plays a specific role in the pathogenesis of inflammatory osteoarthritis and other inflammatory joint diseases, directly affecting chondrocyte survival and function by regulating inflammatory signaling pathways. Currently, no specific neutralizing antibodies against sIL-17RD have been reported, limiting effective intervention against sIL-17RD-mediated inflammatory responses. Furthermore, while existing anti-IL-17A antibodies have shown therapeutic effects in some inflammatory diseases, they cannot directly block the biological activity of sIL-17RD, thus limiting their application in treating sIL-17RD-related inflammatory osteoarthritis. Summary of the Invention

[0006] To address the technical problem that existing technologies lack specific neutralizing antibodies against soluble interleukin-17 receptor D (sIL-17RD), which cannot effectively block sIL-17RD-mediated inflammatory responses and result in a lack of effective treatments for inflammatory diseases such as osteoarthritis, this invention provides a neutralizing nanobody against soluble interleukin-17 receptor D, its preparation method, and its application.

[0007] In a first aspect, the present invention provides a neutralizing nanobody targeting soluble interleukin-17 receptor D (sIL-17RD).

[0008] Furthermore, the heavy chain variable region (VHH) of the nanobody includes a heavy chain complementarity-determining region 3 (CDR-H3) that specifically binds to sIL-17RD. The amino acid sequence of CDR-H3 is 10 amino acids long and contains 2-6 lysine (K) residues. The nanobody can specifically bind to sIL-17RD and neutralize its biological activity.

[0009] Preferably, the amino acid sequence of CDR-H3 is selected from KQKHKNRSWT (SEQ ID NO.1), TKKKKKKPKI (SEQ ID NO.2), MKKKKNHTTT (SEQ ID NO.3), KPKKKKKRKP (SEQ ID NO.4) or a sequence that has at least 80% identity with any of the above sequences.

[0010] Furthermore, the heavy chain variable region (VHH) of the nanobody also includes heavy chain complementarity determination region 1 (CDR-H1) and heavy chain complementarity determination region 2 (CDR-H2), and the combination of CDR-H1 and CDR-H2 corresponds to and matches the corresponding CDR-H3 sequence.

[0011] Specifically, when CDR-H3 is SEQ ID NO.1, CDR-H1 is RIFSYYR (SEQ ID NO.5), and CDR-H2 is DISATGATTY (SEQ ID NO.6). Specifically, when CDR-H3 is SEQ ID NO.2, CDR-H1 is RIFSKYE (SEQ ID NO.7), and CDR-H2 is SSISSEGGTTN (SEQ ID NO.8). Specifically, when CDR-H3 is SEQ ID NO.3, CDR-H1 is FIFSTYP (SEQ ID NO.9), and CDR-H2 is DIYNSGGNTY (SEQ ID NO.10). Specifically, when CDR-H3 is SEQ ID NO.4, CDR-H1 is LTFIRNR (SEQ ID NO.11), and CDR-H2 is SINHSGASTY (SEQ ID NO.12).

[0012] Furthermore, the nanobody is selected from one or more of CQ5, CQ13, CQ11 and CQ6.

[0013] Furthermore, the amino acid sequence of the CQ5 is shown in SEQ ID NO.13, and the nucleotide sequence encoding the CQ5 is shown in SEQ ID NO.17.

[0014] SEQ ID NO.13: MAQVQLQESGGGLVQAGGSLRLSCAASGRIFSYYRMGWFRQAPGKEREWVADISATGATTYYADSVKGRFTISRDDNAKNTVYLQMNSLKPEDTAVYYCAKQKHKNRSWTYWGQGTQVTVSS SEQ ID NO.17: ATGGCCCAGGTTCAGCTGCAGGAAAGTGGTGGTGGCCTGGTTCAGGCAGGTGGTAGTCTGCGTCTGAGTTGCGCAGCCAGTGGCCGTATTTTTAGCTATTATAGGATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATGGGTGGCGGATATTTCAGCGACCGGCGCCACTACCT ATTACGCCGATAGCGTTAAAGGCCGTTTTACCATTAGTCGTGATAATGCAAAAAACACCGTGTATCTGCAGATGAATAGCCTGAAACCGGAAGATACCGCCGTGTATTGTGCCAAGCAGAAGCATAAGAATCGGTCGTGGACTTACTGGGGTCAGGGCACCCAGGTTACCGTTAGCAGC Furthermore, the amino acid sequence of CQ13 is shown in SEQ ID NO.14, and the nucleotide sequence encoding CQ13 is shown in SEQ ID NO.18.

[0015] SEQ ID NO.14: MAQVQLQESGGGLVQAGGSLRLSCAASGRIFSKYEMGWFRQAPGKEREFVASISSEGGTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATKKKKKKPKIYWGQGTQVTVSS SEQ ID NO.18: ATGGCCCAGGTTCAGCTGCAGGAAAGTGGTGGTGGCCTGGTTCAGGCAGGTGGTAGTCTGCGTCTGAGTTGCGCAGCCAGTGGCCGTATTTTTAGCAAGTATGAGATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAACGTGAATTTGTGGCGTCTATTAGTTCGGAAGGCGGAACTACCA ATTACGCCGATAGCGTTAAAGGCCGTTTTACCATTAGTCGTGATAATGCAAAAAACACCGTGTATCTGCAGATGAATAGCCTGAAACCGGAAGATACCGCCGTGTATTATTGTGCCACGAAGAAGAAGAAGAAGAAGCCGAAGATTTACTGGGGTCAGGGCACCCAGGTTACCGTTAGCAGC Furthermore, the amino acid sequence of CQ11 is shown in SEQ ID NO.15, and the nucleotide sequence encoding CQ11 is shown in SEQ ID NO.19.

[0016] SEQ ID NO.15: MAQVQLQESGGGLVQAGGSLRLSCAASGFIFSTYPMGWFRQAPGKEREWVADIYNSGGNTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAMKKKKNHTTTYWGQGTQVTVSS SEQ ID NO.19: ATGGCGCAGGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTATTTTTAGCACCTATCCGATGGGCTGGTTTCGCCAGGCGCCGGGCAAAGAACGCGAATGGGTGGCGGATATTTATAACAGCGGCGGCAACACCT ATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGATGAAAAAAAAAAAAAACCATACCACCACCTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC Furthermore, the amino acid sequence of CQ6 is shown in SEQ ID NO.16, and the nucleotide sequence encoding CQ6 is shown in SEQ ID NO.20.

[0017] SEQ ID NO.16: MAQVQLQESGGGLVQAGGSLRLSCAASGLTFIRNRMGWFRQAPGKEREWVASINHSGASTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAKPKKKKKRKPYWGQGTQVTVSS SEQ ID NO.20: ATGGCGCAGGTGCAGCTGCAGGAAAGCGGCGGCGGCCTGGTGCAGGCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCCTGACCTTTATTCGCAACCGCATGGGCTGGTTTCGCCAGGCGCCGGGCAAAGAACGCGAATGGGTGGCGAGCATTAACCATAGCGGCGCGAGCACCT ATTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGAAACCGAAAAAAAAAAAACGCAAACCGTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC Furthermore, the nanobody may also be a functional variant of CQ5, CQ13, CQ11 and / or CQ6 and / or a derivative thereof.

[0018] Specifically, the functional variants and / or their derivatives are obtained through engineering modifications of CQ5, CQ13, CQ11, and / or CQ6, wherein the engineering modifications include, but are not limited to: (1) Conservative substitution of one or more amino acid residues; (2) Amino acid insertions or deletions at non-critical sites; (3) Modify the frame region (FR) to improve stability or expression efficiency; (4) Introducing or removing potential glycosylation sites; (5) It can be fused with other functional proteins or peptides to form fusion proteins.

[0019] The antibody variants obtained through the above modifications still retain their specific binding ability and neutralizing function to sIL-17RD.

[0020] Furthermore, the functional variants and / or their derivatives are functional variants, their derivatives, and / or their sequence fragments that are obtained through reasonable biotechnological means and still retain the ability to substantially bind sIL-17RD.

[0021] In some embodiments, the functional variants and / or their derivatives are polypeptides that, based on the amino acid sequences SEQ ID NO. 13-16, have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with SEQ ID NO. 13-16, provided that they retain binding activity and / or neutralizing activity against sIL-17RD; the above variants and / or their derivatives may be obtained by conserved or non-conserved amino acid substitution, truncation or elongation (usually not exceeding 10 amino acids), or internal insertion / deletion (usually not exceeding 5 amino acids), preferably by techniques such as site-directed mutagenesis or DNA shuffling.

[0022] In some embodiments, the functional variants and / or their derivatives are variants containing conserved amino acid substitutions. These conserved substitutions refer to replacements between amino acids with similar side-chain properties and functions, such as: interchanges between aliphatic amino acids (glycine, alanine, valine, leucine, isoleucine); interchanges between hydroxy amino acids (serine, threonine); interchanges between acidic amino acids (aspartic acid, glutamic acid); interchanges between amide amino acids (asparagine, glutamine); interchanges between basic amino acids (lysine, arginine, histidine); and interchanges between aromatic amino acids (phenylalanine, tyrosine, tryptophan). Such substitutions generally do not significantly alter the three-dimensional conformation and binding properties of the antibody.

[0023] In some embodiments, the functional variants and / or their derivatives are CDR region transplantation variants, which are chimeric or humanized antibody variants formed by transplanting one, two, or three CDR regions (as defined by Kabat, Chothia, or IMGT) of the aforementioned antibodies (such as CQ5, CQ13, CQ6, and CQ11) onto the framework region of another nanobody or conventional antibody, as long as they retain specificity against sIL-17RD.

[0024] In some embodiments, the functional variants and / or their derivatives are antibody fragments and fusion proteins that contain any functional fragment of the antibody-antigen binding site of the present invention, including but not limited to a single heavy chain variable region (VHH), a VHH fused with a crystallizable fragment (Fc) (producing a monovalent or multivalent VHH-Fc fusion protein), a VHH fused with albumin or an albumin-binding domain to extend half-life, a VHH fused with a reporter molecule (such as a fluorescent protein, enzyme, or radionuclide) for diagnostic purposes, and a bifunctional molecule fused with other therapeutic proteins (such as cytokines or toxins).

[0025] In some embodiments, the functional variants and / or their derivatives are multivalent and multispecific antibodies, which are homotype multivalent antibodies (such as bivalent and trivalent anti-sIL-17RD nanobodies) formed by linking two or more VHH domains of the present invention through linkers, or bispecific or multispecific antibodies formed by linking them with VHH or scFv of other targets (such as inflammatory factors or their receptors, such as TNF-α, IL-6R, IL-1β, etc.); the above design can simultaneously block multiple inflammatory pathways and produce synergistic therapeutic effects.

[0026] Furthermore, the present invention also provides a nucleic acid molecule encoding the neutralizing nanobody, said nucleic acid molecule being an isolated or recombinant polynucleotide (DNA or RNA) encoding the anti-sIL-17RD nanobody or a variant thereof as described in any of the preceding claims, including but not limited to genes having the nucleotide sequences shown in SEQ ID NO. 13-16 and encoding CQ5, CQ13, CQ11 and / or CQ6; further comprising a polynucleotide capable of hybridizing with the above sequences under stringent hybridization conditions and encoding a protein with sIL-17RD binding activity, wherein the stringent hybridization conditions are well known to those skilled in the art, for example, hybridization at 65°C in 6×SSC, 0.5% SDS, 5×Denhardt's solution, 100 μg / mL denatured salmon sperm DNA, followed by washing in 2×SSC, 0.1% SDS at 65°C, and then in 0.2×SSC, 0.1% SDS. The SDS is washed at 65°C; it also includes codon-optimized nucleic acid sequences that encode the same amino acid sequence and have higher translation efficiency in target host cells such as E. coli, yeast, and CHO cells.

[0027] Furthermore, the present invention also provides a recombinant expression vector comprising the nucleic acid molecule, wherein the recombinant expression vector is selected from prokaryotic expression vectors, yeast expression vectors, insect cell expression vectors, and / or mammalian cell expression vectors, and comprises effectively linked polynucleotides as described in the present invention, wherein "effectively linked" means that the polynucleotide sequence is linked to one or more regulatory sequences in a manner that allows its expression; the vector may be a prokaryotic expression vector (e.g., pET series, pBV series), a yeast expression vector (e.g., pPICZ series), an insect cell expression vector (e.g., baculovirus vector), or a mammalian cell expression vector (e.g., pcDNA series, pCHO series); the vector contains necessary regulatory elements, such as promoters (e.g., T7 promoter, CMV promoter, EF-1α promoter), enhancers, ribosome binding sites (RBS), transcription termination signals, polyadenylation signals (polyA), and selection marker genes (e.g., antibiotic resistance gene Amp). + Kan + Zeocin +The vector can also be designed for secretory expression, containing signal peptide sequences (such as pelB, OmpA, Igκ, etc.) to secrete the expressed antibody into the periplasmic space or culture supernatant, facilitating purification.

[0028] Furthermore, the present invention also provides transformed or transfected host cells containing the recombinant expression vector described in the present invention, or whose genome integrates the polynucleotides described in the present invention; the host cells can be prokaryotic cells, such as Escherichia coli strains, such as BL21(DE3), TG1, HB2151, etc., suitable for rapid and low-cost production; the host cells can be eukaryotic cells, such as yeast cells, such as Pichia pastoris and Saccharomyces cerevisiae. The host cells can be cerevisiae, suitable for large-scale fermentation and post-translational modification; the host cells can also be mammalian cells, such as Chinese hamster ovary cells (CHO), human embryonic kidney cells (HEK293), and mouse myeloma cells (NS0, SP2 / 0), which can complete complex humanized glycosylation and are closest to the post-translational processing of natural human antibodies, making them suitable for the production of therapeutic antibodies; the host cells can also be insect cells, such as Sf9 and Sf21 cells, which can achieve high-level expression using a baculovirus expression system; the above host cells can express and produce biologically active anti-sIL-17RD nanobodies under suitable culture conditions.

[0029] Furthermore, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the anti-sIL-17RD nanobody described herein, its variants, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, excipients, or diluents; wherein the therapeutically effective amount refers to the dosage that, at the necessary dose and time, can achieve the prevention, treatment, or improvement of the target disease or symptoms. The specific dosage needs to be determined through clinical trials based on factors such as the patient's age, weight, disease severity, and route of administration. For example, in intra-articular injection for osteoarthritis, a single dose may range from 10 μg to 1 mg per joint; pharmaceutically acceptable carriers include, but are not limited to, buffers (phosphates, citrates, acetates, histidine buffers, etc.), isotonic agents (sodium chloride, glycerol, mannitol, glucose, etc.), stabilizers (sucrose, trehalose, and other sugars, amino acids such as arginine, glycine, etc., surfactants such as polysorbate 80, poloxamer 188, etc.), preservatives (benzyl alcohol, parabens), and lyophilization protectants (mannitol, sucrose, trehalose, etc.); the dosage form of the pharmaceutical composition may be formulated in various forms depending on the route of administration, including but not limited to injections, topical formulations, inhalations, and implants.

[0030] Furthermore, the pharmaceutical compositions of the present invention can be used alone or in combination with other therapeutic agents to produce synergistic or additive effects. The compositions may contain one or more other active ingredients, or can be administered sequentially or simultaneously with other drugs, including in combination with analgesics and anti-inflammatory drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs, such as celecoxib, diclofenac), acetaminophen; in combination with disease-modifying antirheumatic drugs such as methotrexate (for rheumatoid arthritis); in combination with biologics such as anti-TNF-α antibodies (adalimumab, etanercept), anti-IL-6R antibodies (tocilizumab), anti-IL-1 agents (anaspirin); in combination with chondroprotective agents such as glucosamine sulfate, chondroitin sulfate; and in combination with physical therapy or surgery.

[0031] A second aspect of the present invention provides a method for preparing the neutralizing nanobody.

[0032] Furthermore, the method includes the following steps: (1) Provide host cells: Provide host cells containing nucleic acids encoding the nanoantibodies of the present invention as described above.

[0033] (2) Culture: The host cells are cultured under conditions suitable for the expression of the nanobody. The culture conditions (such as temperature, pH, dissolved oxygen, inducer concentration and time) are optimized according to the host cell type and vector system.

[0034] (3) Induced expression: For systems using inducible promoters (such as T7 / lac, pBAD, methanol-induced AOX1), add inducers (such as IPTG, arabinose, methanol) at appropriate cell densities to initiate high-level expression.

[0035] (4) Harvesting and lysis: After culture, cells are collected by centrifugation or filtration. For intracellular expression, cells need to be disrupted (e.g., by sonication, high-pressure homogenization, or enzymatic digestion); for secretory expression, the culture supernatant is collected.

[0036] (5) Separation and purification: Separate and purify the target nanobody from cell lysate or culture supernatant. Purification methods may include: Affinity chromatography: using tags on antibodies (such as His tags, Flag tags, c-myc tags) for metal chelation chromatography (Ni-NTA, Co-NTA) or antibody affinity chromatography; or using the unique properties of nanobodies for purification.

[0037] Ion exchange chromatography: purification based on the charge interaction between the antibody and the medium.

[0038] Hydrophobic interaction chromatography: purification based on surface hydrophobicity.

[0039] Size exclusion chromatography, also known as gel filtration, is used for final purification and desalting, and can assess the aggregation state. It typically employs a two-step or multi-step chromatographic combination strategy, such as affinity chromatography combined with ion exchange chromatography, to obtain a high-purity product.

[0040] (6) Preparation: The purified antibody is dialyzed or ultrafiltered in an appropriate buffer (such as PBS, histidine buffer), and stabilizers (such as sucrose, trehalose, surfactants) may be added. Finally, the antibody is filtered to remove bacteria and prepared as stock solution.

[0041] In some embodiments, the present invention also covers the preparation of said nanobodies or fragments thereof by chemical synthesis methods (such as solid-phase peptide synthesis), particularly for shorter derivatives or labeled peptides for specific research purposes.

[0042] A third aspect of the invention provides the use of the above-described neutralizing nanobody or a pharmaceutical composition comprising the thereof in the preparation of a medicament for the prevention and / or treatment of inflammatory osteoarthritis.

[0043] Furthermore, the neutralizing nanobody inhibits the expression of sIL-17RD, suppresses the P2rx7-NLRP3 inflammasome-mediated chondrocyte pyroptosis pathway, reduces chondrocyte death, promotes chondrocyte proliferation, and maintains extracellular matrix homeostasis, thereby protecting articular cartilage, reversing cartilage degeneration, and / or alleviating pain.

[0044] Furthermore, the aforementioned neutralizing nanobodies or pharmaceutical compositions containing them can be used to treat related diseases, including but not limited to osteoarthritis, particularly inflammatory and progressive osteoarthritis. Furthermore, the aforementioned neutralizing nanobodies or pharmaceutical compositions containing them can alleviate synovitis, inhibit cartilage degradation, relieve pain, and improve joint function, thus exhibiting disease-modifying effects.

[0045] Furthermore, for joint diseases, the preferred route of administration for the above-mentioned neutralizing nanobodies or pharmaceutical compositions containing them is local administration, such as intra-articular injection, which can directly act on the lesion, increase the local drug concentration, and reduce systemic exposure and side effects.

[0046] Furthermore, the frequency of administration of the above-mentioned neutralizing nanobody or pharmaceutical composition containing it may be once every one and a half months, or as needed depending on disease activity.

[0047] The beneficial effects of this invention are as follows: (1) Compared with the prior art, this invention provides for the first time a specific neutralizing nanobody targeting sIL-17RD, which can specifically bind to soluble interleukin-17 receptor D, exhibiting high affinity and specificity; this antibody effectively blocks the sIL-17RD-mediated inflammatory signaling pathway and significantly reduces the expression levels of inflammatory factors such as TNF-α and IL-6. Experimental results show that the antibody of this invention significantly alleviates joint inflammation and pathological damage in the MMT osteoarthritis mouse model, improves the degree of cartilage degeneration, reduces the content of inflammatory factors in the joint cavity, and reduces the infiltration of inflammatory cells and the severity of synovitis; it has good in vivo stability and biological activity, and is suitable for intra-articular drug delivery, providing a new therapeutic target and treatment method for inflammatory diseases such as osteoarthritis, and has clear clinical application value.

[0048] (2) The neutralizing nanobody provided by this invention has the following significant clinical effects: ① Significant anti-inflammatory effect: In vitro, it can inhibit the production of pro-inflammatory factors such as IL-6 and TNF-α in macrophages and chondrocytes and promote the expression of IL-10; in a mouse model of osteoarthritis, it can reduce the levels of sIL-17RD, IL-6 and TNF-α in synovial fluid, alleviate synovitis and inflammatory cell infiltration, and its anti-inflammatory effect is better than triamcinolone and comparable to celecoxib.

[0049] ② Superior analgesic effect: In the osteoarthritis model, the analgesic effect based on gait analysis was significantly better than that of celecoxib and triamcinolone, mainly achieved by downregulating P2RX7 levels.

[0050] ③ Powerful cartilage protection: It can reduce cartilage OARSI score, increase the number of chondrocytes, and maintain cartilage homeostasis by inhibiting P2RX7-NLRP3-mediated chondrocyte pyroptosis, promoting chondrocyte proliferation, and inhibiting MMP13-mediated matrix degradation. Its effect is significantly better than celecoxib and triamcinolone. At the same time, in terms of protecting subchondral bone, it can reduce subchondral bone TRAP-positive cells, inhibit bone loss, and further reduce pain.

[0051] ④ The drug has significant advantages: it can be expressed and purified in E. coli at low cost with low endotoxin levels; it has a small molecular weight, strong tissue penetration, and is more likely to penetrate the cartilage matrix; the half-life in the mouse joint cavity is 7.6 days, and the estimated half-life in the human joint cavity is about 15–23 days, resulting in a long dosing cycle and good compliance; even with a high dose of 1.25 mg / kg, after 6 intra-articular injections of CQ5, no pathological changes were observed in major organs, proving the safety of CQ5 nanobody. Attached Figure Description

[0052] Figure 1 The bio-selection process of phage nanobodies targeting human sIL-17RD protein, in which... Figure 1A represents the purification results of sIL-17RD protein; Figure 1 B represents the phage enrichment status after four rounds of screening; Figure 1 C represents the binding affinity of the four-round screening library to the sIL-17RD protein; Figure 1 D represents positive monoclonal phages 1-24 named CQ after four rounds of screening.

[0053] Figure 2 Sequence comparison and plasmid construction schemes for CQ5, CQ13, CQ11, and CQ6 nanobodies, among which... Figure 2 A shows the amino acid sequence comparison results of CQ5, CQ13, CQ11, and CQ6 nanobodies; Figure 2 B is a schematic diagram of the nanobody plasmid construction model.

[0054] Figure 3 Expression, purification, and affinity analysis of nanobodies targeting human sIL-17RD CQ5, CQ13, CQ11, and CQ6, among which... Figure 3 A shows the results of molecular sieve purification and Coomassie Brilliant Blue staining of CQ5 nanobodies; Figure 3 B shows the results of molecular sieve purification and Coomassie Brilliant Blue staining of CQ13 nanobodies; Figure 3 C represents the results of molecular sieve purification and Coomassie Brilliant Blue staining of CQ6 nanobodies; Figure 3 D represents the results of molecular sieve purification and Coomassie Brilliant Blue staining of CQ11 nanobodies; Figure 3 E represents the EC50 results of affinity between CQ5, CQ13, CQ11, and CQ6 nanobodies and sIL-17RD protein.

[0055] Figure 4 The nanobody targeting sIL-17RD reduced the levels of sIL-17RD, TNF-α and IL-6 in the supernatant of TNF-α-induced macrophages and significantly promoted the production of IL-10. Figure 4 A-4C shows the levels of sIL-17RD protein (A), IL-6 (B), and TNF-α (C) in the supernatant of primary macrophages derived from bone marrow of IL-17RDKO mice, 24 hours after the addition of empty vector plasmid, GFP, and IL-17RD gene, followed by TNF-α restimulation with and without the addition of CQ5 and CQ13 nanobodies. 4D-4F shows the inhibition rate of sIL-17RD production (D), inhibition rate of TNF-α production (E), and activation rate of IL-10 production (F) in the supernatant after 24 hours of stimulation of primary macrophages derived from bone marrow of mice with different concentrations of CQ5 nanobodies and TNF-α, as detected by ELISA.

[0056] Figure 5The sIL-17RD-targeting nanobody reduced the levels of sIL-17RD, TNF-α, and IL-6 in the supernatant of TNF-α-induced chondrocytes. Figure 5 A-5C represents primary chondrocytes from IL-17RDKO mice. The levels of sIL-17RD protein (A), IL-6 (B), and TNF-α (C) in the supernatant were measured 24 hours after TNF-α restimulation with and without the addition of CQ5 and CQ13 nanobodies.

[0057] Figure 6 The study compared the effects of intra-articular injection of CQ5 and CQ13 nanobodies on sIL-17RD levels, inflammatory factor levels, improvement in cartilage damage, reduction in osteoarthritis scores, alleviation of synovial inflammation, and reduction in synovitis scores in a mouse model of osteoarthritis. Figure 6 A is a schematic diagram of the process of intra-articular injection of CQ5 and CQ13 nanobodies for the treatment of osteoarthritis. Figure 6 B represents the level of sIL-17RD in the joint lavage fluid after intra-articular injection of CQ5 and CQ13 nanobodies; Figure 6 C represents the protein levels of inflammatory factors IL-6 and IL-1β in the joint lavage fluid after intra-articular injection of CQ5 and CQ13 nanobodies; Figure 6 D represents the H&E staining results of knee joint sections from four mouse osteoarthritis models after treatment with CQ5 and CQ13 nanobodies to evaluate synovial inflammation. Figure 6 E represents the synovitis score for each mouse in the four groups; 6F represents the cartilage damage assessment results of knee joint sections from the four groups of mice with osteoarthritis after treatment with CQ5 and CQ13 nanobodies using Safranin-Fix-Green staining; and 6G represents the cartilage damage score for each mouse in the four groups. Figure 6 H-6I is a schematic diagram of a single intra-articular injection of nanobody in mice and sample collection, as well as the detection results of the half-life of CQ5 nanobody in the synovial fluid of mice after nanobody injection.

[0058] Figure 7 CQ5 nanobody significantly reduced pain indices in mice with osteoarthritis, among which Figure 7 A is a schematic diagram of the positive control model of intra-articular injection of CQ5 nanobody, celecoxib and triamcinolone in the MMT osteoarthritis mouse model; Figure 7 B represents the representative results of the mouse gait experiment; Figure 7 C represents the statistical results of the average stride length on the left side, average stride length on the right side, average footprint intensity, average footprint area, and hind leg contact time in the mouse gait model experiment.

[0059] Figure 8 Intra-articular injection of CQ5 nanobodies reduced synovial inflammation and cartilage damage in a mouse model of osteoarthritis. Figure 8 A shows the H&E staining results of the knee joint of MMT mice after administration of CQ5 nanobody; Figure 8 B shows the Safranin-Fix Green staining results of the knee joint of MMT mice after nanobody administration; Figure 8 C represents the results of synovial inflammation score and number of inflammatory cells infiltrating the knee joint of MMT model mice after treatment with nanobodies; Figure 8 D represents the OARSI score and chondrocyte count of the knee joint in MMT model mice after nanobody treatment.

[0060] Figure 9 Intra-articular injection of CQ5 nanobody reduced the levels of sIL-17RD, TNF-α, and IL-6 in the synovial fluid of osteoarthritis model mice. Figure 9 A represents the sIL-17RD protein level in the synovial fluid of MMT model mice after nanobody treatment; Figure 9 B represents the IL-6 protein level in the synovial fluid of MMT model mice after nanobody treatment; Figure 9 C represents the TNF-α protein level in the synovial fluid of MMT model mice after nanobody treatment.

[0061] Figure 10 Intra-articular injection of CQ5 nanobody reduced pyroptosis of chondrocytes in a mouse model of osteoarthritis. Figure 10 A shows the results of Casp-1 / DAPI staining on knee joint sections of MMT mice after nanobody treatment; Figure 10 B shows the pyroptosis marker N-GSDMD / DAPI staining results of knee joint sections from MMT mice after nanobody treatment. Figure 10 C represents the percentage of active Casp-1 positive cells in the knee joint of MMT model mice after nanobody treatment and the average fluorescence intensity of active Casp-1. Figure 10 D represents the percentage of active N-GSDMD-positive cells in the knee joint of MMT model mice after nanobody treatment and the average fluorescence intensity of active N-GSDMD.

[0062] Figure 11 Intra-articular injection of CQ5 nanobodies reduced pro-inflammatory responses in chondrocytes, extracellular matrix degradation, and expression of P2RX7-NLRP3 inflammasome-related proteins in osteoarthritis model mice. Figure 11 A represents the levels of Cox2 and Il6 mRNA in the articular cartilage tissue of MMT model mice after treatment with nanobodies; Figure 11 B represents the level of Mmp13 mRNA in the articular cartilage tissue of MMT model mice after treatment with nanobodies. Figure 11C represents the mRNA levels of P2RX7-NLRP3 inflammasome-related genes, including P2rx7, Nlrp3, Asc, and Casp1, in the articular cartilage tissue of MMT model mice after nanobody treatment.

[0063] Figure 12 CQ5 nanobody inhibits TNF-α-induced P2RX7-NLRP3 inflammasome-mediated pyroptosis in chondrocytes. 12A-12D involved knocking down P2RX7 in C28 chondrocytes, affecting TNF-α-induced sIL-17RD-mediated P2RX7 expression, pyroptosis marker N-GSDMD and its total protein levels, and IL-1β levels in the supernatant. 12E-12F involved adding the NLRP3 inhibitor Dapansutrile to C28 chondrocytes, affecting TNF-α-induced sIL-17RD-mediated pyroptosis marker N-GSDMD and its total protein levels. 12G-12I involved adding the control protein GFP, CQ5 nanobody, celecoxib, and triamcinolone to C28 chondrocytes, affecting TNF-α-induced sIL-17RD-mediated pyroptosis marker N-GSDMD and its total protein levels.

[0064] Figure 13 CQ5 nanobody reverses P2RX7-mediated inhibition of chondrocyte proliferation. 13A-13C show the effect of P2RX7 knockdown in C28 chondrocytes on TNF-α-induced sIL-17RD-mediated Ki67-positive chondrocyte proliferation; 13A shows Ki67 / DAPI counterstaining; 13B shows statistical results of Ki67-positive chondrocytes; 13C shows Q-PCR detection in C28 cells. P2RX7 Knockdown efficiency; 13D-13E promotes chondrocyte proliferation in osteoarthritis model mice by intra-articular injection of CQ5 nanobodies, among which... Figure 13 D represents the Ki67 / DAPI immunofluorescence staining results of the knee joint of MMT model mice after nanobody treatment; Figure 13 E represents the percentage of Ki67-positive chondrocytes.

[0065] Figure 14 Intra-articular injection of CQ5 nanobodies reduced the number of subchondral osteoclasts in a mouse model of osteoarthritis. Figure 14 A shows the TRAP staining results of the knee joint of MMT model mice after nanobody treatment; Figure 14 B represents the statistical results of the number of osteoclasts in A.

[0066] Figure 15Tissue safety evaluation of intra-articular injection of CQ5 nanobody: H&E staining analysis of heart, liver, spleen, lung, kidney and brain of MMT model osteoarthritis mice after 6 injections of high dose CQ5 (1.25 mg / kg) and control GFP protein. Detailed Implementation

[0067] For ease of understanding, the following explains some key terms in this embodiment: Soluble interleukin-17 receptor D (sIL-17RD) refers to the protein form of interleukin-17 receptor D (IL-17RD) that is released from the cell membrane or secreted into the extracellular space through mechanisms such as proteolytic cleavage or selective splicing. sIL-17RD is a soluble circulating protein or localized cytokine present in bodily fluids and possesses active biological functions, such as enhancing the activation of inflammatory signaling pathways like NF-κB and promoting the production and release of pro-inflammatory cytokines, thereby amplifying and maintaining the inflammatory response.

[0068] Nanobodies (Nb) are used interchangeably with heavy chain single-domain antibodies (VHH), specifically referring to antibody variable domains derived from camels that naturally lack the light chain and the first constant region (CH1). These nanobodies consist of a single polypeptide chain of approximately 110-130 amino acid residues, with a molecular weight of approximately 12-15 kDa, and retain full antigen-binding capacity. These nanobodies exhibit high solubility, high stability, low immunogenicity, and excellent tissue penetration.

[0069] The heavy chain variable region (VHH) is the core functional domain of the nanobody, responsible for recognizing and binding antigens. The VHH consists of multiple complementarity-determining regions (CDRs) and framework regions (FRs), where CDRs directly participate in antigen binding, while FRs provide a structural scaffold.

[0070] The heavy chain complementarity-determining region 3 (CDR-H3) is a key loop region in the nanobody VHH that has a highly variable sequence and interacts directly with the antigen. The CDR-H3 is typically longer and more conformationally extended than that of conventional antibodies, enabling it to penetrate and bind to antigenic epitopes that are difficult for conventional antibodies to reach.

[0071] Specific binding refers to the strength and selectivity of the interaction between a nanobody or its variant and the target antigen sIL-17RD. This binding means that the nanobody binds to sIL-17RD with significantly higher affinity than to unrelated molecules. This specific binding can be verified using techniques such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR).

[0072] Neutralizing biological activity refers to the ability of nanobodies to inhibit or block the biological function of sIL-17RD. This neutralization effect may be achieved through mechanisms such as steric hindrance, conformational changes, or accelerated clearance, thereby effectively inhibiting sIL-17RD-mediated signaling pathway activation and reducing the expression levels of inflammatory factors. This neutralizing activity can be evaluated through cellular function experiments.

[0073] Example 1 This embodiment provides a method for screening neutralizing nanobodies targeting soluble interleukin-17 receptor D (sIL-17RD).

[0074] The nanobodies can be obtained in various ways. For example, heavy chain antibody genes can be isolated from peripheral blood lymphocytes of camelids through immunization, followed by screening using a phage display library. Alternatively, fully synthetic or semi-synthetic antibody libraries can be used to screen for nanobodies that can bind to sIL-17RD through in vitro screening techniques such as phage display, yeast display, or ribosome display. In this embodiment, a fully synthetic nanobodies phage library is used for screening. During the screening process, human sIL-17RD protein can be coated on a solid phase, and then the library can be panned to enrich clones with strong binding ability.

[0075] The heavy chain variable region (VHH) of the nanobody includes a heavy chain complementarity-determining region 3 (CDR-H3) that specifically binds to sIL-17RD. CDR-H3 is a key region in the VHH structure that directly interacts with the antigen, and its sequence diversity plays a decisive role in binding specificity and affinity. The CDR-H3 can be designed and optimized using methods such as random mutation or directed evolution to obtain binding ability to sIL-17RD. For example, a VHH library containing a randomized CDR-H3 region can be constructed, and clones with binding activity can be obtained through screening.

[0076] The CDR-H3 amino acid sequence has specific characteristics, with its length limited to 10 amino acids. This length range of CDR-H3 can be achieved in various ways. For example, during the construction of a VHH library, oligonucleotide primers can be designed and synthesized to introduce random sequences into the gene region encoding CDR-H3, and the length of these random sequences can be controlled to be within 10 amino acids. Alternatively, CDR-H3 sequences with the specified length range can be screened from the heavy chain antibody genes of natural camel species.

[0077] Furthermore, the amino acid sequence of the CDR-H3 contains 2-6 consecutive lysine (K) residues. The introduction of these consecutive lysine residues can be achieved through gene synthesis or site-directed mutagenesis. For example, when designing a random sequence for CDR-H3, consecutive lysine coding sequences can be intentionally introduced at specific positions. Alternatively, for a screened VHH sequence, PCR-mediated site-directed mutagenesis can be used to introduce or adjust the number and position of lysine residues in the CDR-H3 region to satisfy the characteristic of 2-6 consecutive lysine residues.

[0078] Therefore, the nanobody can specifically bind to sIL-17RD and neutralize its biological activity. This specific binding ability can be verified through various in vitro experiments, such as detecting the binding signal between the nanobody and sIL-17RD using ELISA and comparing it with the binding signal of irrelevant proteins. The neutralization of its biological activity can be assessed through cellular functional experiments, such as observing the inhibitory effect of the nanobody on the release of TNF-α-induced macrophage inflammatory factors or activation of signaling pathways in a sIL-17RD-induced macrophage inflammation model. Specifically, in an in vitro cell culture system, sIL-17RD and the nanobody are co-incubated, and then changes in intracellular or extracellular inflammation-related markers are detected to evaluate the neutralizing effect of the nanobody.

[0079] Specifically as follows: 1. The amplification process of the fully synthesized nanobody library is as follows: a) The phage library was stored at -80°C; b) 50 μL of library (10) in an Erlenmeyer flask 11 (pfu / mL) + 30 mL 4 YT + 30 mL 4% glucose (preheated to 37℃ before adding library and 60 μL Amp); c) Shake at 37℃ and 230 rpm until OD600 = 0.4-0.6 (approximately 1 hour; the bacterial concentration at OD600 = 0.4-0.6 is approximately 10). 7 -10 8 about); d) Aliquot the bacterial culture into two 50 mL centrifuge tubes using a 5 mL pipette, adding 10 mL of the solution to each tube. 10 M13K07 (multiple of infection 20:1; M13K07 was stored at -80℃, and the titer after freezing needs to be re-determined). e) Let stand at 37℃ for 45 min, centrifuge at 3000g at 4℃ for 10 min, discard the supernatant, and resuspend the precipitate in 60 mL of 2 YT-AK (Amp 100 μg / mL + Kana 50 μg / mL); f) Shake overnight at 30℃ and 230 rpm for 12-15 hours; g) On the second day, dispense the bacterial culture into two 50 mL sterile centrifuge tubes using a pipette; h) Centrifuge at 4℃, 15000 rpm for 30 min, transfer the supernatant to two new 50 mL sterile centrifuge tubes, add 1 / 4 volume of 20% PEG / NaCl to the supernatant, mix by inverting, let stand at 4℃ for 4 h, centrifuge at 4℃, 15000 rpm for 30 min. i) Discard the supernatant, resuspend the precipitate in 1 mL PBS, and transfer it to a 1.5 mL EP tube; j) Centrifuge at 4℃ and 13000 rpm for 20 min; k) Transfer the supernatant to a new 1.5 mL EP tube, add 1 / 4 volume of 20% PEG / NaCl to the supernatant, and let stand at 4℃ for 10 min; l) Centrifuge at 4℃, 13000 rpm for 10 min, discard the supernatant, and resuspend the precipitate in 200 μL PBS; 2. The process of phage amplification is as follows: a) Mix 100 μL M13K07 + 900 μL TG1 (OD=0.4-0.6), inoculate at 37℃ for 45 min; b) Add the infected bacterial culture to 50 mL of 2YT, shake at 37°C and 230 rpm for 1 h, then add 51 μL of KANA and shake overnight at 230 rpm and 30°C. c) On the second day, the bacterial solution was aliquoted into two 50 mL sterile centrifuge tubes, with each tube containing 25 mL of bacterial solution. The tubes were then centrifuged at 4°C and 5000 rpm for 30 min. d) Transfer the supernatant to two new 50 mL sterile centrifuge tubes, add 6.25 mL of 20% PEG / NaCl, and incubate at 4°C for 2 h; e) Centrifuge at 4℃ and 5000 rpm for 30 min, discard the supernatant completely, resuspend the precipitate in 1 mL PBS, transfer to a sterile 1.5 mL EP tube (all EP tubes for this step are pre-cooled on ice), and centrifuge at 4℃ and 13000 rpm for 20 min. f) Transfer the supernatant to a new 1.5 mL EP tube, add 300 μL of 20% PEG / NaCl, and incubate at 4°C for 10 min. g) 4℃ 13000 rpm 10 min, discard the supernatant, and resuspend the precipitate in 200 μL of PBS. 3. The phage library enrichment process is as follows: a) Antigen coating: The antigen protein ( Figure 1 A, human sIL-17RD protein) was diluted to 100 μg / mL with CBS buffer, 100 μL of antigen protein per well, and incubated overnight at 4°C; b) Wash: Discard coating solution, add 200 μL 0.1% PBST / well, wash 4 times for 1 min each time; c) Closure: 100 μL 5% skim milk powder / well incubated at 37℃ for 2 h; d) Library pre-incubation: Mix the library with 5% skim milk powder and incubate at room temperature by rotation for 1 h; e) Wash: Discard the blocking solution, add 200 μL 0.1% PBST / well, wash 4 times for 1 min each time; f) Library incubation: 100 μL of pre-incubated library / well, incubated at room temperature for 1 h, then incubated at 37℃ for 1 h; g) Wash: Discard the library, 200 μL 0.1% PBST / well, wash 5 times for 1 min each time; h) Library elution: 100 μL elution buffer / well, room temperature for 8 min; i) Library neutralization: Add the eluted library to 335 μL of neutralization solution; k) Add all the eluted library to 8 mL of TG1 (OD=0.4-0.6) and let stand at 37°C for 45 minutes; l) Take 100 μL of diluted bacterial solution and plate it to determine the library titer. Centrifuge the remaining bacterial solution at 3000 g at 4℃ for 20 min. Discard the supernatant completely. Resuspend the bacterial solution precipitate in 200 μL of 2 YT and plate it on two YT-AG plates. Incubate overnight at 37℃ with the plates inverted. m) On the second day, add 2 mL of 2 YT to the plate, scrape the bacterial culture off with a cell scraper and add it to a 50 mL centrifuge tube, centrifuge at 3000 g, 4℃ for 20 min; n) Discard the supernatant completely, resuspend the precipitate in 2 mL of 2YT, and add a portion of the bacterial culture to 30 mL of pre-warmed 4YT + 30 mL of 4% glucose to make the OD600 of the liquid < 0.1. The required library volume can be calculated from the OD value. After adding the library, add Amp, and the subsequent steps are the same as for library amplification. After 4 rounds of phage library panning (the panning antigen coating concentration and elution conditions are detailed in Table 1), the phages bound to sIL-17RD were effectively enriched, reaching 21-fold (the enrichment factor is detailed in Table 1). Figure 2 B).

[0080] The conditions for phage enrichment are listed in Table 1: Table 1. Phage enrichment conditions

[0081] 4. The phage ELISA experiment is as follows: After successful phage enrichment, single clones were randomly selected for phage ELISA identification. The specific process is as follows: a) The night before, the antigen was coated with 500 ng / well. Two wells were set up for the experimental group and two wells for the control group. The control group was coated with CBS and the experimental group was coated with antigen. b) Discard the coating solution, wash the plate, add 200 μL of 0.1% PBST to each well, wash three times for 1 min each time; c) 100 μL of 5% BSA (prepared with PBS) per well, block at room temperature for 2 hours; d) Discard the sealing solution, wash the plate, and repeat as above; e) Add 100 μL of phage supernatant per well and incubate at 37°C for 1 hour (seal the plate with sealing film; it is not necessary to place it in a humidifier). Remember the order of sample addition. f) Discard the phage supernatant, wash the plate, add 200 μL of 0.1% PBST to each well, and wash three times for 1 min each time; g) Add 100 μL of secondary antibody per well (the secondary antibody dilution solution is 0.1% BSA in PBS) and incubate at 37°C for 1 hour; h) Discard the secondary antibody, wash the plate, add 200 μL of 0.1% PBST to each well, and wash three times for 1 min each time; i) Add 100 μL of colorimetric reagent per well and develop color in the dark for 15-30 min; j) Add 100 μL of stop solution per well and measure OD450.

[0082] Our comparison of the phage binding sIL-17RD results across the four rounds revealed that, after enrichment, the phage binding sIL-17RD in the fourth round was more than twice that of the negative control (see details). Figure 1 C).

[0083] Next, the phages from the fourth round of enrichment were selected, and positive single clones were screened using ELISA. We selected 24 clones, labeled CQ1 to CQ24. ELISA results showed that the binding affinity of all 24 single clones to sIL-17RD was more than 10-fold higher than that of the negative control (see details). Figure 1 D).

[0084] Example 2 This embodiment provides a method for constructing and expressing an expression vector for a neutralizing nanobody targeting sIL-17RD.

[0085] 1. Nanobody phage sequencing and sequence alignment analysis: Next, CQ5, CQ6, CQ11, and CQ13, with ELISA binding folds exceeding 20, were selected for gene sequencing. Amino acid sequence alignment analysis was performed on the sequencing results, as shown below. Figure 2 As shown in Figure A. During the comparison, we found that the nanobodies targeting sIL-17RD have the following characteristics: Besides the nanobodies' backbone, CDR-1, CDR-2, and CDR-3 share the following characteristics: They have similar sequence features. Including: CDR-3 contains 10 amino acids and multiple consecutive K motifs (2-6); CDR-1 contains the IFS*Y motif; CDR-2 contains the VA*I motif, etc.

[0086] 2. Prokaryotic expression process of nanobodies: The target fragment VHH sequence was amplified by PCR and then inserted into the prokaryotic expression plasmid pDB146 to construct the recombinant expression plasmid. Protein expression was then performed using the BL21 prokaryotic expression system (see the construction flowchart for details). Figure 2 B); a) Slice BL21 Escherichia coli containing pDB146 plasmid from -80℃ onto LB+Amp plates and incubate overnight at 37℃; b) Pick a single clone into 8 mL of LB + Amp (1:1000) medium and shake until saturated (37℃, 240 rpm). c) Propagation: Take 4 mL of saturated bacterial culture and incubate in 800 mL LB (Amp) at 37℃ until OD600 = 0.8 (0.6-1.0) (>3h, measure OD at 3h); d) IPTG induction (final concentration 0.1 mM, i.e., 1 M IPTG - 1:10000 added), 16℃, 230 rpm, induction for 18 h.

[0087] Example 3 This embodiment provides a prokaryotic expression and purification method for sIL-17RD neutralizing nanobodies, as detailed below: a) Collect cells by centrifuging at 4000 rpm for 10 min (note whether the supernatant is pure; if it is not pure, it indicates that the centrifugation was incomplete). b) Resuspend the cells in 30 mL of pre-cooled sterile ddH2O into a new 50 mL tube. Centrifuge again at 4000 rpm for 10 min. c) Resuspend the precipitate obtained in the previous step in 30 mL of pre-cooled Buffer A (30 mM Tris-HCl, pH 8.0, 20% Sucrose) (during this step, the cell clumps should feel relatively loose and easily dispersed). Add 300 μL of 100 mM EDTA, pH 8.0 (final concentration 1 mM), and pour into a clean (sterilized and rinsed with double-distilled water) conical flask. Stir magnetically at room temperature for 10 min at low speed, just enough to keep the rotor rotating. After magnetic stirring, pour the liquid from the conical flask back into a 50 mL tube. Centrifuge at 4°C, 10000 g, for 10 min, and carefully remove the supernatant. d) The cells were completely resuspended in 30 mL of pre-cooled 5 mM MgSO4 and poured into a washed Erlenmeyer flask. The Erlenmeyer flask was placed in a container filled with ice (such as a large beaker), and the mixture was slowly stirred with a magnetic rotor on the ice for 10 min. During this process, the target protein was released into the solution. e) After magnetic stirring, pour the liquid from the conical flask back into the 50 mL tube. Incubate at 10000 g for 10 min at 4°C. Replace with a standard centrifuge rotor. Transfer the supernatant to a new 50 mL tube, labeled Lysates. Add 1.5 mL of 1 M NaH₂PO₄, 3.6 mL of 2.5 M NaCl, and 150 μL of 2 M imidazole to the collected supernatant, approximately 30 mL of Lysates. f) Take 300 μL of Ni beads suspension and wash three times with lysis buffer. Add 1 mL of lysis buffer each time, mix by inverting, centrifuge at 5000 g for 1 min, and discard the supernatant; g) Add the washed Ni beads to a 50 mL tube containing lysates and incubate at 4°C for 2 h by rotation. h) Pre-cool the large centrifuge to 4°C. Centrifuge at 3000 g for 5 min. Transfer the supernatant to a new 50 mL centrifuge tube; this supernatant is the effluent FL. i) Add 15 mL of washing buffer to a Lysates tube (still containing beads) and pour it into a clean chromatography column. When the liquid in the column is almost completely drained, add another 15 mL of washing buffer to a 50 mL tube and pour it into the column. Repeat this washing process three times. j) Add 300 μL of Elution buffer to elute, repeat 4 times (total 1.2 mL), and collect the eluent in an enzyme-free EP tube.

[0088] k) After the sample is collected and concentrated, it is subjected to Western blot and Coomassie Brilliant Blue staining.

[0089] The results are as follows Figure 3 A, Figure 3 B, Figure 3 C and Figure 3 As shown in D, CQ5, CQ13, CQ6, and CQ11 nanobody proteins with high purity and concentration were obtained after purification.

[0090] Example 4 This embodiment provides an EC50 affinity analysis method for sIL-17RD neutralizing nanobodies. The specific methods for analyzing the binding of CQ5, CQ6, CQ13, and CQ11 to the sIL-17RD protein are as follows: a) Dilute sIL-17RD protein to 4 μg / mL 24 h in advance and add it to a high affinity microplate at a rate of 100 μL / well. Place the microplate in a humidified chamber and coat it at low temperature overnight. 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 anti-M13-HRP (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 colorimetric reagent, incubate at room temperature in the dark for 15 min, then add 2 M H2SO4 to stop the color development, and measure OD450 with a microplate reader. ii) Use Log 10 [Nanobody (nM)] was fitted with OD450, and the EC50 affinity values ​​were calculated respectively.

[0091] The results are as follows Figure 3 E, the results indicate that the nanobody CQ5 (EC 50 = 0.308 μM) has a high affinity for sIL-17RD, while the nanobody CQ13 (EC) 50 = 6.151 μM), CQ6 (EC 50 = 5.406 μM) and CQ11 (EC50 The affinity of the IL-17RD protein is relatively weak (9.874 μM).

[0092] Example 5 This embodiment provides an in vitro activity verification method for neutralizing nanobodies targeting sIL-17RD.

[0093] 1. Replacement in IL-17RD knockout macrophages IL17RD Gene analysis was conducted on CQ5 and CQ13 nanobodies to neutralize TNF-α-induced endogenous sIL-17RD levels and reduce inflammatory cytokine levels. a) Seed macrophages derived from primary bone marrow of IL-17RD knockout mice into plates 24 hours in advance; b) Electroporate back the plasmid expressing GFP and human IL-17RD genes (pcDNA3.1 vector), and continue to culture the transfected cells overnight; c) Pre-treat the cells 2 hours before the next day: add CQ5 and CQ13 nanobodies to the cell supernatant at a final concentration of 1 μg / mL; d) After 2 h, TNF-α was added for stimulation, and the cell supernatant was collected after 24 h. e) ELISA was used to detect the levels of sIL-17RD, IL-6 and TNF-α in the supernatant.

[0094] The results are as follows Figure 4 As shown in Figure A, human cells were reintroduced into macrophages that had IL-17RD knocked out. IL17RD Gene stimulation with TNF-α significantly promoted endogenous sIL-17RD levels, while treatment with CQ5 and CQ13 significantly reduced sIL-17RD levels. Correspondingly, in macrophages with IL-17RD knockout, human [recombinant human IL-17RD] was added... IL17RD Gene stimulation and TNF-α stimulation significantly increased the levels of pro-inflammatory cytokines IL-6 and TNF-α, while the addition of CQ5 and CQ13 significantly reduced the production of IL-6 and TNF-α cytokines (see details). Figure 4 (B-4C). Among the two nanobodies, CQ5 was superior to CQ13 nanobodies in reducing sIL-17RD, IL-6, and TNF-α (see details). Figure 4 A-4C).

[0095] 2. In primary bone marrow-derived macrophages from C57BL / 6 mice, the effects of CQ5 neutralizing nanobody on TNF-α-induced endogenous sIL-17RD and inflammatory factors were analyzed: a) Extraction and induction of primary macrophages derived from mouse bone marrow, the specific procedures are as follows: 1. 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 antibodies, PBS, and red blood cell lysis buffer were then added. 2. Place the separated femur and tibia into a mortar, add 5 mL of culture medium, and grind. Do not rotate or press, as this will break the cells. Take the suspension of culture medium and bone marrow cavity into a 15 mL centrifuge tube and centrifuge at 1200 rpm for 5 min. 3. 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. 4. Centrifuge at 1200 rpm for 5 min. White cell pellet will be visible at the bottom of the tube. Resuspend the cells in 3 mL of culture medium and mix well. Seed the cells into Pretri plates, 10 mL of culture medium per plate, and add M-CSF. After three days, add 5 mL of culture medium. The desired cells will be obtained around the fifth day. Subsequent treatment with antibodies or proteins will be performed. b) Seed the primary bone marrow-derived macrophage line from C57BL / 6 mice into plates 24 h in advance. c) Electroporate back the plasmid expressing GFP and human IL-17RD genes (pcDNA3.1 vector), and continue to culture the transfected cells overnight; d) Pre-treat the cells 2 hours before the next day: add different concentrations of CQ5 nanobodies to the cell supernatant; e) After 2 h, TNF-α was added for stimulation, and the cell supernatant was collected after 24 h. f) ELISA was used to detect the levels of sIL-17RD, TNF-α, and IL-10 in the supernatant; g) Log 10 Based on the concentrations of CQ5 (ng / mL), sIL-17RD, TNF-α, and IL-10, the inhibition rates of sIL-17RD and TNF-α, and the activation rate of IL-10 were calculated.

[0096] The results are as follows Figure 4 DE showed that CQ5 significantly inhibited TNF-α-induced macrophage secretion of sIL-17RD and the production of pro-inflammatory factor TNF-α, while promoting the production of anti-inflammatory factor IL-10.

[0097] 3. Replacement in IL-17RD knockout chondrocytes IL17RD Gene analysis was conducted on CQ5 and CQ13 nanobodies to neutralize TNF-α-induced endogenous sIL-17RD levels and reduce inflammatory cytokine levels. a) Dissociation and culture of primary chondrocytes: 1. Animal handling and disinfection: After euthanizing 8-week-old male C57BL / 6 mice, they were disinfected by immersing them in 75% ethanol for 10 minutes; 2. Tissue separation: Under aseptic conditions, the soft tissues around the femoral head and tibial plateau are carefully dissected under an anatomical microscope; 3. Cartilage processing: Collect cartilage tissue, cut it into small fragments, and wash it twice with phosphate-buffered saline (PBS) containing 1% penicillin / streptomycin; 4. Primary digestion: Add 0.25% trypsin at 5 times the tissue volume and digest at 37°C for 30 minutes; 5. Washing: After centrifuging at 200g for 5 minutes, discard the supernatant and wash the tissue pellet with PBS; 6. Secondary digestion: Add 2% type II collagenase solution prepared with complete culture medium at a ratio of 1:3, and digest overnight at 37°C; 7. Cell collection: After centrifugation at 200g, collect the supernatant containing free chondrocytes. Repeat digestion of the remaining cartilage fragments under the same conditions until no new cells are separated. 8. Cell suspension treatment: Combine all cell suspensions, centrifuge at 1200g, resuspend the cells in complete culture medium, and then seed them into 25 cm² culture flasks; 9. Cell culture: Place the cells in a humidified incubator at 37°C and 5% CO2 for culture; 10. Cell Expansion and Use: Primary chondrocytes were allowed to adhere and expand to 80% confluence. To ensure phenotypic stability, subsequent experiments used only first-generation (P1) and second-generation (P2) cells.

[0098] b) Seed primary chondrocytes from IL-17RD knockout mice into plates 24 hours in advance; c) Electroporate back the plasmid expressing GFP and human IL-17RD genes (pcDNA3.1 vector), and continue to culture the transfected cells overnight; d) Pre-treat the cells 2 hours before the next day: CQ5 and CQ13 nanobodies were added to the cell supernatant at a final concentration of 1 μg / mL; e) After 2 h, TNF-α was added for stimulation, and the cell supernatant was collected after 24 h. f) ELISA was used to detect the levels of sIL-17RD, IL-6 and TNF-α in the supernatant.

[0099] The results are as follows Figure 5 As shown, human cells were reintroduced into primary chondrocytes with IL-17RD knocked out. IL17RDGene stimulation with TNF-α significantly promoted endogenous sIL-17RD levels, while treatment with CQ5 and CQ13 significantly reduced sIL-17RD levels. Correspondingly, in chondrocytes with IL-17RD knockout, human [recombinant human IL-17RD] was added... IL17RD Gene stimulation and TNF-α stimulation significantly promoted the levels of IL-6 and TNF-α pro-inflammatory cytokines, while the addition of CQ5 and CQ13 significantly reduced the production of IL-6 and TNF-α inflammatory cytokines.

[0100] Example 6 This embodiment provides a method for pharmacodynamic evaluation and half-life determination of a mouse model of osteoarthritis treated with sIL-17RD neutralizing nanobody. Details are as follows: 1. In a mouse model of MMT (medial collateral ligament transection + medial meniscectomy) osteoarthritis, evaluate the effects of CQ5 and CQ13 on synovitis, cartilage damage, etc. a) C57BL / 6 mice were divided into the following groups: 1. Sham-operated group (n=4); 2. MMT+GFP control protein (0.25 mg / kg) (n=6); 3. MMT+CQ5 (0.25 mg / kg) (n=6); 4) MMT+CQ13 (0.25 mg / kg) (n=6). The specific design is as follows: Figure 6 A; b) Perform a sham surgery or medial collateral ligament transection + medial meniscectomy (MMT) osteoarthritis model on day 0. Specifically: Weigh the C57BL / 6 mice before anesthesia and calculate the required amount of anesthetic based on weight. Anesthetize the mice using a 1 mL sterile syringe at a dosage of 1 g / 10 μL. After catching the mice, place them 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. After anesthesia, shave the hair from the webbed feet to the joints on the hind limbs, and clean away any excess hair with 75% ethanol. Use forceps to pick up a sterile cotton ball, soak it in iodine, and disinfect the exposed skin of the mouse. Routinely select a midline incision at the knee joint, flex the knee at 45°, and make an incision about 1.0 cm deep on the medial side of the patellar ligament. Incise the superficial fascia layer by layer 0.5 cm above the joint space. Begin cutting the vastus medialis muscle at a depth of 1 cm, extending to the medial side of the tibial insertion of the patellar ligament. Continue cutting until you reach the femur. When making the incision in the lower half of the muscle, the knee should be flexed, avoiding the medial collateral ligament. 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 flexion. Avoid cleaning the infrapatellar bursa as it may cause bleeding 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. Separate the medial collateral ligament in the eversion position, and after transversely cutting it with a sharp scalpel, enter the medial joint space in extreme flexion and external rotation. Separate the meniscus from anterior to medial and then to the posterior edge. The location is very deep. Carefully cut the posterior horn of the medial meniscus, taking care to protect the cartilage tissue and the anterior and posterior cruciate ligaments. Irrigate the joint, return the patella to its original position, and suture the incision layer by layer. Clean the blood near the wound promptly; use tweezers to take a sterile 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 any breathing difficulties; if any occur, provide immediate resuscitation.

[0101] c) Subsequently, the mice were injected into the joint cavity every seven days at the dosage described in a).

[0102] d) The mice were euthanized on day 33, and the joint fluid and tibia were collected and cryopreserved.

[0103] e) Arthritis-like symptoms were analyzed using ELISA to assess sIL-17RD, TNF-α, and IL-6 levels.

[0104] f) Tibial bone fixation followed by embedding, sectioning, H&E staining, and safranin-fast green staining, etc.

[0105] g) Statistical analysis of H&E staining results and analysis of synovitis scores; statistical analysis of safranin-fixed green staining and analysis of OARSI grading, etc.

[0106] 2. Detection of sIL-17RD and inflammatory factor levels in synovial fluid This study detected the levels of inflammatory factors involved in the experiment. Specifically, the levels of sIL-17RD, TNF-α, IL-6, and IL-10 in the samples were measured using a commercially available double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit. The procedure is briefly described below. The instructions for each kit are similar, and ELISA detection is mentioned in many places throughout the text, so it will not be described in detail again: a) Dilute the capture antibody to 2.0 μg / mL with phosphate-buffered saline (PBS), add 100 μL of the diluted capture antibody to each well of the ELISA plate, seal the plate and incubate 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.

[0107] The results are as follows Figure 6 As shown in B-6C, compared to the control sham surgery, the levels of sIL-17RD, TNF-α, and IL-6 in the synovial fluid of MMT model mice were significantly increased, while they were significantly decreased in the CQ5 and CQ13 treatment groups. This indicates that the nanobodies CQ5 and CQ13 can significantly inhibit the levels of sIL-17RD, TNF-α, and IL-6 in the joint cavity of MMT model mice. Comparing the treatment effects of CQ5 and CQ13, we found that CQ5 was still better than CQ13 in reducing the levels of sIL-17RD and the inflammatory factors TNF-α and IL-6 in vivo.

[0108] 3. Histopathological analysis of the knee joint This embodiment describes the pathological analysis of the knee joint tissue of MMT model mice. The specific procedures are as follows: 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.

[0109] The degree of cartilage degeneration was assessed according to the modified Pritzker's OARSI (International Society for the Study of Osteoarthritis) grading-staging system. The grading criteria (cartilage structural damage) are as follows: 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.

[0110] 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.

[0111] 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).

[0112] 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”.

[0113] Experimental results are as follows Figure 6 D-6G, H&E staining, and synovitis scoring results revealed that CQ5 and CQ13 significantly inhibited MMT-induced synovitis. Figure 6 D-6E); Safranin-Fix Green staining and OARSI grading results showed that CQ5 and CQ13 significantly inhibited MMT-induced cartilage degeneration (D-6E); Figure 6 (F-6G). Compared with CQ5 and CQ13, CQ5 showed better inhibition of synovitis and cartilage degeneration. Based on the results of in vitro and in vivo experiments, we subsequently selected CQ5 neutralizing nanobodies for further functional validation experiments.

[0114] 4. Half-life analysis of CQ5 neutralizing nanobody after a single injection This embodiment tested the half-life of the neutralizing nanobody CQ5 after a single intra-articular administration in mice. The specific procedure is as follows: a) Dilute the nanobody to the working concentration using sterile PBS.

[0115] b) Fix the mice and inject the antibody into their joint cavity. The dosage is usually 0.5 mg / kg, and the volume is generally 10 µL. Record the accurate time and dosage of administration.

[0116] c) Time point (see details) Figure 6 H): Joint fluid was extracted from the corresponding anesthetized mice at 6 hours, 12 hours, 24 hours (1 day), 3 days, 5 days and 7 days after administration.

[0117] d) Immediately store the synovial fluid sample in a -80°C freezer to avoid repeated freeze-thaw cycles.

[0118] e) CQ5 is purified with a His tag, so the CQ5-His in the synovial fluid sample was detected using the His-tag enzyme-linked immunosorbent assay kit.

[0119] f) Reading: Immediately after the test is completed, use an ELISA reader to measure the absorbance of each well at a wavelength of 450 nm.

[0120] i) Calculate the CQ5 content in the synovial fluid sample by measuring the absorbance of each well based on the standard curve and at a wavelength of 450 nm.

[0121] The results are as follows Figure 6 As shown in Figure I, the half-life of the CQ5 nanobody after intra-articular injection is 7.6 days.

[0122] Example 7 This embodiment provides a method for detecting the analgesic effect of the sIL-17RD neutralizing nanobody CQ5 in a mouse model of osteoarthritis. Details are as follows: This embodiment performs behavioral detection (gait analysis), and the specific operation is as follows: a) C57BL / 6 mice were grouped as follows, with the specific design scheme as follows: Figure 7 A; 1. Sham surgery group, 5 animals; 2. MMT+GFP control protein (intra-articular injection, 0.25 mg / kg), 5 animals; 3. Low-dose MMT+CQ5 group (intra-articular injection, MMT+CQ5-L, 0.05 mg / kg), 5 animals; 4. Medium-dose MMT+CQ5 group (intra-articular injection, MMT+CQ5-M, 0.25 mg / kg), 5 animals; 5. High-dose MMT+CQ5 group (intra-articular injection, MMT+CQ5-H, 1.25 mg / kg), 5 animals; 6. MMT + Celecoxib, 26 mg / kg, administered by gavage daily starting on day 5, 5 animals; 7. MMT + triamcinolone acetonide, 0.75 mg / kg, intra-articular injection on days 3, 17, and 31, 5 animals; b) Start adaptive training 3 days before the formal test; place the mice in a simulated gait channel and let them walk freely for 5 minutes, and continue training for 3 consecutive days; c) A pre-run will be conducted on the day of the test to eliminate invalid trajectories (such as stopping midway, jumping, turning); the experimental environment will be kept constant: dark throughout the test, background noise <45 dB, and room temperature 22–25°C.

[0123] d) Use an automated gait analysis system (CatWalk XT system) for detection; set channel parameters: length ≥ 100 cm, width ≤ 5 cm to ensure the mouse walks in a straight line.

[0124] e) The mouse is placed at the starting end, and its effective gait sequence as it traverses the passage is automatically recorded.

[0125] f) Define the criteria for a valid run: a continuous walking distance ≥20 cm, speed fluctuation <50%, and at least 3 complete step cycles; record 3–5 valid runs for each mouse and take the average as the final data.

[0126] The results are as follows Figure 7 B Figure 7 As shown in Figure C, compared with the control sham-operated group, MMT osteoarthritis mice exhibited reduced stride length on both sides, weakened average footprint intensity, reduced average footprint area, and significantly shortened hind leg ground contact time. After injection of three doses of CQ5, both the medium and high dose groups improved MMT-induced symptoms, while the low dose group showed partial improvement. In addition, compared with the oral analgesic celecoxib and the intra-articular injection of the nonsteroidal anti-inflammatory drug triamcinolone, the medium and high doses of CQ5 were significantly better in improving MMT-induced gait changes. These results indicate that CQ5 has a better analgesic effect.

[0127] Example 8 This embodiment provides a method for treating synovitis and cartilage damage in a mouse model of osteoarthritis by comparing the sIL-17RD neutralizing nanobody CQ5 with clinically used osteoarthritis drugs celecoxib and triamcinolone. Details are as follows: a) For C57BL / 6 mice, an MMT model was established, and the injection protocol was as before. The mice were grouped as follows. 1. Sham surgery group, 5 animals; 2. MMT+GFP control protein (intra-articular injection, 0.25 mg / kg), 5 animals; 3. Medium-dose MMT+CQ5 group (intra-articular injection, MMT+CQ5, 0.25 mg / kg), 5 animals; 4. MMT + Celecoxib, 26 mg / kg, administered by gavage daily starting on day 5, 5 animals; 5. MMT + triamcinolone acetonide, 0.75 mg / kg, intra-articular injection on days 3, 17, and 31, 5 animals; b) Histopathological analysis of the knee joint, including H&E and synovitis scores, number of inflammatory cells, safranin-fast green staining and OARSI grading, and chondrocyte count, see Example 6, Part 3.

[0128] The results are as follows Figure 8As shown in A-8D, intra-articular injection of CQ5 significantly improved MMT-induced synovitis (including increased synovitis score and increased inflammatory cell infiltration) and cartilage degeneration (including increased OARSI grade and decreased chondrocyte number). Compared with the positive control drugs celecoxib and triamcinolone, CQ5 was comparable to celecoxib in inhibiting synovitis and superior to triamcinolone, with a significant difference. Compared with the positive control drugs celecoxib and triamcinolone, CQ5 nanobody was better than celecoxib in inhibiting cartilage damage, but the difference was not statistically significant. It was superior to triamcinolone, with a significant difference.

[0129] Example 9 This embodiment provides a method for analyzing the sIL-17RD and inflammatory factors in the synovial fluid of a mouse model of osteoarthritis by comparing the sIL-17RD neutralizing nanobody CQ5 with clinically used osteoarthritis drugs celecoxib and triamcinolone. Details are as follows: a) For C57BL / 6 mice, an MMT model was established, and the injection protocol was as before. The mice were grouped as follows. 1. Sham surgery group, 5 animals; 2. MMT+GFP control protein (intra-articular injection, 0.25 mg / kg), 5 animals; 3. Medium-dose MMT+CQ5 group (intra-articular injection, MMT+CQ5, 0.25 mg / kg), 5 animals; 4. MMT + Celecoxib, 26 mg / kg, administered by gavage daily starting on day 5, 5 animals; 5. MMT + triamcinolone acetonide, 0.75 mg / kg, intra-articular injection on days 3, 17, and 31, 5 animals; b) Method for detecting the content of sIL-17RD and inflammatory factors in synovial fluid, as described in Example 6, Section 2.

[0130] The results are as follows Figure 9As described in A-9C, compared to the control sham surgery, the levels of sIL-17RD, TNF-α, and IL-6 in the synovial fluid of MMT model mice were significantly increased, while they were significantly decreased in the CQ5 treatment group. This indicates that the nanobody CQ5 can significantly inhibit the levels of sIL-17RD, TNF-α, and IL-6 in the joint cavity of MMT model mice. Comparing the effects of CQ5 with the positive control drugs celecoxib and triamcinolone acetonide, we found that CQ5 reduced the levels of sIL-17RD and inflammatory factors TNF-α and IL-6 in vivo, which was still better than intra-articular injection of triamcinolone acetonide and comparable to long-term oral celecoxib. In addition, besides the significant inhibition of sIL-17RD levels by the CQ5 neutralizing nanobody, the positive control drugs celecoxib and triamcinolone acetonide also reduced the level of sIL-17RD in the synovial fluid. These results suggest that MMT induces a pro-inflammatory environment, promoting the production of sIL-17RD, an effect that is inhibited by anti-inflammatory drugs.

[0131] Example 10 This embodiment provides a method for detecting pyroptosis of chondrocytes in a mouse model of osteoarthritis by comparing the sIL-17RD neutralizing nanobody CQ5 with clinically used osteoarthritis drugs celecoxib and triamcinolone. Details are as follows: a) For C57BL / 6 mice, an MMT model was established, and the injection protocol was as before. The mice were grouped as follows. 1. Sham surgery group, 5 animals; 2. MMT+GFP control protein (intra-articular injection, 0.25 mg / kg), 5 animals; 3. Medium-dose MMT+CQ5 group (intra-articular injection, MMT+CQ5, 0.25 mg / kg), 5 animals; 4. MMT + Celecoxib, 26 mg / kg, administered by gavage daily starting on day 5, 5 animals; 5. MMT + triamcinolone acetonide, 0.75 mg / kg, intra-articular injection on days 3, 17, and 31, 5 animals; b) Immunofluorescence staining of joint sections for pyroptosis markers cleaved N-terminal GSDMD (N-GSDMS) and upstream active Caspase-1 (active Casp-1), the specific procedure is as follows: 1. Immerse the sections 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; 2. 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. 3. 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. 4. 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; 5. 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). 6. Remove the slides from 4℃ 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.

[0132] 7. 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 for 5 minutes each time in the dark.

[0133] 8. 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.

[0134] The results are as follows Figure 10 As shown, compared with celecoxib and triamcinolone, the CQ5 nanobody significantly reduced the levels of active Casp-1 and N-GSDMD, indicating that CQ5 is significantly more effective than celecoxib and triamcinolone in inhibiting MMT-induced chondrocyte pyroptosis.

[0135] Example 11 This embodiment provides a method for detecting chondrocyte inflammation, extracellular matrix degradation, and pyroptosis markers in a mouse model of osteoarthritis, comparing it with celecoxib and triamcinolone, both clinically used drugs for osteoarthritis. The specific details are as follows: a) For C57BL / 6 mice, an MMT model was established, and the injection protocol was as before. The mice were grouped as follows. 1. Sham surgery group, 5 animals; 2. MMT+GFP control protein (intra-articular injection, 0.25 mg / kg), 5 animals; 3. Medium-dose MMT+CQ5 group (intra-articular injection, MMT+CQ5, 0.25 mg / kg), 5 animals; 4. MMT + Celecoxib, 26 mg / kg, administered by gavage daily starting on day 5, 5 animals; 5. MMT + triamcinolone acetonide, 0.75 mg / kg, intra-articular injection on days 3, 17, and 31, 5 animals; b) RNA extraction and detection from cartilage tissue are as follows: 1. Cartilage tissue was transferred to 600 μL Trizol-Reagent and then to an RNA-free EP tube; 2. Add 120 μL of chloroform, shake vigorously, centrifuge at 12000 g for 15 min at 4 °C, take the upper aqueous phase containing RNA (about 250 μL), and add 350 μL of 70% ethanol diluted with DEPC water. 3. 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; 4. 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. Dissolve in treated DEPC water, volume 15 μL. 5. RNA concentration and 260 / 280 were determined using NanoDrop. After concentration determination, reverse transcription was performed according to the kit instructions to obtain cDNA, which was then detected by RT-Q-PCR. Results are as follows: Figure 11 As shown, compared with celecoxib and triamcinolone, the CQ5 nanobody treatment group had significantly lower levels of cartilage tissue. Cox2, Il-6, Mmp13 The mRNA levels of these drugs were significantly reduced, with effects comparable to long-term oral celecoxib and superior to intra-articular triamcinolone. Furthermore, the P2RX7-NLRP3 inflammasome-related gene in the CQ5 nanobody treatment group included… P2rx7 , Nlrp3 , Asc , Casp1 The mRNA level was also significantly reduced. These results indicate that CQ5 reduces cartilage inflammation, inhibits extracellular matrix degradation in chondrocytes, and suppresses chondrocyte pyroptosis by reducing the P2RX7-NLRP3 inflammasome-related gene.

[0136] Example 12 This embodiment provides an in vitro method for verifying P2rx7-mediated sIL-17RD-induced pyroptosis in chondrocytes and for inhibiting pyroptosis using the sIL-17RD-neutralizing nanobody CQ5. Details are as follows: a) The C28 chondrocyte cell line was seeded onto a plate the day before; b) On the second day, C28 cells were transfected with siRNAs from the control and P2RX7 cells via liposomes and cultured for another 24 h. Alternatively, nanobodies and sIL-17RD protein can be incubated overnight at 4 °C at a molar ratio of 1:1; the cells can be pretreated 2 h in advance the next day: sIL-17RD protein is added to the cell supernatant at a final concentration of 2 μg / mL, and the incubated nanobodies, positive control drugs celecoxib and triamcinolone are added to the treatment group; or the cells can be pretreated 2 h in advance: sIL-17RD protein and NLRP3 inhibitor Dapansutrile are added. c) After TNF-α for 24 hours, cell supernatant was collected and ELISA was performed to analyze the cleavage of secreted IL-1β; at the same time, cell lysate was collected and immunoblot was performed on pyroptosis-related markers N-GSDMD and total GSDMD protein, P2RX7.

[0137] The results are as follows Figure 12 As shown, knockdown of P2RX7 inhibits TNF-α-induced sIL-17RD-mediated chondrocyte pyroptosis and IL-1β secretion. Figure 12 A-12D); the NLRP3 inhibitor Dapansutrile significantly inhibited TNF-α-induced sIL-17RD-mediated chondrocyte pyroptosis (A-12D); Figure 12 The CQ5 nanobody significantly inhibited TNF-α-induced sIL-17RD-mediated chondrocyte pyroptosis. Furthermore, compared to celecoxib and triamcinolone, CQ5's inhibitory effect on TNF-α-induced sIL-17RD-mediated chondrocyte pyroptosis was superior to both positive control drugs. These results indicate that the CQ5 neutralizing nanobody inhibits TNF-α-induced P2RX7-NLFR3 inflammasome-mediated chondrocyte pyroptosis.

[0138] Example 13 This embodiment provides an in vitro method for verifying P2rx7-mediated inhibition of chondrocyte proliferation by sIL-17RD, and an in vivo method for promoting chondrocyte proliferation using the sIL-17RD-neutralizing nanobody CQ5. Details are as follows: 1. Verify that P2rx7 mediates sIL-17RD's inhibition of chondrocyte proliferation: a) The C28 chondrocyte cell line was seeded onto a plate the day before; b) On the second day, C28 cells were transfected with siRNAs of control and P2RX7 cells via liposomes and cultured for another 24 h. On the second day, the cells were pretreated 2 h in advance: sIL-17RD protein was added to the cell supernatant at a final concentration of 2 μg / mL. c) After TNF-α stimulation for 48-72 h, cells were collected, and the methods for fixation, Ki67 fluorescence staining, and DAPI counterstaining are detailed in the fluorescence staining section of Example 10. 2. CQ5, a neutralizing nanobody targeting sIL-17RD, promotes chondrocyte proliferation. a) For C57BL / 6 mice, an MMT model was established, and the injection protocol was as before. The mice were grouped as follows. 1. Sham surgery group, 3 animals; 2. MMT+GFP control protein (intra-articular injection, 0.25 mg / kg), 3 animals; 2. Medium-dose MMT+CQ5 group (intra-articular injection, MMT+CQ5, 0.25 mg / kg), 3 animals; b) For details on cartilage tissue fixation, Ki67 fluorescent staining, DAPI counterstaining, etc., please refer to the fluorescent staining section under Example 10.

[0139] The results are as follows Figure 13 As shown, knocking down P2RX7 reverses TNF-α-induced sIL-17RD-mediated inhibition of chondrocyte proliferation ( Figure 13 (A-13C) indicates that TNF-α-induced sIL-17RD-mediated inhibition of chondrocyte proliferation is mediated through P2RX7.

[0140] Compared with the control group GFP, CQ5 nanobody significantly promoted cartilage proliferation in MMT mice. Figure 13 These results indicate that the CQ5 neutralizing nanobody promotes chondrocyte proliferation by neutralizing sIL-17RD and inhibiting the TNF-α-P2RX7 pathway in chondrocytes.

[0141] Example 14 This embodiment provides a method for detecting subchondral osteoclast formation in a mouse model of osteoarthritis using the sIL-17RD neutralizing nanobody CQ5. Details are as follows: a) For C57BL / 6 mice, an MMT model was established, and the injection protocol was as before. The mice were grouped as follows. 1. Sham surgery group, 5 animals; 2. MMT+GFP control protein (intra-articular injection, 0.25 mg / kg), 5 animals; 2. Medium-dose MMT+CQ5 group (intra-articular injection, MMT+CQ5, 0.25 mg / kg), 5 animals; b) After collecting, fixing, embedding, and sectioning the tibia, refer to the commercial TRAP staining kit for the TRAP staining method for TRAP staining and DAPI counterstaining.

[0142] The results are as follows Figure 14 As shown, compared with the control GFP group, intra-articular injection of CQ5 significantly reduced the number of TRAP-positive osteoclasts in the subchondral bone. This result demonstrates that CQ5 has a protective effect on the subchondral bone in osteoarthritis.

[0143] Example 15 This embodiment provides a method for evaluating the tissue safety of intra-articular injection of CQ5 nanobodies. Details are as follows: a) For C57BL / 6 mice, an MMT model was established, and the injection protocol was as before. The mice were grouped as follows. 1. MMT+GFP control protein (intra-articular injection), 5 animals; 2. Medium-dose MMT+CQ5 group (intra-articular injection, CQ5, 1.25 mg / kg), 5 animals; b) After intra-articular injection of GFP and CQ5 nanobodies 6 times, the heart, liver, spleen, lung, kidney and brain of MMT model osteoarthritis mice were collected, fixed and embedded, and the H&E staining results are detailed in item 3 of Example 6.

[0144] Analysis results as follows Figure 15 As shown, after six intra-articular injections of the high-dose CQ5 nanobody, similar to the GFP group, there were no significant pathological changes in the heart, liver, spleen, lungs, kidneys, and brain, indicating the high safety of CQ5 nanobody treatment.

[0145] By combining the above screening, expression, and functional verification methods, we can not only obtain nanobodies with high affinity for soluble interleukin-17 receptor D, but also effectively screen antibody molecules with clear neutralizing functions, thereby ensuring the biological effectiveness of the obtained nanobodies in subsequent applications.

[0146] Therefore, the above method can be used stably and reliably to prepare the neutralizing nanobody described in this invention.

[0147] The experimental results of the above embodiments demonstrate that the neutralizing nanobody described in this invention can effectively block the inflammatory signaling pathway mediated by soluble interleukin-17 receptor D, significantly reduce the expression levels of inflammatory factors, and alleviate inflammatory responses and pathological damage in related tissues. Therefore, the nanobody described in this invention has clear application value in the preparation of drugs for the prevention and / or treatment of diseases related to soluble interleukin-17 receptor D dysfunction, and is particularly suitable for inflammatory diseases such as osteoarthritis.

[0148] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A neutralizing nanobody targeting soluble interleukin-17 receptor D (sIL-17RD), characterized in that, The heavy chain variable region (VHH) of the nanobody includes a heavy chain complementarity-determining region 3 (CDR-H3) that specifically binds to sIL-17RD. The amino acid sequence of CDR-H3 is characterized by being 10 amino acids in length and containing multiple lysine residues (K, 2-6). The nanobody can specifically bind to sIL-17RD and neutralize its biological activity.

2. The neutralizing nanobody according to claim 1, characterized in that, The amino acid sequence of the CDR-H3 is selected from any of the following sequences or has at least 80% identity with any of the following sequences; (1) KQKHKNRSWT (SEQ ID NO.1); (2) TKKKKKKPKI (SEQ ID NO.2); (3) MKKKKNHTTT (SEQ ID NO.3); (4)KPKKKKKRKP (SEQ ID NO. 4).

3. The neutralizing nanobody according to claim 2, characterized in that, The heavy chain variable region (VHH) of the nanobody further includes heavy chain complementarity-determining region 1 (CDR-H1) and heavy chain complementarity-determining region 2 (CDR-H2), and the combinations of CDR-H1 and CDR-H2 correspond as follows: When CDR-H3 is SEQ ID NO.1, CDR-H1 is RIFSYYR (SEQ ID NO.5), and CDR-H2 is DISATGATTY (SEQ ID NO.6). When CDR-H3 is SEQ ID NO.2, CDR-H1 is RIFSKYE (SEQ ID NO.7), and CDR-H2 is SSISSEGGTTN (SEQ ID NO.8). When CDR-H3 is SEQ ID NO.3, CDR-H1 is FIFSTYP (SEQ ID NO.9), and CDR-H2 is DIYNSGGNTY (SEQ ID NO.10). When CDR-H3 is SEQ ID NO.4, CDR-H1 is LTFIRNR (SEQ ID NO.11), and CDR-H2 is SINHSGASTY (SEQ ID NO.12).

4. The neutralizing nanobody according to any one of claims 1-3, characterized in that, The nanobody is selected from CQ5, CQ13, CQ11 and CQ6, the amino acid sequence of CQ5 is shown in SEQ ID NO.13, the amino acid sequence of CQ13 is shown in SEQ ID NO.14, the amino acid sequence of CQ11 is shown in SEQ ID NO.15 and the amino acid sequence of CQ6 is shown in SEQ ID NO.

16.

5. The neutralizing nanobody according to claim 4, characterized in that, The nucleotide sequence encoding CQ5 is shown in SEQ ID NO.17, the nucleotide sequence encoding CQ13 is shown in SEQ ID NO.18, the nucleotide sequence encoding CQ11 is shown in SEQ ID NO.19, and the nucleotide sequence encoding CQ6 is shown in SEQ ID NO.

20.

6. A nucleic acid molecule encoding a neutralizing nanobody as described in any one of claims 1-5, characterized in that, The nucleic acid molecule is in the form of DNA or RNA, preferably a DNA molecule that has been codon-optimized for use in prokaryotic or eukaryotic host expression systems.

7. A recombinant expression vector, characterized in that, The recombinant expression vector is selected from prokaryotic expression vectors, yeast expression vectors, insect cell expression vectors and / or mammalian cell expression vectors, and contains the nucleic acid molecules as described in claim 6.

8. A host cell, characterized in that, The host cell comprises the recombinant expression vector as described in claim 7, or has the nucleic acid molecule as described in claim 6 integrated into its genome, and the host cell is selected from Escherichia coli, Pichia pastoris, HEK293 cells, CHO cells and / or their derived cell lines.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of the neutralizing nanobody as described in any one of claims 1-5 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, excipients or diluents.

10. A method for preparing neutralizing nanobodies as described in any one of claims 1-5, characterized in that, The method includes the following steps: (1) Provide human-derived sIL-17RD; (2) Screening nanobodies from the antibody library that can specifically bind to the sIL-17RD; (3) The selected nanobodies are functionally validated to obtain target nanobodies that can neutralize sIL-17RD.

11. Use of the neutralizing nanobody as described in any one of claims 1-5 in the preparation of a medicament for the prevention and / or treatment of inflammatory osteoarthritis.

12. The application according to claim 11, characterized in that, The neutralizing nanobody reduces synovitis by inhibiting the expression and function of sIL-17RD, reduces chondrocyte death by inhibiting the P2rx7-NLRP3 inflammasome-mediated pyroptosis pathway, promotes chondrocyte proliferation, and maintains extracellular matrix homeostasis, thereby protecting articular cartilage, reversing cartilage degeneration, and alleviating pain.

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